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WO2025012429A2 - Dispositif et procédés d'enrichissement et d'isolement de molécules d'acide nucléique - Google Patents

Dispositif et procédés d'enrichissement et d'isolement de molécules d'acide nucléique Download PDF

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Publication number
WO2025012429A2
WO2025012429A2 PCT/EP2024/069813 EP2024069813W WO2025012429A2 WO 2025012429 A2 WO2025012429 A2 WO 2025012429A2 EP 2024069813 W EP2024069813 W EP 2024069813W WO 2025012429 A2 WO2025012429 A2 WO 2025012429A2
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WIPO (PCT)
Prior art keywords
particles
nucleic acid
magnetic field
particle retaining
unit
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PCT/EP2024/069813
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WO2025012429A3 (fr
Inventor
Gerd Lüdke
Ralf Himmelreich
Andreas Boos
Wolfgang Maurer
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Curetis GmbH
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Curetis GmbH
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Publication of WO2025012429A3 publication Critical patent/WO2025012429A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/30—Combinations with other devices, not otherwise provided for
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications

Definitions

  • the present invention relates to methods for enriching and isolating nucleic acid molecules using e.g. magnetizable particles.
  • the present relates to a device for processing nucleic acid molecules using e.g. magnetizable particles.
  • Known methods of isolating nucleic acid molecules from complex starting materials like whole blood, blood serum, urine, feces, or cell culture material usually comprise lysis of biological material by a detergent in the presence of protein degrading enzymes, followed by several extractions with organic solvents, e.g., phenol and/or chloroform, ethanol precipitation and dialysis of the nucleic acids.
  • organic solvents e.g., phenol and/or chloroform
  • ethanol precipitation e.g., ethanol precipitation and dialysis of the nucleic acids.
  • a pathogen e.g. a virus or a bacterium
  • a nucleic acid amplification method for example the utmost sensitive polymerase- chain-reaction
  • the present inventors have developed a procedure which allows the dispersion, enrichment, and isolation of nucleic acid molecules with magnetizable particles or other particles.
  • magnetizable particles such as magnetizable beads were used for mixing components of a composition.
  • the present inventors have rather used these magnetizable particles for both, the homogenization and collection of nucleic acid molecules. This may allow the purification of nucleic acid molecules in high yields and quality, especially automated.
  • the present invention relates to a method for enriching nucleic acid molecules comprising the steps of
  • composition which comprises the following components: a sample material comprising nucleic acid molecules, a nucleic acid molecule binding buffer, magnetizable particles which are capable of binding nucleic acid molecules, and a magnetizable stirring element,
  • the present invention relates to a method for isolating nucleic acid molecules comprising the steps of
  • the present invention relates to a particle retaining device for processing nucleic acid molecules using particles, comprising: at least one receiving space for a fluidic system comprising at least one particle retaining portion, at least one particle retaining unit configured to apply an external magnetic field to the particle retaining portion in order to provide retaining forces to the particles within the particle retaining portion which retain the particles within the particle retaining portion, a fluid transport unit configured to transport fluid through the particle retaining portion, and a control unit configured to control the at least one particle retaining unit and the fluid transport unit in a fluid transport operation state/mode of the device such that the at least one particle retaining unit retains the particles within the at least one particle retaining portion when the fluid transport unit transports a fluid through the particle retaining portion.
  • the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (TUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.
  • the term “about” indicates a certain variation from the quantitative value it precedes.
  • the term “about” allows a ⁇ 5% variation from the quantitative value it precedes, unless otherwise indicated or inferred.
  • the use of the term “about” also includes the specific quantitative value itself, unless explicitly stated otherwise. For example, the expression “about 80°C” allows a variation of ⁇ 4°C, thus referring to range from 76°C to 84°C.
  • the at least one substance may be a biological substance, such as a microbiological substance.
  • the term “at least one substance”, according to the present invention, refers to a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule).
  • the at least one substance comprises or consists of a nucleic acid molecule (e.g. DNA or RNA molecule) from a microbial species such as bacteria or viruses.
  • a nucleic acid molecule e.g. DNA or RNA molecule
  • a microbial species such as bacteria or viruses.
  • a substance specifically a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule) (from a microbial species), which is associated with a particular disease or condition or with a specific disease or condition stage.
  • a nucleic acid molecule e.g. DNA or RNA molecule
  • microbial species from a microbial species
  • the at least one substance e.g. nucleic acid molecule such as DNA or RNA molecule
  • the at least one substance which is enriched and/or isolated with the device of the present invention is preferably labelled with a detectable dye specifically fluorescence marker/probe such as fluorophore.
  • the nucleic acid molecule such as DNA or RNA molecule as the at least one substance is labelled with a detectable dye specifically fluorescence marker/probe such as TaqMan probe.
  • a TaqMan probe is a hydrolysis probe that is designed to increase the specificity of quantitative PCR.
  • the at least one substance is part of a sample such as biological sample as described herein. If the sample contains cellular material and the at least one substance (e.g. nucleic acid molecule such as DNA or RNA molecule) is contained therein, the cellular material needs to be lysed first in order to release the at least one substance (e.g. nucleic acid molecule such as DNA or RNA molecule) from the cells. Subsequently, the at least one substance (e.g. nucleic acid molecule such as DNA or RNA molecule) is isolated from the cell debris and then purified. In case the sample already contains the at least one substance (e.g.
  • nucleic acid molecule such as DNA or RNA molecule
  • a PCR reaction is preformed to amplify the nucleic acid molecule such as DNA or RNA molecule before detection.
  • the PCR reaction is preferably conducted in the presence of a TaqMan probe.
  • the RNA is first transcribed into cDNA (complementary DNA) before the amplification reaction is performed.
  • a TaqMan probe consists of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3 ’-end.
  • fluorophores e.g. 6- carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET
  • quenchers e.g. tetramethylrhodamine, acronym: TAMRA
  • the quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Forster resonance energy transfer (FRET).
  • TaqMan probes are designed such that they anneal within a nucleic acid such as DNA region amplified by a specific set of primers.
  • TaqMan probes can be conjugated to a minor groove binder (MGB) moiety, dihydrocyclopyrroloindole tripeptide (DPI3), in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature (T m ) due to increased stabilization of van der Waals forces.
  • MGB minor groove binder
  • DPI3 dihydrocyclopyrroloindole tripeptide
  • the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus, relieving the quenching effect and allowing fluorescence of the fluorophore.
  • fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of nucleic acid such as DNA template present in the PCR. This signal can then be detected with a corresponding detection unit.
  • nucleic acid molecule encompasses DNA as well as RNA molecules, both in any possible configuration, i.e. in the form of double- stranded (ds) nucleic acid, or in the form of single-stranded (ss) nucleic acid, or as a combination thereof (in part ds or ss).
  • RNA molecule may be mRNA, tRNA, rRNA, or a mixture thereof.
  • cell lysis refers to a technique that destroys and/or disrupts cells for the purpose of analyzing the contents of the cells, such as analyzing the at least one substance (e.g. nucleic acid molecule such as DNA or RNA molecule) contained in the cells.
  • the cells may be mammalian cells and/or microbial cells, such as bacterial and yeast cells.
  • lysate refers to the product of enzymatic, osmotic, and/or mechanical disruption of the cell membranes of a cell population.
  • Cell lysates are widely used for the isolation of cellular components such as nucleic acid molecules like DNA or RNA molecules, proteins, or whole organelles.
  • a lysate is produced in order to detect, analyze, and/or quantify the at least one substance, specifically the substance comprising or consisting of a nucleic acid molecule comprised in the cell population.
  • disease refers to an abnormal condition that affects the body of an individual.
  • a disease is often construed as a medical condition associated with specific symptoms and signs.
  • the term “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one’s perspective on life, and one’s personality.
  • the at least one substance is preferably associated with infectious diseases, inflammatory diseases, sepsis, autoimmune diseases, cancer diseases (or simply cancer), or any combinations thereof.
  • infectious disease refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease. Said diseases are caused by a virus, a bacterium, a fungus, and/or a parasite. The substance that causes an infectious disease can also be designated as pathogen.
  • pathogen refers to a virus, a bacterium, a fungus, and/or a parasite that can cause an infectious disease. It is then a pathogenic agent or substance.
  • the infectious disease is a respiratory disease such as pneumonia like hospitalized pneumonia, an implant or tissue infection, an intra-abdominal infection, or a urinary tract infection or a blood stream infection (e.g. if no other source of infection may be localized) or a CNS infection (meningitis/encephalitis).
  • respiratory disease such as pneumonia like hospitalized pneumonia, an implant or tissue infection, an intra-abdominal infection, or a urinary tract infection or a blood stream infection (e.g. if no other source of infection may be localized) or a CNS infection (meningitis/encephalitis).
  • respiratory disease such as pneumonia like hospitalized pneumonia, an implant or tissue infection, an intra-abdominal infection, or a urinary tract infection or a blood stream infection (e.g. if no other source of infection may be localized) or a CNS infection (meningitis/encephalitis).
  • respiratory disease refers to any disease affecting the respiratory system.
  • respiratory diseases as used herein include (i) obstructive lung diseases, (ii) restrictive lung diseases, (iii) respiratory tract infections, such as upper respiratory tract infections, e.g., common cold, sinusitis, tonsillitis, otitis media, pharyngitis, or laryngitis, and lower respiratory tract infections, e.g., pneumonia, (iv) respiratory tumors, e.g., small cell lung cancer, non-small cell lung cancer (e.g., adenocarcinoma, large cell undifferentiated carcinoma), other lung cancers such as carcinoid, Kaposi’s sarcoma, or melanoma, lymphoma, head and neck cancer, mesothelioma, and cancer metastasis in the lung such as from breast cancer, colon cancer, prostate cancer, germ cell cancer, and renal cell carcinoma, (v) pleural cavity diseases, e.g., empyema and mesothelioma, and (vi)
  • respiratory diseases that can be diagnosed using molecular diagnostics, preferably using nucleic acid amplification and analysis methods.
  • respiratory tract infections such as infections with pathogens, e.g., bacteria, viruses, yeast, or fungi, preferably yeast or bacteria, and respiratory tumors are preferred respiratory diseases in the context of the present invention.
  • Particularly more preferred respiratory diseases in the context of the present invention are pneumonias, in particular pneumonias caused by infections with pathogens, such as bacterial, viral, fungal, parasitic, atypical, community-acquired, healthcare-associated, hospital-acquired, ventilator-acquired pneumonia, or severe acute respiratory syndrome, tuberculosis, bronchitis, pathogenic infections during cystic fibrosis or chronic obstructive pulmonary disease (COPD), and a respiratory tumor.
  • pathogens such as bacterial, viral, fungal, parasitic, atypical, community-acquired, healthcare-associated, hospital-acquired, ventilator-acquired pneumonia, or severe acute respiratory syndrome, tuberculosis, bronchitis, pathogenic infections during cystic fibrosis or chronic obstructive pulmonary disease (COPD), and a respiratory tumor.
  • pathogens such as bacterial, viral, fungal, parasitic, atypical, community-acquired, healthcare-associated, hospital
  • the respiratory pathogens preferably include Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Stenotrophomonas maltophilia, Haemophilus parainfluenzae, Escherichia coli, Enterococcus faecalis, Serratia marcescens, Haemophilus parahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecium, Klebsiella oxytoca, Pseudomonas fhiorescens, Neisseria meningitidis, Streptococcus pyogenes, Pneumocystis jirovecii, Klebsiella pneumoniae, Legionella pneumophil
  • the hospitalized pneumonia is selected from the group consisting of hospitalized community-acquired pneumonia (hCAP), hospital-acquired (or nosocomial) pneumonia (HAP), ventilator-associated pneumonia (VAP), healthcare-associated pneumonia (HCAP), and severe community-acquired pneumonia (SCAP),
  • hCAP hospitalized community-acquired pneumonia
  • HAP hospital-acquired (or nosocomial) pneumonia
  • VAP ventilator-associated pneumonia
  • HCAP healthcare-associated pneumonia
  • SCAP severe community-acquired pneumonia
  • the implant or tissue infection is selected from the group consisting of burn wound infections, cardiology-associated infections, catheter-associated infections, deep skin and tissue infections, diabetic foot infections, orthopedic implant infections, implant infections and surgical site infections,
  • the intra-abdominal infection is selected from the group consisting of acute abdomen, ascites, cholecystitis, diverticulitis, peritonitis, and surgical site infections,
  • the urinary tract infection is selected from the group consisting of catheter-associated urinary tract infection, complicated cystitis, urosepsis, and pyelonephritis.
  • inflammatory disease refers to a disease in which the immune system attacks and/or damages the body’s own tissues, resulting in an inflammation.
  • the inflammatory disease is selected from the group consisting of atherosclerosis, an autoimmune disease, allergy, asthma, a coeliac disease, glomerulonephritis, hepatitis, and an inflammatory bowel disease.
  • inflammation may be part of the normal healing process.
  • the substance (under test) may be related to an inflammation resulting from normal healing process or from an inflammatory disease.
  • sepsis refers to a life-threatening complication of a wide variety of infectious diseases. Sepsis arises when the body’s response to an infection causes injury to its own tissues and organs. Sepsis is usually caused by an inflammatory immune response triggered by the infection. Most commonly, the infection is bacterial, but it may also be fungal, viral, or protozoan. Disease severity partly determines the outcome. The risk of death from sepsis is as high as 30%, from severe sepsis as high as 50%, and from septic shock as high as 80%.
  • cancer disease refers to or describe the physiological condition in an individual that is typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, lung cancer, preferably non-smallcell lung carcinoma (NSCLC) or small-cell lung carcinoma (SCLS), breast cancer, cervical cancer, gastric cancer, bladder cancer, skin cancer, nasopharyngeal cancer, neuroendocrine cancer, colon cancer, urothelial cancer, liver cancer, ovarian cancer, esophageal cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, renal cancer, head and neck cancer, brain cancer, lymphatic cancer, blood cancer, squamous cell cancer, laryngeal cancer, retina cancer, prostate cancer, uterine cancer, testicular cancer, bone cancer, lymphoma, and leukemia.
  • cancer also encompasses cancer metastases.
  • a substance specifically a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule) that is indicative of a microorganism.
  • a nucleic acid molecule e.g. DNA or RNA molecule
  • exemplary microorganisms include but are not limited to a bacterium, virus, fungus and protozoa.
  • the substance can be a pathogen.
  • Substances such as pathogens that can be enriched and/or isolated with the device of the present invention include, but are not limited to, Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus ho minis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influnzae, Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.
  • MSRA methicillin-resistant Staphylococcus aureus
  • Staphylococcus aureus Staphylococcus ho minis
  • Enterococcus faecalis Pseudomonas aeruginosa
  • Substances that can be enriched and/or isolated with the device of the present invention also encompass substances responsible for a variety of sexually transmitted diseases selected from the following: gonorrhea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), Chlamydia (Chlamydia trachomatis), nongonococcal urethritis (Ureaplasm urealyticum), yeast infection (Candida albicans), chancroid (Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSV type I & II), HIV I, HIV II and hepatitis A, B, C, G, as well as hepatitis caused by TTV (torque teno virus).
  • the substance comprises or consists of a nucleic acid molecule from the above microorganism.
  • the substance specifically the substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule), which is enriched and/or isolated with the device of the present invention can specifically be designated as analyte.
  • a nucleic acid molecule e.g. DNA or RNA molecule
  • the at least one substance may be part of a fluid, e.g. a liquid or a gas.
  • the fluid may be a fluidic sample.
  • the fluidic sample may be a processed, non-processed, native (as removed from the body or from another source) or not yet processed fluidic sample.
  • the fluidic sample may be any medium which is suitable to accommodate the at least one substance.
  • the sample handled, tested, or analyzed with the device of the invention can be of any origin or nature, for example of biological, chemical natural, synthetic, or semi-synthetic origin. The invention is, thus, not limited to any specific sample origin. Any sample suspected to contain the at least one substance, specifically the substance comprising or consisting of a nucleic acid molecule, can be used in conjunction with the device of the invention.
  • the sample is a biological sample (a processed, non-processed, native or not yet processed biological sample). More preferably, the biological sample is a bodily sample (a processed, non-processed, native or not yet processed bodily fluid). Even more preferably, the bodily sample is a bodily fluid (a processed, non-processed, native or not yet processed bodily fluid) or bodily tissue (a processed, non-processed, native or not yet processed bodily tissue) sample. Specifically, the bodily tissue sample is a liquified bodily tissue sample.
  • a processed biological sample is based on/derived from a biological material.
  • the processed biological sample is a lysed sample or an extracted sample.
  • the biological sample as describe herein preferably may comprise cells and said cells may contain, in turn, a substance comprising or consisting of a nucleic acid molecule.
  • the biological sample may represent a culture medium or a culture supernatant, e.g. microbial culture medium, microbial growth medium.
  • the culture medium or the culture supernatant may comprise prokaryotes (bacteria, viruses) or eukaryotes, especially prokaryotic (bacteria, viruses) or eukaryotic pathogens.
  • the bodily sample such as bodily fluid or bodily tissue sample may be incorporated directly into the device of the present invention without further processing.
  • the bodily sample such as bodily fluid or bodily tissue sample may also be pretreated before incorporation into the device of the present invention.
  • the choice of pretreatments will depend on the type of bodily sample such as bodily fluid or bodily tissue used sample and/or the nature of the substance. For instance, where the substance is present at low level in the bodily fluid or bodily tissue, the bodily fluid or bodily tissue can be concentrated via any conventional means to enrich the sub stance/ analyte.
  • Methods of concentrating the sub stance/ analyte include but are not limited to drying, evaporation, centrifugation, filtering, sedimentation, precipitation, and amplification.
  • the substance/analyte is a nucleic acid molecule (e.g. DNA or RNA molecule)
  • it can be extracted using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. ("Molecular Cloning: A Laboratory Manual"), or using nucleic acid binding resins following the accompanying instructions provided by manufactures.
  • the substance/analyte is a molecule present on or within e.g.
  • a cell having a cell nucleus or within an entity having no nucleus extraction can be performed using lysing agents including but not limited to denaturing detergent such as SDS (sodium dodecyl sulfate) or non-denaturing detergent such as thesit, sodium deoxylate, triton X-100, and tween-20.
  • denaturing detergent such as SDS (sodium dodecyl sulfate) or non-denaturing detergent such as thesit, sodium deoxylate, triton X-100, and tween-20.
  • the bodily tissue sample may be, for example, liquefied before incorporation into the device.
  • bodily sample refers to any sample that is derived from the body of an individual. Especially, the term “bodily sample” refers to any sample that is derived from the body of an individual and comprises a substance, specifically a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule).
  • a nucleic acid molecule e.g. DNA or RNA molecule.
  • the term “bodily sample” encompasses a bodily fluid sample and a bodily tissue sample.
  • tissue sample refers to any tissue sample that is derived from the body of an individual.
  • tissue sample refers to any sample that is derived from tissue of an individual and comprises a substance, specifically a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule).
  • Said bodily tissue sample encompasses a skin flake, skin biopsy, hair follicle, biopsy tissue, tissue explant, and tissue section.
  • the bodily tissue sample also encompasses tumor tissue sample.
  • Said bodily tissue sample may be removed from a patient or (control) subject by conventional biopsy techniques. It is preferred that the bodily tissue sample is a liquified bodily tissue sample.
  • bodily fluid sample refers to any fluidic sample that is derived from the body of an individual.
  • the term “bodily fluid sample” refers to any fluidic sample that is derived from fluid of an individual and comprises a substance, specifically a substance comprising or consisting of a nucleic acid molecule (e.g. DNA or RNA molecule).
  • a nucleic acid molecule e.g. DNA or RNA molecule.
  • any bodily fluid suspected to contain the at least one substance can be used in conjunction with the subject device and system.
  • the body fluid sample may be a respiratory sample, a blood sample, an urine sample, a sputum sample, a breast milk sample, a cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, a gastric juice sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.
  • CSF cerebrospinal fluid
  • cerumen earwax
  • the disease may be a respiratory disease.
  • the bodily sample such as a bodily fluid or bodily tissue sample is preferably taken for the purpose of a scientific test, such as for diagnosing a disease, e.g. a respiratory disease, for example, by detecting and/or identifying a pathogen or the presence of a tumor marker in a bodily sample which is preferably relevant for the diagnosis of a respiratory disease.
  • a bodily sample in the context of the present invention comprises cells, for example, pathogens or cells of the individual the bodily sample originated from, for example, tumor cells.
  • the preferred bodily samples are samples that are relevant for the diagnosis of a respiratory disease.
  • Such bodily samples may be respiratory samples, i.e. bodily samples derived from the respiratory tract, and non-respiratory samples, i.e. bodily samples that are not derived from the respiratory tract.
  • the respiratory tract in the context of the present invention preferably comprises the nose, nasal passages, paranasal sinuses, throat, pharynx, voice box, larynx, trachea, bronchi, bronchioles, and lungs, including respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.
  • respiratory samples in the context of the present invention are sputum, pus (e.g., pus from the paranasal cavity), bronchial secretion, tracheal secretion, endotracheal secretion, bronchial aspirates, tracheal aspirates, endotracheal aspirates, bronchial lavage, bronchoalveolar lavage (BAL), bronchial swab, nasopharyngeal swab, laryngeal swab, and lung biopsies.
  • Preferred non-respiratory samples used in the present invention are relevant for the diagnosis of respiratory diseases.
  • Preferred examples of non-respiratory samples in the context of the present invention are blood, pus, pleural fluid, pleural punctates, gastric juice, gastric aspirates, and drainages or punctate fluids from other body locations.
  • the term “individual”, as used herein, refers to any subject whose sample comprises a substance which may be enriched and/or isolated with the device of the present invention.
  • the individual is preferably an animal, more preferably a mammalian animal including a human being.
  • an individual in the context of the present invention may be a mouse, rat, guinea-pig, rabbit, cat, dog, goat, sheep, pig, cow, horse, or human, preferably a human.
  • the individual may be a patient, wherein the term “patient” refers to an individual suffering from a disease or condition, or being suspected of suffering from a disease or condition.
  • nucleotide refers to an organic molecule consisting of a nucleoside and a phosphate.
  • a nucleotide is composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates.
  • the four nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used in place of thymine.
  • the nucleotide serves as monomeric unit of nucleic acid molecules, such as deoxyribonucleotide acid (DNA) or ribonucleotide acid (RNA).
  • DNA deoxyribonucleotide acid
  • RNA ribonucleotide acid
  • the nucleotide is a molecular building-block of DNA and RNA.
  • the DNA molecule may be a double stranded DNA, a genomic DNA, or a complementary DNA (cDNA) molecule.
  • the RNA molecule may be a messenger RNA (mRNA), a small nucleolar RNA (snoRNAs), a ribosomal RNA (rRNA), or a transfer RNA (tRNA) molecule.
  • mRNA messenger RNA
  • snoRNAs small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • nucleotide sequence or “polynucleotide” are interchangeably used herein and refer to single-stranded and double-stranded polymers of nucleotide monomers, including without limitation, 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, e.g., H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • a nucleotide sequence or polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof and may include nucleotide analogs.
  • a “nucleic acid amplification method”, in the context of the present invention, is any molecular biological technique that is suitable for amplifying, i.e. multiplying, a nucleic acid, wherein the amplification may be linear or exponential.
  • nucleic acid amplification methods are polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA), multiple displacement amplification (MDA), Q-beta replicase amplification, and loop-mediated isothermal amplification.
  • the amplification method may be specific for a certain nucleic acid such as a specific gene or a fragment thereof, or may be universal such that all or a specific type of a nucleic acid, such as mRNA, is amplified universally.
  • the skilled person may design oligonucleotide primers which specifically hybridize to the nucleic acid of interest and use these primers in a PCR experiment.
  • a “nucleic acid analysis method” in the context of the present invention is any method that allows for detection and/or identification of a specific nucleic acid, wherein the term “detection” also comprises the quantitative determination of a nucleic acid.
  • the detection and/or identification may be based on specific amplification, for example, by the amplification of a specific DNA fragment using oligonucleotide primers specific for said DNA fragment in the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the detection and/or identification may also be achieved without amplification, for example, by sequencing the nucleic acid to be analyzed or by sequence specific hybridization, for example, in the context of a microarray experiment. Sequencing techniques and microarraybased analysis are well known procedures in the field. The sequencing includes next generation sequencing.
  • the nucleic acid to be isolated, amplified, detected, analyzed and/or identified may be DNA such as double stranded DNA, genomic DNA, or complementary DNA (cDNA).
  • the RNA may be messenger RNA (mRNA), small nucleolar RNA (snoRNAs), ribosomal RNA (rRNA), or transfer RNA (tRNA).
  • the nucleic acid amplification and/or analysis method is a polymerase chain reaction (PCR) or a reverse transcription polymerase chain reaction (RT-PCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • the PCR is selected from the group consisting of digital PCR, realtime PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the digital PCR may be digital droplet PCR or digital partition PCR.
  • PCR polymerase chain reaction
  • a PCR reaction may be carried out in a single test tube or chamber simply by mixing DNA (deoxyribonucleic acid) with a set of reagents and performing thermal cycles. Thereby, the following steps are repeated several times thereby doubling the number of double DNA strands in each cycle:
  • DNA polymerase thermoostable polymerase
  • PCR reverse-transcriptase-PCR
  • qRT-PCR or RT-qPCR real time quantitative PCR
  • inverse PCR irt-PCR
  • immune PCR immune PCR
  • agglutination-PCR e.g. PCR-Test (reverse-transcriptase-PCR), qRT-PCR or RT-qPCR (real time quantitative PCR), inverse PCR, irt-PCR (immunoquantitative real time PCR), immune PCR, agglutination-PCR.
  • RT-PCR reverse transcription polymerase chain reaction
  • a master mix For a PCR reaction, a master mix is used.
  • the master mix contains dNTPs (deoxyribose nucleoside triphosphate), e.g.
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguanosine TP
  • dTTP deoxythymidine TP
  • dCTP deoxy cytidineTP
  • Taq DNA polymerase enzymes MgCh, stabilizers, and enhancers in a reaction buffer.
  • Master mix, specific primers and/or universal primers and probes for the detection/amplification of the at least one substance and the at least one substance have to be brought together to perform the PCR reaction.
  • the term “probe”, as used herein, may refer to detectable “auxiliary” molecules or other materials, e.g. using optical detection or optical detection units.
  • the probe may comprise a Taqman probe.
  • a Taqman probe is a hydrolysis probe that is designed to increase the specificity of quantitative PCR.
  • the probe may have fluorescence characteristics in order to ease or to admit optical detection.
  • double marked (double labeled) probes with quenchers may be used, especially in order to enhance the substances under tests that may be tested within one chamber.
  • only one color may be detected using e.g. intercalating dyes or other dyes.
  • the PCR system may have applications in a broad range of molecular biology and biotech lab experiments, including cloning (or synthesis of specific DNA fragments), sequencing, genotyping, nucleic acid synthesis, gene expression, generation of NGS (next generation sequencing) libraries, and mutagenesis.
  • a PCR master mix may specifically help researchers and scientists to enhance their PCR assay performance by providing a spectrum of benefits, including saving time and reducing the chances of any errors/cross-contamination in preparing PCR formulations. They are often utilized in routine or high-yielding PCR.
  • PCR master mixes are available in liquid and lyophilized forms.
  • the liquid form mix is required to be stored at a temperature between -20°C to +4°C and is typically cheaper than the lyophilized or freeze-dried mixes.
  • Lyophilized PCR master mixes can be stored at ambient temperatures for a longer period. Moreover, they are easy to transport and while running PCR, the solution only needs to be reconstituted in the buffer solution, which comes with the master mix.
  • Master mixes for real-time PCR may further include at least one probe, especially a fluorescent compound and/or fluorescence enhancing compound and/or fluorescence suppression compound or molecule.
  • a probe especially a fluorescent compound and/or fluorescence enhancing compound and/or fluorescence suppression compound or molecule.
  • only one dye may be used within one chamber, e.g. an intercalating dye.
  • the probe may be provided separate from the master mix, e.g. within a detection chamber, e.g. within the (PCR) disc wheel mentioned below.
  • enriching nucleic acid molecules refers to a technique which allows the accumulation of nucleic acid molecules in a composition.
  • Nucleic acid molecule extraction and enrichment are the first steps of any amplification experiment no matter what kind of amplification is used to detect the nucleic acid molecule, e.g. a specific pathogen. Efficient nucleic acid molecule extraction and enrichment is essential to obtain good results using any molecular test.
  • the optimal extraction and enrichment method should fulfill the following conditions: speed, short working time, cost-effectiveness, high sensitivity and specificity, good reproducibility, and safety for environment, operator, etc.
  • the nucleic acid molecules are enriched or processed using e.g. magnetizable particles.
  • nucleic acid molecules refers to a technique which allows the separation of nucleic acid molecules from a composition.
  • the nucleic acid molecules are isolated using e.g, magnetizable particles.
  • nucleic acid molecule binding buffer refers to a buffer which is designed for use in the cleaning and concentration of nucleic acid molecules. This buffer allows and/or facilities efficient binding of nucleic acid molecules, e.g. to a solid support such as magnetizable particles, and recovery of nucleic acid molecules from enzymatic reactions and/or impure samples comprising nucleic acid molecules.
  • the nucleic acid binding buffer may contain a chaotropic salt (e.g. guanidinium isothiocyanate).
  • the nucleic acid binding buffer is preferably a buffer comprising a chaotropic agent and alcohol.
  • the chaotropic agent is selected from the group consisting of an agent comprising thiocyanate ions, iodine ions, perchlorate ions, nitrate ions, bromine ions, chlorine ions, acetate ions, fluorine ions, and sulfate ions, and/or the alcohol is selected from the group consisting of ethanol and isopropanol.
  • non-nucleic acid molecule binding buffer refers to a buffer which is designed to reduce or even prevent binding of nucleic acid molecules, e.g. to a solid support such as magnetizable particles. In this way, the nucleic acid molecules may be isolated from a composition.
  • the non-nucleic acid binding buffer (elution, release, de-binding), as used herein, is preferably a buffer selected from the group consisting of water, TRIS buffer having a pH of between 6 to 9, e.g. 6, 7, 8, or 9, NaOH buffer having a pH of about 9, and ammoniumchloride buffer having a pH of about 7.5.
  • Known methods of isolating nucleic acid molecules from complex starting materials like whole blood, blood serum, urine, feces, or cell culture material usually comprise lysis of biological material by a detergent in the presence of protein degrading enzymes, followed by several extractions with organic solvents, e.g., phenol and/or chloroform, ethanol precipitation and dialysis of the nucleic acids.
  • organic solvents e.g., phenol and/or chloroform
  • ethanol precipitation e.g., ethanol precipitation and dialysis of the nucleic acids.
  • a pathogen e.g. a virus or a bacterium
  • a nucleic acid amplification method for example the utmost sensitive polymerase- chain-reaction
  • the present inventors have developed a procedure which allows the dispersion, enrichment, and isolation of nucleic acid molecules with e.g. magnetizable particles.
  • magnetizable particles such as magnetizable beads were used for mixing components of a composition.
  • the present inventors have rather used these magnetizable particles for both, the homogenization and collection of nucleic acid molecules. This allows the purification of nucleic acid molecules in high yields and quality].
  • the present invention relates to a method for enriching nucleic acid molecules comprising the steps of:
  • composition which comprises the following components: a sample material comprising nucleic acid molecules, a nucleic acid molecule binding buffer, magnetizable particles which are capable of binding nucleic acid molecules, and a magnetizable stirring element,
  • This step may be optionally if e.g. a comparably round processing chamber is used and/or if the particles are included in the mixing/stirring by turbulences or other physical processes within the processing chamber.
  • the nucleic acid molecules are DNA or RNA molecules.
  • the DNA molecule may be a double stranded DNA, a genomic DNA, or a complementary DNA (cDNA) molecule.
  • the RNA molecule may be a messenger RNA (mRNA), a small nucleolar RNA (snoRNAs), a ribosomal RNA (rRNA), or a transfer RNA (tRNA) molecule.
  • mRNA messenger RNA
  • snoRNAs small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • the sample material comprising nucleic acid molecules may be selected from the group consisting of an aqueous solution, a processed sample, and a biological sample, preferably a cell lysate, a microbial culture, or a processed, e.g. lysed, biological sample.
  • the composition may be provided in step (i) in a container or reaction vessel.
  • the composition may be a liquid composition such as an aqueous composition.
  • the composition provided in step (i) may be a dispersion, e.g. an aqueous dispersion.
  • the nucleic acid binding buffer provided in step (i) may be a buffer that creates conditions that allow binding of nucleic acid molecules to particles.
  • the nucleic acid binding buffer may be a buffer comprising a chaotropic agent and alcohol.
  • the chaotropic agent may be selected from the group consisting of an agent comprising thiocyanate ions, iodine ions, perchlorate ions, nitrate ions, bromine ions, chlorine ions, acetate ions, fluorine ions, and sulfate ions
  • the alcohol may be selected from the group consisting of ethanol and isopropanol.
  • the chaotropic agent comprising thiocyanate ions may be guanidinium (iso)thiocyanate or guanidiniumchloride.
  • the nucleic acid molecule binding buffer comprises maleic acid, sodium hydroxide, Tween 20, and guanidine hydrochloride, e.g. Qiagen AL.
  • the generation of the magnetic field in step (ii) magnetizes the particles and magnetizes the stirring element, e.g. a stirring bar. This may allow the adherence of the magnetized particles at the stirring element, e.g. the stirring bar. Thus, the magnetized particles may adhere to the magnetized stirring element due to magnetic forces.
  • the external magnetic field may be generated by a permanent magnet, which may be more preferably located at a defined distance from the particles. Even more preferably, the permanent magnet may be located at a distance of e.g. between 1 mm (millimeter) to 5 mm from the particles .
  • the magnetic field may be generated by an electromagnet.
  • the magnetic flux density B/magnetic induction may be a measure of the density of magnetic field lines passing through a given area. It describes the intensity of the magnetic field and how strongly it can affect magnetic material.
  • the magnetic flux density at a point depends on the strength of the magnetic source (such as magnet or an electric current) as well as the distance from the source. The closer the distance of the particles and/or stirring element to the magnetic source, the higher the magnetic flux density/magnetic induction.
  • the stirring element in step (iii) may rotate/move with a speed of preferably between > 1600 rotations per minute (rpm) and ⁇ 3000 rpm, of more preferably between > 1800 and ⁇ 2500 rpm or between > 1850 and ⁇ 2500 rpm, of even more preferably between > 2000 and ⁇ 2500 rpm.
  • the stirring element in step (iii) moves with a speed of preferably > 2000 rotations per minute (rpm).
  • the centrifugal force may be stronger than the force adhering the particles to the stirring element so that the particles and the other components comprised in the composition can be mixed with each other.
  • step (iii) has the effect of homogenizing the components of the composition and the effect of dispersing the particles within the composition.
  • the generation of the magnetic field and the application of centrifugal forces separates/isolates the particles, e.g. from each other and/or from the optional stirring element. Accordingly, no particle clot formation occurs which leads to the subsequent enrichment of nucleic acid molecules at the particles in high yield and quality (step (iv)).
  • the density B of the magnetic field in step (iv) may be preferably reduced within a particle retaining chamber or a within a processing chamber by increasing/extending the distance between the permanent magnet and the particles, or by shielding the permanent magnet from the particles, or otherwise, e.g. decreasing the electrical power of or switching off an electromagnet.
  • the distance between the permanent magnet and the particles may be increased/extended by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in step (iv) may be reduced within a particle retaining chamber or a within a processing chamber to less than 0.1 T or to less than 0.05 T.
  • the shielding of the permanent magnet may be achieved by using shielding materials, e.g. comprising MU metal (comprising or consisting of nickel, alloy of nickel and other metal, e.g. iron, etc.), soft iron, etc.
  • shielding materials e.g. comprising MU metal (comprising or consisting of nickel, alloy of nickel and other metal, e.g. iron, etc.), soft iron, etc.
  • the magnetic field density B of the magnetic field in optional step (v) may be preferably increased within a particle retaining chamber or a within a processing chamber by decreasing/shortening the distance between the permanent magnet and the particles, e.g. beads, or by removing the shielding of the permanent magnet.
  • the distance between the permanent magnet and the particles may be decreased/shortened by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in optional step (v) may be, for example, increased within a particle retaining chamber or a within a processing chamber to a value within the range of 0.5 T to 1.8 T, 0.8 T to 1.6 T or of 1 T to 1.5 T. Due to this process, the particles, e.g. beads having the nucleic acid molecules adhered thereto are allowed to adhere to the stirring element.
  • the density B of the magnetic field in step (iv) may be preferably reduced by decreasing the power of the electromagnet or switching off the electromagnet.
  • the magnetic flux density of the electromagnet may be decreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in step (iv) may be reduced within a particle retaining chamber or a within a processing chamber to 0 T or a comparably low value. Due to this process, the nucleic acid molecules are allowed to adhere to the particles. In other words, due to this process, the nucleic acid molecules are collected by the particles.
  • the intensity (B) of the magnetic field in optional step (v) may be preferably increased by increasing the power of the electromagnet or switching on the electromagnet.
  • the magnetic flux density of the electromagnet may be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in optional step (v) may be, for example, increased within a particle retaining chamber or a within a processing chamber to a value within the range of 0.5 T to 2.5 T, 0.8 to 2.3 T or of 1 T to 2 T. Due to this process, the particles having the nucleic acid molecules adhered thereto are allowed to adhere to the stirring element.
  • the nucleic acid molecules adhere to the particles e.g. due to ionic interactions. Ionic interactions may arise from electrostatic attraction between two groups of opposite charge. These bonds may be formed between positively charged side chains and negatively charged groups.
  • the magnetizable particles which are capable of binding nucleic acid molecules are preferably coated with a sorbent material.
  • the sorbent material may be selected from the group consisting of SiCh crystals, amorphous silicon oxide and glass powder, alkylsilica, aluminum silicate (zeolite), or, activated silica with -NH2. While the nucleic acid molecules are negatively charged, the sorbent material may be positively charged, so that ionic interactions between the negatively charged groups and the positively charged groups occur.
  • the magnetizable particles may have a diameter of between 0.1 pm and 20 pm, preferably of between 0.5 pm and 15 pm, and more preferably of between 1 pm and 10 pm, e.g. of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pm.
  • at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or even 100% of the particles comprised in the composition are within this range.
  • the method for enriching nucleic acid molecules may comprise the steps of:
  • composition which comprises the following components: a sample material comprising nucleic acid molecules, a nucleic acid molecule binding buffer comprising a chaotropic agent and an alcohol, magnetizable particles coated with a sorbent material which are capable of binding nucleic acid molecules, and a magnetizable stirring element,
  • the method for enriching nucleic acid molecules may comprise the steps of:
  • composition which comprises the following components: a sample material comprising nucleic acid molecules, a nucleic acid molecule binding buffer comprising a chaotropic agent and an alcohol, magnetizable particles coated with a sorbent material which are capable of binding nucleic acid molecules, and a magnetizable stirring element,
  • the composition may be comprised in a reaction vessel/container.
  • the magnetic field may be preferably generated by a magnet located outside the reaction vessel/container, which magnet may be particularly movable in X, Y, and/or Z direction.
  • the magnetic field may be generated in step (ii) so that the particles, e.g. bead comprised in the composition adhere to the stirring element as well as to the side of the reaction vessel/container facing the magnet.
  • the increase of the magnetic flux density B in optional step (v) may allow adherence of the particles having the nucleic acid molecules adhered thereto to the stirring element as well as to the side of the reaction vessel/container facing the magnet.
  • the method mentioned above may result in the enrichment of nucleic acid molecules at the particles or optionally at the stirring element to which the particles having the nucleic acid molecules adhered are attached.
  • the present invention relates to a method for isolating nucleic acid molecules comprising the steps of:
  • the nucleic acid molecules are DNA or RNA molecules.
  • the DNA molecule may be a double stranded DNA, a genomic DNA, or a complementary DNA (cDNA) molecule.
  • the RNA molecule may be a messenger RNA (mRNA), a small nucleolar RNA (snoRNAs), a ribosomal RNA (rRNA), or a transfer RNA (tRNA) molecule.
  • mRNA messenger RNA
  • snoRNAs small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • the non-nucleic acid binding buffer may be a buffer that creates conditions that allow elution of nucleic acid molecules from the particles.
  • This buffer can also be designated as nucleic acid molecule elution buffer.
  • the non-nucleic acid molecule binding buffer (nonNAMbb) may be selected from the group consisting of water, TRIS buffer having a pH of between 6 to 9, NaOH buffer having a pH of about 9, and ammoniumchloride buffer having a pH of about 7.5.
  • the method for isolating nucleic acid molecules comprises the steps of:
  • the stirring element in step (c) may rotate/move with a speed of preferably between > 1000 rpm or > 1600 rotations per minute (rpm) and ⁇ 3 000 rpm, of more preferably between > 1800 and ⁇ 2500 rpm or between > 1850 and ⁇ 2500 rpm, of even more preferably between > 2000 and ⁇ 2500 rpm.
  • the stirring element may rotate/move with a speed of preferably > (greater than) 2000 rotations per minute (rpm).
  • the centrifugal force may be stronger than the force adhering the particles having the nucleic acid molecules adhered thereto to the stirring element. Thereby, the particles are dispersed in the non-nucleic acid molecule binding buffer, whereby the nucleic acid molecules are removed from the particles.
  • stirring may be made within a range of 0.5 seconds to 10 seconds.
  • a lower rotation speed may be compensated by a longer rotation duration and vice versa.
  • the intensity of the magnetic field in optional step (d) may be preferably increased by decreasing/shortening the distance between the permanent magnet and the particles, or by removing the shielding of the permanent magnet.
  • the distance between the permanent magnet and the particles may be decreased/ shortened by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in optional step (d) is, for example, increased at the particles to a value in the range of 0.5 T to 1.8 T, 0.8 t to 1.6 T or 1 T to 1.5 T if a permanent magnet is used. Due to this process, the (unloaded) particles (free of nucleic acid molecules) adhere to the stirring element.
  • the density B of the magnetic field in optional step (d) may be preferably increased by increasing the power of the electromagnet or switching on the electromagnet.
  • the power of the electromagnet may be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • the magnetic flux density B/magnetic induction in optional step (d) may be, for example, increased within a particle retaining chamber or a within a processing chamber to a value within the range of 0.5 T to 2.5 T or of 0.8 T to 2.3 T or 1 T to 2 T. Due to this process, the (unloaded) particles (free of nucleic acid molecules) may adhere to the stirring element.
  • the separation/removal of the nucleic acid molecules within the fluid from the particles may be achieved not only via magnetic separation (very fast) but also via sedimentation, filtration or centrifugation.
  • the method comprises after step (i) and before step (ii) the following steps:
  • the stirring element may rotate/move with a speed of preferably between > 1000 rpm or > 1600 rotations per minute (rpm) and ⁇ 3000 rpm, of more preferably between > 1800 and ⁇ 2500 rpm or between > 1850 and ⁇ 2500 rpm, of even more preferably between > 2000 and ⁇ 2500 rpm.
  • the stirring element in step (iii) rotates/moves with a speed of preferably > 2000 rotations per minute (rpm).
  • the centrifugal force may be stronger than the force adhering the particles having the nucleic acid molecules adhered thereto to the stirring element.
  • the nucleic acid molecules are removed from the stirring element. They are dispersed in the (first) washing buffer, whereby the particles are washed.
  • the magnetic flux density B at the particles may be increased to a value in the range of 0.5 T to 1.8 T or 0.8 T to 1.6 T or 1 T to 1.5 T if a permanent magnet is used or to a value in the range of 0.5 T to 2.5 T, 0.8 T to 2.3 T or 1 T to 2 T if an electromagnet is used.
  • the particles having the nucleic acid molecules adhered thereto are allowed to adhere to the stirring element.
  • the method steps (a, b, and c) mentioned above may be repeated (see d, e, and f), specifically under the same conditions, e.g. at least once, twice, etc.
  • the first washing buffer differs from the one further washing buffer in salt and alcohol content. More specifically, the first washing buffer has a higher salt content and a lower alcohol content than the one further washing buffer.
  • the first washing buffer comprises between 10% and 40%, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40%, salt and between 30 to 60%, e.g. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60%, alcohol.
  • the one further washing buffer may be a second, or third washing buffer.
  • the second washing buffer comprises between 70 to 80%, e.g.
  • alcohol and/or the third washing buffer comprises between 90 to 100%, e.g. 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% alcohol, e.g. mass percent or volume percent.
  • a particle retaining device for processing nucleic acid molecules using particles is provided.
  • the particle retaining device should operate in a simple way and/or should have a simple construction.
  • the particle retaining device should fulfill at least two different functions, preferably a plurality of functions, e.g. retaining the particles, e.g. beads within a particle retaining portion and/or releasing the particles from a retaining state and/or perform at least one mixing function, etc.
  • the particle retaining device may comprise at least one, several or all of
  • At least one receiving space for a fluidic system comprising at least one particle retaining portion, and/or
  • At least one particle retaining unit configured to apply an external magnetic field to the particle retaining portion in order to provide retaining forces to the particles within the particle retaining portion which retain the particles within the particle retaining portion, and/or
  • a fluid transport unit configured to transport fluid through the particle retaining portion, and/or
  • a control unit configured to control the at least one particle retaining unit and the fluid transport unit in a fluid transport (or e.g. a fluid change) operation state of the device such that the at least one particle retaining unit retains the particles or at least a majority of the particles (e.g. at least 90 percent or at least 95 percent of the particles, e.g. by number or by weight) within the at least one particle retaining portion when the fluid transport unit transports a fluid through the particle retaining portion.
  • a fluid transport or e.g. a fluid change
  • the technical effect of the device may be that the particles may be retained at a specific location of the fluidic system, e.g. within the particle retaining portion and that the fluid may be pumped through the particle retaining portion at the same time.
  • the particles and/or an optional stirring element or other holding element is transported from one chamber to another chamber of the fluidic system.
  • This may allow to use a simple fluidic system and/or an essentially closed fluidic system.
  • the device may allow a high degree of automation for retaining the particles, e.g. during a transportation of a fluid within the particle retaining portion or another portion of the fluidic system, e.g. the complete change of fluid may be automated, e.g. without involving manual steps of operation staff.
  • the fluidic system may comprise or may consist of e.g. a plastic material.
  • the fluidic system itself may have no or only a minor influence to the magnetic field that is externally applied, especially by the particle retaining unit.
  • the fluid transport unit may be configured to transport a fluid through the fluidic system, e.g. a liquid or through parts of the fluidic system
  • the fluid transport unit may comprise a pump or at least a part of a pump, e.g. a driving unit of a peristaltic pump (may result only in minor damage of pumped particles) or any other appropriate pump, e.g. centrifugal, diagonal, radial pump, etc.
  • the particles may have a uniform shape or a non-uniform shape, e.g. as resulting from sinter processes. Thus, the particles may look agglomerated, caked or baked. Other shapes are e.g. beads, balls, spheres, ellipsoids, etc.
  • the material of the particles may be a magnetizable material in order to enable collection of the particles at or on a stirring element and/or at the wall of the particle retaining portion, e.g. as a “conglomerate” which may follow the field lines of the external magnetic field, e.g. in a static state or even in a dynamic state, e.g. if the external magnetic field is moved and/or rotated.
  • the magnetizable particles may comprise or may consist of a ferromagnetic material with p (relative permeability) in the range of 100 to 10000 or 300 to 10000, e.g. iron oxides.
  • the remanence or the remanent magnetization may be low such that the particles lose their magnetic field as soon as the external magnetic field is removed.
  • the remanent magnetization may be low, e.g. after at most 1 second or after at most 0.1 second after removal of the external magnetic field to give only two examples.
  • An optional magnetizable (auxiliary) element e.g. a stirring bar
  • the auxiliary element may have at least one or several of the following fimction(s): stirring element, magnetic force mediation element, magnetizable element in order to provide an additional internal magnetic source, e.g. for retaining of the particles. It may be possible to release magnetized particles from a magnetized auxiliary element by rotation of the magnetized auxiliary element, e.g. high speed rotation resulting in centrifugal forces acting on the particles on the rotating auxiliary element and/or shear forces between the particles and a liquid or other fluid which comprises the particles and the rotating auxiliary element. Magnetizable particles in the neighborhood of the magnetized auxiliary element may be magnetized by this element. However, other particles may be magnetized selectively using an external magnetic field.
  • the auxiliary element may be magnetizable, i.e. comprising or consisting of a ferromagnetic material with p (relative permeability) e.g. in the range of 100 to 10000 or 300 to 10000, e.g. iron oxides, soft iron, mu metal, cobalt alloy, nickel alloy, etc.
  • p relative permeability
  • the particles and the optional auxiliary element are de-magnetizable, e.g. by removing an external magnetic field this may allow “floating” of the particles within a dispersion, e.g. for binding of molecules to the particles or for de-binding (release) of molecules from the particles, e.g. depending on characteristics of a liquid (e.g. a buffer solution) which comprises the particles.
  • a liquid e.g. a buffer solution
  • the receiving space may be a retaining space if the carrier or cartridge is mechanically or otherwise coupled to, e.g. inserted into the particle retaining device.
  • the receiving space may be configured to receive a carrier that carries the fluidic system.
  • the carrier may be a cartridge, e.g. a cartridge that is essentially closed to all sides. Thus, there may be only some valves that allow ventilation and/or that may be opened if needed.
  • the carrier may carry the particle retaining portion, e.g. a chamber, especially a processing chamber or a channel between two structures of the fluidic system, e.g. between two buffer chambers, or between a buffer chamber and a processing/reaction chamber, etc.
  • the carrier may carry further structures of the fluidic system, e.g. a switching portion enabling to connect different portions of the fluidic system selectively, at least one test chamber for performing a test, e.g. a PCR test.
  • retaining of particles may be performed independent of the usage of a stirring element.
  • other steering methods may be used, e.g. using a rotatable element that extends through a wall of the particle retaining portion or of another portion of the fluidic system, e.g. using a gasket to have a tight fluidic connection.
  • the rotatable element may have a coupling portion for coupling to a driving unit of the device.
  • membrane based stirring solutions may be used, e.g. providing a closed system, especially at the stirring location.
  • using an element for several purposes may be advantageous, e.g. using the auxiliary element for stirring and for retaining of the particles in different operation states/modes of the device.
  • the auxiliary element may be much larger than the particles, e.g. having a maximum extension that is at least by factor 10 or by factor 100 larger than a maximum extension of the particles or of an average maximum extension of the particles.
  • the upper limit of the factor may be 1000 or 10000 to give only two examples. To give a specific example, if the maximum extension of the particles is 10 micron the maximum extension of the auxiliary element may be in the range of 1 mm (millimeter) to 3 mm.
  • the at least one particle retaining unit may comprise at least one of, at least two of or all three of:
  • a z-axis module configured to move the magnetic field along a z-axis transversally towards or away from the particle retaining portion, and/or
  • a y-axis module configured to move the magnetic field along a y-axis laterally relative to the particle retaining portion, wherein the y-axis may be arranged perpendicular or about perpendicular relative to the z-axis, e.g. in the direction of gravity, and/or
  • An x-axis module configured to move the magnetic field along an x-axis laterally relative to the particle retaining portion, wherein the x-axis may be arranged perpendicular or about perpendicular relative to the z-axis and/or to the y-axis, e.g. perpendicular to the direction of gravity.
  • a movement along the z direction may increase or decrease the magnetic flux density within the particle retaining portion, e.g. within a processing chamber (e.g. mixing) or within a reaction chamber (e.g. chemical or biochemical reaction).
  • a processing chamber e.g. mixing
  • a reaction chamber e.g. chemical or biochemical reaction
  • a movement in the x direction may be used for a change of the particle retaining unit and/or of the mixing unit between different particle retaining portions, e.g. between processing or reaction chambers arranges side by side, e.g. as seen in insertion direction of a cartridge into the device.
  • a movement in the y direction may allow to adjust the height within one particle retaining portion, e.g. chamber, especially depending on a filling level or based on other issues.
  • - A movement in the y direction may allow a change of the particle retaining unit and/or of the mixing unit between chambers that are arranged one over the other.
  • - Sophisticated collection and/or stirring schemes may be implemented within one chamber using only an x movement, only a y movement or a combination thereof.
  • control unit may be configured to control the at least one particle retaining unit such that in a particle collecting operation state/mode of the device:
  • the at least one particle retaining unit may be arranged adjacent to the particle retaining portion, e.g. in order to apply a high magnetic flux density B to the particle retaining portion.
  • the magnetic field may apply a magnetic flux density B within the particle retaining portion that may be strong enough to generate retaining forces that retain at least a majority of the particles (e.g. more than 90 percent by number or more than 95 percent by number) within the particle retaining portion.
  • the magnetic field may not be rotated or may be only rotated with a rotation speed of at most 20 rpm. Thus, particles that are already “caught” retained in the magnetic field even if there is a slight rotation. There may be an additional translation or other movement of the magnetic field. However, in other embodiments no additional translation or other movement of the magnetic field is used during particle collection, e.g. depending of the overall volume of the particle retaining portion, e.g. processing chamber.
  • the particle collecting operation step may be performed before the fluid transport operation state of the device in order to retain at least a majority of the particles within the particle retaining portion during the following fluid transport, e.g. liquid transport.
  • the particles may be retained preferably at a wall of the particle retaining portion, e.g. at at least one wall and/or at a magnetizable element arranged within the particle retaining portion, e.g. a stirring element or other auxiliary element.
  • An example of a particle collect configuration is described below with reference to figure 8.
  • the retaining forces may be based on magnetic attraction.
  • magnetic dipoles of the particles may align to the external magnetic field lines.
  • a magnetic south pole may be attracted to the opposite dipole, i.e. magnetic north pole of the external magnet field and vice versa.
  • the retaining force may be increased by usage of an optional magnetizable element that aligns to the field lines of the external magnetic field or by usage of a magnetizable force mitigation element that creates a secondary larger magnetic dipole within the particle retaining space under the influence of the external magnetic field.
  • the fluid transport unit may not be operated to transport a fluid through the particle retaining portion until the majority or all of the particles have been collected and/or are retained by the external magnetic field of the particle retaining unit.
  • control unit may be configured to control the at least one particle retaining unit such that in the particle collecting operation state/mode of the device:
  • the at least one particle retaining unit may be positioned at a first lateral position relative to the particle retaining portion in order to collect a first part of the majority of the particles or of all particles, and/or
  • the at least one particle retaining unit may be positioned at a second lateral position relative to the particle retaining portion in order to collect a second part of the majority of the particles or of all particles, wherein the second lateral position may be different from the first lateral position.
  • the distance may be in the range of 1 mm to 5 mm.
  • At least three positions or at least four positions or at least five positions within the particle retaining portion may be used in order to collect the majority of the particles or all particles, e.g. beads.
  • the number of position may be less than 100 or less than 20 to give only two examples for possible upper limits.
  • the positions may be selected due to fluid levels, e.g. liquid levels within a channel/chamber or due to other conditions. Examples for collecting schemes are illustrated in figures 9 and 10. These examples are described in more detail below.
  • the waiting time may allow to attract particles within a specific radius, e.g. particles that are dispersed in a dispersion.
  • positions with no additional waiting time i.e. the particles are collected within the particle retaining space using a continuous movement of the magnetic field, e.g. a movement that is different from a rotation of the magnetic field.
  • slight rotation may ease alignment of the (e.g. induced) magnetic dipoles of the particles and following collection of the particles at an auxiliary element and/or at the wall of the particle retaining portion.
  • the at least three positions may be located at a lower portion of the particle retaining portion, at a medium portion of the particle retaining portion and at a higher (upper) portion of the of the particle retaining portion.
  • the main points or portions are used to collect particles in order to make sure that the majority of the particles or all particles are retained or caught by the external magnetic field.
  • control unit may be configured to control the at least one particle retaining unit such that in the particle collecting operation state the particles are collected within the particle retaining portion from below to above with regard to gravity in the normal operation mode of the device.
  • many particles may be collected at a lower portion first. Thereafter, particles at an upper portion may be collected.
  • control unit may be configured to control the at least one particle retaining unit such that in the particle collecting operation state the particles are collected within the particle rotation portion from above to below with regard to gravity in the normal operation mode of the device. Particles that have not been caught at upper positions may have sunk due to gravity in the meantime and may still be collected at a lower position.
  • control unit may be configured to control the at least one particle retaining unit such that in the particle collecting operation state the particles are collected within the particle retaining portion above a fluid level. This may allow to collect also particles that have been catapulted or centrifuged to upper portions of the particle retaining portion, e.g. of a processing chamber.
  • control unit may be configured to control the at least one particle retaining unit such that in a mixing state of the device:
  • the at least one particle retaining unit is positioned at a first lateral position relative to the particle retaining portion in order to perform a mixing operation within the particle retaining portion using a first rotation axis, and
  • the at least one particle retaining unit is positioned at a second lateral position relative to the particle retaining portion in order to perform a mixing operation within the particle retaining portion using a second rotation axis, wherein the second lateral position is different from the first lateral position.
  • the distance may be in the range of 1 mm to 5 mm.
  • the first rotation axis may be different with regard to the second rotation axis.
  • the first rotation axis may be parallel to or about parallel to the second rotation axis.
  • the maximal extension of the particle retaining portion may be less than 4 cm, less than 3 cm (centimeter) or less than 2.5 cm.
  • mixing may be performed using different lateral positions of the particle retaining unit (or mixing unit if no particle retaining step is performed before mixing) and/or using different lateral positions of the rotation axes, e.g. of an external magnetic field. This may result in excellent mixing of the fluid, e.g. too high homogeneity of particles distributed in the fluid, e.g. of beads and/or of molecules (e.g. NAMs) that should bound to the particles.
  • the mixing operation state/mode may be a dynamic mixing mode as described below, a static mixing mode as described below or a combined dynamic/static mode. However, other appropriate mixing modes may be used as well.
  • control unit may be configured to control the at least one particle retaining unit such that in a dynamic mixing state/mode of the device:
  • the magnetic field may enable to transmit a mixing force to the particles and/or to a magnetizable element, preferably a stirring element arranged within the particle retaining portion.
  • the magnetic field may be rotated with a rotation speed above 1000 rpm or above 1500 rpm, and
  • the at least one particle retaining unit may be moved in order to move the magnetic field in addition to the rotation continuously laterally within the particle retaining portion,
  • the dynamic mixing state may be performed before the fluid transport operation state of the device in order to mix a fluid within the particle retaining portion, e.g. in order to enable later binding of molecules within a fluid to the particles which are within the same fluid as the molecules, especially binding of nucleic acid molecules (NAM).
  • NAM nucleic acid molecules
  • mixing may be possible without using a separate auxiliary element, e.g. without using a stirring bar.
  • the particles may e.g. be moved in circles near the wall of particle retaining portion, e.g. a processing chamber.
  • the majority of the particle conglomerate may be moved within the particle retaining portion by moving the magnetic field, e.g. in addition to a rotation of the magnetic field.
  • good stirring may be possible without using an internal stirring element.
  • usage of an internal stirring element may enhance stirring.
  • a rotation of the magnetic field above 1000 rpm or above 1500 rpm (rounds per minute) may allow to use centrifugal forces and/or shear forces in order to distribute the particles evenly within a fluid, e.g. to form a “cloud” of the particles.
  • the particles may be released from the wall of the particle retaining portion and/or from an auxiliary element, e.g. stirring bar that is arranged within the particle retaining portion due to the centrifugal forces and/or due to shear forces and/or due to turbulences.
  • This mixing mode may be a dynamic mixing mode, see e.g. figures 4 and 6 as described below in more detail.
  • the at least one particle retaining unit may be arranged at a transversal distance from the particle retaining portion that enables to transmit a mixing force to the particles and/or to a magnetizable element arranged optionally within the particle retaining portion.
  • One continuous mixing step e.g. during this time the same fluid, e.g. liquid may be hold within the particle retaining portion, e.g. a chamber in which dynamic stirring/mixing takes place.
  • One continuous mixing step may have a duration within the range of 10 seconds to 10 minutes or within the range of 1 minute to 5 minutes.
  • the dynamic mixing state may be performed preferably after the particle collecting operation state, e.g. in order to achieve later high binding yields of the molecules and/or to prevent damage of molecules already bound to the particles, especially during at least one washing step. Particles from tiny or “dead” angles (comers) may be collected. Agglomerates or clusters of particles may not be formed due to the prior collection of the particles, etc.
  • no immediate previous particle collection operation state/step may be performed before the mixing step, e.g. dynamic mixing. Due to the high rotation speed homogenous mixing may be achieved even if not all particles are collected on an optional stirring element before mixing. Thus, no prior particle collection may be necessary in round chambers or in chambers comprising a round bottom portion.
  • the particle retaining portion may be a fluid chamber that is adapted to mixing. Movement of a maximum of the magnetic flux density within the particle retaining portion may be possible, e.g. a movement that is different from rotation of the magnetic field.
  • the fluid transport unit may not be operated during dynamic mixing in order to transport a fluid through the particle retaining portion.
  • at least one axis module may be used that may move the at least one particle retaining unit laterally along a respective axis or at least two axis modules may be used which move the at least one particle retaining unit laterally along a respective axis, e.g. simultaneously.
  • excellent dynamic mixing results may be possible, e.g. in preparation for particle collection and/or particle retaining.
  • Dynamic mixing may be programmed easier, e.g. using a “sub routine” or a “sub program” without specifying each single mixing position by a user of the “sub routine”.
  • other parameters may be specified, e.g. a mixing region and/or a mixing duration.
  • control unit may be configured to control the at least one particle retaining unit such that in a static mixing state/mode of the device:
  • the magnetic field may enable to transmit a mixing force to the particles and/or to a magnetizable element arranged within the particle retaining portion,
  • the at least one particle retaining unit may be positioned at a first lateral (e.g. static) position relative to the particle retaining portion that enables to transmit a mixing force to the particles and/or to a magnetizable element arranged within the particle retaining portion,
  • the magnetic field may be rotated at the first lateral (e.g. static) position with a rotation speed above 1000 rpm or above 1500 rpm for at least 0.5 seconds, for at least 1 second or for at least 2 seconds, and
  • the at least one particle retaining unit may be positioned thereafter at a second lateral (static) position that may be different from the first lateral position, wherein the second lateral (static) position enables to transmit a mixing force to the particles and/or to a magnetizable element, preferably a stirring element arranged within the particle retaining portion,
  • the magnetic field may be rotated at the second lateral (static) position with a rotation speed above 1000 rpm or above 1500 rpm for at least 0.5 seconds, for at least 1 seconds or for at least 2 seconds.
  • a rotation of the magnetic field above 1000 rpm or above 1500 rpm (rounds per minute) may allow to use centrifugal forces and/or shear forces in order to distribute the particles evenly within a fluid, e.g. to form a “cloud” of the particles.
  • the particles may be released from the wall of the particle retaining portion and/or from an auxiliary element, e.g. stirring bar that is arranged within the particle retaining portion.
  • the static mixing mode is illustrated in figure 5 as described below.
  • the static mixing state may be performed before the fluid transport operation state of the device in order to mix a fluid within the particle retaining portion.
  • the at least one particle retaining unit may be arranged at a transversal distance from the particle retaining portion that enables to transmit a mixing force to the particles and/or to a magnetizable element arranged within the particle retaining portion.
  • the same fluid e.g. liquid may remain within a chamber in which static stirring takes place.
  • Stirring may be performed with or without a further stirring element, e.g. without a force mediation element in addition to particles.
  • the static mixing state may be performed preferably after the particle collecting operation state mentioned above. Particles from tiny or “dead” angles (corners) may be collected. Agglomerates or clusters of particles may not be formed due to the prior collection of the particles, etc.
  • no immediate particle collection operation state/step may be performed before the mixing step, e.g. static mixing. Due to the high rotation speed homogenous mixing may achieved even if not all particles are collected on an optional stirring element before mixing.
  • the static mixing state may be performed preferably after the dynamic mixing state, e.g. in order to mix at positions which have not reached by dynamic mixing.
  • the static mixing state/mode may be performed before a dynamic mixing state, e.g. in order to remove particles from “dead” edges/angles or dead “corners” before starting dynamic mixing which may not reach these positions.
  • the fluid transport unit may not be operated to transport a fluid through the particle retaining portion during static mixing.
  • At least one axis module may be used that may move the at least one particle retaining unit laterally along a respective axis or at least two axis modules may be used which move the at least one particle retaining unit laterally along a respective axis, e.g. simultaneously.
  • excellent static mixing results may be possible, e.g. in preparation for particle collection and/or particle retaining.
  • Static mixing may be used to make sure that mixing at specified positions is performed. These positions may be known to be relevant for the overall mixing result. There may be positions within the particle retaining portion, e.g. within a processing chamber that may not be reached by specifying regions for the dynamic mixing mode.
  • the rotation speed of the magnetic field may be controlled by the control unit to such a value that centrifugal forces and/or shear forces between the particles and the fluid within the particle retaining portion are generated to the particles that result in release of the particles form at least one, from at least two of or from all of:
  • the at least one particle retaining unit may be configured to rotate the magnetic field preferably with a rotation speed of at least 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm, 2000 rpm or at least 2100 rpm.
  • the rotation speed may be less than 2500 rpm or less than 3000 rpm to give only two examples for an upper limit.
  • the effort for providing rotation speeds below 2500 rpm may be reasonable, e.g. the costs for an electric or electronic high speed motor. These values may be valid for the dynamic mixing operation state of the device and/or for the static mixing state of the device and/or for other operation states of the device using rotation of the magnetic field.
  • An optional magnetizable force mediator element may be used within the particle retaining portion, e.g. in order to enhance stirring.
  • the particles, e.g. beads and/or the auxiliary stirring element may form a conglomerate within the particle retaining portion, especially at lower rotation speeds, e.g. less than 1000 rpm.
  • a continuous cloud of particles may be formed at higher rotation speeds.
  • homogenous distribution of the particles within the mixing fluid may be reached, e.g. for enhancing the yield in a following binding step of molecules to the beads, for enhancing the efficiency of at least one washing step performed after binding and/or for enhancing the yield in a later de-binding step (release of the molecules from the particles).
  • Centrifugal forces and/or forces due to shear forces and/or due to turbulences within the fluid may allow homogenous distribution, e.g. for higher viscosity of liquid and/or in the presence of magnetic fields that influence the magnetizable particles.
  • the conglomerate may be “dissolved” at higher rotation speeds.
  • control unit may be configured to control the at least one particle retaining unit such that in a first particle release state/mode of the device:
  • the value of the magnetic flux density B e.g. the maximum value within the particle retaining portion may be decreased from a value that retains or that is able to retain the particles within the particle retaining portion, preferably at a wall thereof and/or at a element to a value at which the particles are released from the wall of the particle retaining portion and/or from a magnetizable element, preferably a stirring element arranged within the particle retaining portion.
  • the magnetic field may not be rotated or may only be rotated with a rotation speed of at most 20 rpm during the first particle release state/mode.
  • the particles may remain in the particle retaining portion but may be released from a wall, e.g. chamber wall and/or magnetizable force mediation element.
  • the particles may move comparably free within the particle retaining portion, e.g. compared to the fluid transport operation state or to the a particle collection operation state mentioned above. No conglomerate of particles may be formed during the first particle release state.
  • binding of molecules to the particles or de-binding (release) of molecules from the particles may be promoted, e.g. depending on the buffer solution surrounding the particles and on the surrounding molecules surrounding the particles in case of binding.
  • figure 7 illustrates a configuration that may be used for binding /de-binding.
  • the first particle release state may be performed after the static mixing state and/or after the dynamic mixing state mentioned above, e.g. in order to have a good/excellent binding yield or a good to excellent de-binding yield.
  • the first particle release state may be performed before the fluid transport operation state of the device.
  • the bounded molecules may not be removed with the fluid during the fluid transport operation state. Otherwise, only the de-bounded molecules may be removed after a de-binding step, e.g. for further processing, especially within a PCR test.
  • Pictures of the particle retaining portion and/or of another portion of the fluidic system may be optionally taken with a camera during the waiting time, e.g. for regulatory purposes.
  • the particle retaining unit may be switched off, e.g. in case of using an electromagnet to generate the magnetic field.
  • the particle retaining unit may be arranged at a transversal distance from the particle retaining portion that may enable to reduce the magnetic field density within the particle retaining portion to a value at which the particles are released from the wall and/or from e.g. a force mitigating element, preferably from a stirring element arranged within the particle retaining portion.
  • the particle retaining unit may be moved laterally away from the particle retaining portion, e.g. using an x and/or y movement away from particle retaining portion in order to decrease the magnetic field at the particles further.
  • the magnetic field may not be rotated or may only be rotated with a rotation speed of at most 20 rpm, e.g. in order to promote binding or de-bending processes.
  • the fluid transport unit may not be operated to transport a fluid through the particle retaining portion during the first particle release state.
  • the value of the magnetic flux density B may be a maximum value at a specific position within the particle retaining portion, e.g. the maximum value during a rotation of the magnetic field. Absolute values may be used as values.
  • control unit may be configured to control the at least one particle retaining unit such that in a second particle release state/mode of the device:
  • the value of the magnetic flux density e.g. the maximum value within the particle retaining portion may be decreased from a value that retains or that is able to retain the particles within the particle retaining portion, preferably at a wall thereof and/or at a magnetizable element to a value at which the particles are released from the wall of the particle retaining portion and/or from e.g. a magnetizable element, preferably a stirring element arranged within the particle retaining portion.
  • the magnetic field may be rotated in the second particle release state with a rotation speed of at least 1000 rpm or at least 1500 rpm.
  • the magnetic flux density of the external magnetic field within the chamber may be too low to magnetize magnetizable particles to a relevant magnetic strength.
  • an auxiliary element e.g. a magnetizable stirring bar may still be magnetized by the external magnetic field.
  • stirring may be still possible using the auxiliary element and the rotating external magnetic field.
  • inertia forces may allow to continue stirring for a short time, e.g. for at least 0.5 seconds or at least one second.
  • the second particle release state (with rotating magnetic field) may be performed before the particle collecting operation state.
  • the second particle release state (with rotating magnetic field) may be performed after the static mixing state and/or after the dynamic mixing state of the same fluid, e.g. liquid that may be used in the second particle release state.
  • the second particle release state (with rotating magnetic field) may be performed before the fluid transport/change operation state of the device that removes liquid that may be used in the second particle release state from the particle retaining portion and/or that optionally fills in a second fluid in the particle retaining portion, preferably a process chamber or reaction chamber.
  • the value of the magnetic flux density B may be e.g. the maximum value at a specific position within the particle retaining portion, e.g. within a processing chamber, especially the maximum value during a rotation of the magnetic field. Absolute values may be used as values.
  • the particle retaining unit may comprise at least one permanent magnet, preferably a ring magnet, or a bar magnet and/or a magnet generating a magnetic flux density within the range of 0.5 T to 1.8 T, 0.8 T (Tesla) to 1.5 T or 1 T to 1.5 T as e.g. measured at the surface of the magnet.
  • a permanent magnet preferably a ring magnet, or a bar magnet and/or a magnet generating a magnetic flux density within the range of 0.5 T to 1.8 T, 0.8 T (Tesla) to 1.5 T or 1 T to 1.5 T as e.g. measured at the surface of the magnet.
  • the transversal movement of the particle retaining unit may be performed within a range of 1 mm distance to an outer surface of the particle retaining portion to a distance of at least 10 mm or of at least 14 mm.
  • the lower distance value may prevent that the magnet or other magnetic source of the particle retaining unit contact the wall, e.g. during high speed rotation and possible vibrations during high speed rotation.
  • the greater distance may allow to decrease the value of the magnetic flux density within the particle retaining portion to an appropriate low value, e.g. in order to enable the first particle release state or the second particle release state as mentioned above.
  • the magnet may comprise or may consist of neodymium, e.g. the magnet may be a power or super magnet.
  • the magnet may have at least one extension that is greater than 1 mm (millimeter) or greater than 5 mm.
  • the maximum extension, e.g. the outer diameter or a length of the magnet on the particle retaining unit may be in the range of 0.5 cm to 2.5 cm or 1 cm to 2 cm, e.g. 1.4 cm.
  • the device may comprise a detection unit that may be configured to detect whether a rotation of the magnetic field results in the rotation of the particles, e.g. beads and/or in the rotation of an auxiliary element within the particle retaining portion, preferably a magnetizable element configured to be used for stirring of the particles.
  • a detection unit may be configured to detect whether a rotation of the magnetic field results in the rotation of the particles, e.g. beads and/or in the rotation of an auxiliary element within the particle retaining portion, preferably a magnetizable element configured to be used for stirring of the particles.
  • the following detection principles may be used, e.g. applied alone or in combination:
  • Photoelectric barrier e.g. using an emitter and a receiver at the same side or on opposite sides of the particle retaining portion or other processing chamber, and/or
  • an indication unit may be configured to provide an indication based on a detection result whether a rotation of the particles and/or of the optional auxiliary element is detected or is not detected.
  • the indication unit may generate a signal via a user interface, e.g. an alarming signal if no rotation occurs within the particle retaining portion despite the rotation of the external magnetic field.
  • the indication unit may store data indicating the result of the detection, e.g. local storage or distributed storage, e.g. within a data cloud. Monitoring a rotation feedback may be advantageous for regulatory purposes, i.e. to get an admission for usage of the device in the health/medical field.
  • a particle retaining system may comprise a particle retaining device according to any one of the preceding claims and an analysis carrier, e.g. a cartridge.
  • the analysis carrier may comprise the fluidic system.
  • the fluidic system preferably a particle retaining portion of the analysis carrier may comprise at least one optional magnetizable element (auxiliary element) that preferably generates an induced magnetic field only under the influence of the magnetic field and/or that does not generate an induced magnetic field in the absence of the magnetic field.
  • auxiliary element optional magnetizable element
  • the fluidic system preferably the particle retaining portion may comprise the particles. At least 50 percent, at least 80 percent or all of the particles may be magnetizable particles which preferably generate an induced magnetic field only under the influence of the magnetic field and/or which do not generate an induced magnetic field in the absence of the magnetic field.
  • the auxiliary element may have at least one extension that may be at least 1 mm or at least 2 mm.
  • the particles may have a maximum extension that may be lower 0,1 mm (corresponds to 100 micron).
  • a magnetizable force mediator element may be able to attract many of the particles, especially to prevent that all particles are attracted to the wall of the fluidic system, chamber or only in the direction of the wall.
  • the magnetic field of the earth may be too week to induce magnetic field in force mediator element to a considerable amount, especially not a magnetic field that is strong enough to attract the particles, especially not with a sufficient strength or in a sufficient amount, e.g. sufficient to collect the particles in a reasonable time, e.g. within less than 10 minutes.
  • Ferromagnetic materials may be used for the auxiliary element (e.g. stirring element) and/or for the particles (e.g. beads).
  • the at least one magnetizable element may be configured to fulfill at least one, at least two or all of the following function(s):
  • a closed carrier (e.g. cartridge) system may be provided, especially closed in the neighborhood of the particle retaining portion/chamber.
  • No gasket may be needed for rotation of stirring element within the particle retaining portion/chamber since the auxiliary element may be rotated by the external magnetic field or only the particles may be rotated by the external magnetic field of the particle retaining portion.
  • the particle retaining device may be used for enriching nucleic acid molecules within a solution.
  • the particle retaining device may be used to perform a method according to any one of the embodiment described above.
  • the usage of the particle retaining device e.g. for performing at least one PCR test or at least 10 PCR tests within one cartridge or at least 20 PCR tests within one cartridge may provide excellent results and/or may allow very cost efficient testing.
  • automatic testing may be possible, e.g. not involving manual movement of a magnet with the beads attached thereto from one reaction or processing chamber, e.g. glass cylinder (receptacle) to another.
  • a method for enriching nucleic acid molecules comprising the steps of:
  • composition which comprises the following components: a sample material comprising nucleic acid molecules (NAM), a nucleic acid molecule binding buffer (NAMbb), magnetizable particles (Be) which are capable of binding nucleic acid molecules (NAM), and a magnetizable stirring element (StBB),
  • NAM nucleic acid molecules
  • NAMbb nucleic acid molecule binding buffer
  • Be magnetizable particles
  • StBB magnetizable stirring element
  • the sample material comprising nucleic acid molecules (NAM) is selected from the group consisting of an aqueous solution, a processed sample, and a biological sample, preferably a cell lysate, a microbial culture, or a processed biological sample.
  • the nucleic acid molecules (NAM) are DNA or RNA molecules.
  • the magnetizable particles (Be) which are capable of binding nucleic acid molecules (NAM) are coated with a sorbent material.
  • the sorbent material is selected from the group consisting of SiCh crystals, amorphous silicon oxide and glass powder, alkylsilica, aluminum silicate (zeolite), or, activated silica with -NH2.
  • the magnetizable particles (Be) have a diameter of between 0.1 pm and 20 pm, preferably of between 0.5 pm and 15 pm, and more preferably of between 1 pm and 10 pm.
  • the stirring element (StBB) in step (iii) moves with a speed of > 2000 rotations per minute (rpm).
  • the intensity (B) of the magnetic field is reduced by increasing/extending the distance between the permanent magnet (Magi, Mag2) and the particles (Be), or by shielding the permanent magnet (Magi, Mag2) from the particles (Be).
  • nucleic acid binding buffer is a buffer comprising a chaotropic agent and alcohol.
  • the chaotropic agent is selected from the group consisting of an agent comprising thiocyanate ions, iodine ions, perchlorate ions, nitrate ions, bromine ions, chlorine ions, acetate ions, fluorine ions, and sulfate ions.
  • a method for isolating nucleic acid molecules comprising the steps of:
  • non-nucleic acid molecule binding buffer (nonNAMbb) is selected from the group consisting of water, TRIS buffer having a pH of between 6 to 9, NaOH buffer having a pH of 9, and ammoniumchloride buffer having a pH of 7,5.
  • Particle retaining device for processing nucleic acid molecules (NAM) using particles (Be), comprising: at least one receiving space (RS2) for a fluidic system (FS) comprising at least one particle retaining portion (PRP), at least one particle retaining unit (PRU) configured to apply an external magnetic field to the particle retaining portion (PRP) in order to provide retaining forces to the particles (Be) within the particle retaining portion (PRP) which retain the particles (Be) within the particle retaining portion (PRP), a fluid transport unit (FTU, P) configured to transport fluid through the particle retaining portion (PRP), a control unit (CUI, CU2) configured to control the at least one particle retaining unit (PRU) and the fluid transport unit (FTU, P) in a fluid transport operation state of the device (D, PRD) such that the at least one particle retaining unit (PRU) retains the particles (Be) within the at least one particle retaining portion (PRP) when the fluid transport unit (FTU, P) transports a
  • ZAM1, ZAM2 configured to move the magnetic field along a z-axis transversally towards or away from the particle retaining portion (PRP), and/or
  • YAM1, YAM2 configured to move the magnetic field along a y- axis laterally relative to the particle retaining portion (PRP), wherein the y-axis is arranged perpendicular or about perpendicular relative to the z-axis
  • an x-axis module (XAM1, XAM2) configured to move the magnetic field along an x- axis laterally relative to the particle retaining portion (PRP), wherein the x-axis is arranged perpendicular or about perpendicular relative to the z-axis and/or to the y-axis.
  • the device (D) according to items 29 or 30, wherein the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a particle collecting operation state of the device (D): the at least one particle retaining unit (PRU) is arranged adjacent to the particle retaining portion (PRP), the magnetic field applies a magnetic flux density (B) within the particle retaining portion (PRP) that is strong enough to generate retaining forces that retain at least a majority of the particles within the particle retaining portion (PRP), the magnetic field is not rotated or is only rotated with a rotation speed of at most 20 rpm, the particle collecting operation step is performed before the fluid transport operation state of the device in order to retain at least a majority of the particles within the particle retaining portion (PRP).
  • the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a particle collecting operation state of the device (D): the at least one particle retaining unit (PRU) is arranged adjacent to the particle retaining portion (
  • the device (D) according to item 31, wherein the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in the particle collecting operation state of the device (D): the at least one particle retaining unit (PRU) is positioned at a first lateral position relative to the particle retaining portion (PRP) in order to collect a first part of the majority of the particles, the at least one particle retaining unit (PRU) is positioned at a second lateral position relative to the particle retaining portion (PRP) in order to collect a second part of the majority of the particles (Be), wherein the second lateral position is different from the first lateral position.
  • the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in the particle collecting operation state of the device (D): the at least one particle retaining unit (PRU) is positioned at a first lateral position relative to the particle retaining portion (PRP) in order to collect a first part of the majority of the particles, the at least one particle
  • control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a mixing state of the device (D): the at least one particle retaining unit (PRU) is positioned at a first lateral position relative to the particle retaining portion (PRP) in order to perform a mixing operation within the particle retaining portion (PRP) using a first rotation axis, the at least one particle retaining unit (PRU) is positioned at a second lateral position relative to the particle retaining portion (PRP) in order to perform a mixing operation within the particle retaining portion (PRP) using a second rotation axis, wherein the second lateral position is different from the first lateral position.
  • the device (D) according to any one of the items 29 to 33, wherein the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a dynamic mixing state of the device (D): the magnetic field enables to transmit a mixing force to the particles (Be) and/or to a magnetizable element (MME) arranged within the particle retaining portion (PRP), the magnetic field is rotated with a rotation speed above 1000 rpm or above 1500 rpm, and that at least one particle retaining unit (PRU) is moved in order to move the magnetic field in addition to the rotation continuously laterally within the particle retaining portion (PRP), the dynamic mixing state is performed before the fluid transport operation state of the device in order to mix a fluid within the particle retaining portion (PRP).
  • the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a dynamic mixing state of the device (D): the magnetic field enables to transmit a mixing force to the particles (Be) and/or to
  • the device (D) according to any one of the items 29 to 34, wherein the control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a static mixing state of the device (D): the magnetic field enables to transmit a mixing force to the particles and/or to a magnetizable element (MME) arranged within the particle retaining portion (PRP), the at least one particle retaining unit (PRU) is positioned at a first lateral position relative to the particle retaining portion (PRP) that enables to transmit a mixing force to the particles and/or to a magnetizable element (MME) arranged within the particle retaining portion (PRP), the magnetic field is rotated at the first lateral position with a rotation speed above 1000 rpm or above 1500 rpm for at least 0.5 seconds or for at least 1 second, and the at least one particle retaining unit (PRU) is positioned thereafter at a second lateral position that is different from the first lateral position, wherein the second lateral position enables to transmit
  • the device (D) according to items 34 or 35, wherein the rotation speed of the magnetic field is controlled by the control unit (CU) to such a value that centrifugal forces and/or shear forces are generated to the particles (Be) that result in release of the particles (Be) form at least one, from at least two of or from all of release from other particles (Be), and/or release from a wall of the particle retaining portion (PRP) that is arranged adjacent to the at least one particle retaining unit (PRU), and/or release from a force mitigation element that is arranged within the particle retaining portion (PRP). 37.
  • control unit CU
  • PRU particle retaining unit
  • control unit (CU) is configured to control the at least one particle retaining unit (PRU) such that in a second particle release state of the device (D): the value of the magnetic flux density (B) within the particle retaining portion (PRP) is decreased from a value that is able to retains the particles (Be) within the particle retaining portion (PRP), and the magnetic field is rotated with a rotation speed of at least 1000 rpm or of at least 1500 rpm.
  • the particle retaining unit comprises at least one permanent magnet, preferably a magnet generating a magnetic flux density within the range of 0.5 T to 2 T as measured at its surface.
  • the device (D) according to any of the preceding items 29 to 37, comprising a detection unit that is configured to detect whether a rotation of the magnetic field results in the rotation of the particles (Be) and/or in the rotation of an auxiliary element (MME) within the particle retaining portion (PRP), preferably a magnetizable element configured to be used for stirring of the particles (Be), and comprising preferably an indication unit configured to provide an indication based on a detection result whether a rotation of the particles and/or of the optional auxiliary element is detected or is not detected.
  • a detection unit that is configured to detect whether a rotation of the magnetic field results in the rotation of the particles (Be) and/or in the rotation of an auxiliary element (MME) within the particle retaining portion (PRP), preferably a magnetizable element configured to be used for stirring of the particles (Be), and comprising preferably an indication unit configured to provide an indication based on a detection result whether a rotation of the particles and/or of the optional auxiliary element is detected or is not detected.
  • Particle retaining system comprising a device according to any one of the preceding items and an analysis carrier (C), wherein the analysis carrier (C) comprises the fluidic system (FS), wherein the particle retaining portion (PRP) comprises at least one magnetizable element (MME) that generates an induced magnetic field (only) under the influence of the magnetic field and/or that does not generate an induced magnetic field in the absence of the magnetic field, wherein the fluidic system (FS) comprises the particles, wherein at least 50 percent, at least 80 percent or all of the particles are magnetizable particles (Be) which generate an induced magnetic field under the influence of the magnetic field and/or which do not generate an induced magnetic field in the absence of the magnetic field.
  • MME magnetizable element
  • the at least one magnetizable element is configured to fulfill at least one, at least two or all of the following function(s): to be used as a second source of a magnetic field in addition to a first source of a magnetic field within the particle retaining unit, to be used as a stirring element (StBB) if rotated by the at least one particle retaining unit (PRU), and/or to be used as a particle collecting element (StBB) if moved by the at least one particle retaining unit (PRU).
  • the at least one magnetizable element MME is configured to fulfill at least one, at least two or all of the following function(s): to be used as a second source of a magnetic field in addition to a first source of a magnetic field within the particle retaining unit, to be used as a stirring element (StBB) if rotated by the at least one particle retaining unit (PRU), and/or to be used as a particle collecting element (StBB) if moved by the at least one particle retaining unit (PRU).
  • Figure 1 a cartridge as an embodiment of an analyte carrier comprising also chemical substances used for preparing and performing a PCR test.
  • Figure 2 a first - more general - embodiment of a positioning unit for a magnetic field generation unit.
  • Figure 3 a second - more specific - embodiment of a positioning unit for a magnetic field generation unit.
  • Figure 4 an arrangement of a processing chamber and of a magnetic field generation unit during a dynamic mixing mode in a first high magnetic flux density operation mode.
  • Figure 5 an arrangement of a magnetic field generation unit relative to a processing chamber during a static mixing mode in the first high magnetic flux density operation mode.
  • Figure 6 a possible mixing path during the dynamic mixing mode.
  • Figure 7 the arrangement of the magnetic field generation unit relative to the processing chamber during a preparation step for the collection of particles, during a binding step of nucleic acid molecules to the beads or during a de-binding (release step) of the molecules from the beads.
  • Figure 8 the arrangement of the magnetic field generation unit relative to the processing chamber during a second high magnetic flux density operation mode used for the collection of particles.
  • Figure 9 four variants of a first embodiment of positions, e.g. stopping positions of a stirring element during the second high magnetic flux density operation mode (collection of particles).
  • Figure 10 a second embodiment (fifth variant) of positions, e.g. stopping positions of a stirring element during the second high magnetic flux density operation mode (collection of particles).
  • Figure 11 a mixing step.
  • Figure 12 a binding step.
  • Figure 13 a particle collecting step whereby nucleic acid molecules attached to the particles.
  • Figure 14 a de-binding step.
  • Figure 15 a particle collecting step whereby no nucleic acid molecules attached to the particles.
  • Figure 1 illustrates a cartridge C as an embodiment of an analyte carrier comprising also chemical substances used for preparing and performing a PCR test.
  • Figure 1 illustrates a cartridge C comprising a first or stationary fluidic system FS1 of a stationary unit 100.
  • Cartridge 10 comprises two movable unit(s) 200, SW, e.g. a switching wheel and DW, 600, e.g. (PCR) disc wheel.
  • the cartridge C may comprise sidewalls SW1 to SW4, especially of the stationary unit 100.
  • the sidewall SW1 may be a lower sidewall in the usage position of the cartridge C.
  • the sidewall SW2 may be a left sidewall (as illustrated in figure 1), e.g. a sidewall that is inserted first into a slot of a device D used to perform a test.
  • the sidewall SW3 may be a right sidewall, e.g. a sidewall that may be inserted into the slot after the side wall SW2 has been inserted.
  • the sidewall SW4 may be an upper sidewall in the usage position of the cartridge C.
  • the cartridge C, and especially the stationary unit 100 may comprise at least two or exactly two large circular apertures 110, 112.
  • the circular aperture 110 may form a receptacle for the movable (rotatable) unit SW, 200 which may comprise a second/movable fluidic system.
  • the second/movable fluidic system may be movable (rotatable) relative to the first fluidic system FS1.
  • the circular aperture 112 may form a receptacle for the movable (rotatable) unit 600, DW which may comprise chambers Cl to Cl l comprising different test auxiliary materials, e.g. primers and probes, preferably labeled or coupled with at least one, at least two or at least three or more than three fluorescence material(s) per chamber Cl to Cl 1, optionally combined with at least one, at least two or at least three quencher(s) (double marked, double labeled), e.g. using FRET (Forster resonant energy transmission), e.g. via molecular dipole coupling (especially non-radiating) or non-FRET principles.
  • test auxiliary materials e.g. primers and probes, preferably labeled or coupled with at least one, at least two or at least three or more than three fluorescence material(s) per chamber Cl to Cl 1, optionally combined with at least one, at least two or at least three quencher(s) (double marked, double labeled), e
  • the movable (rotatable) unit DW, 600 may be movable (rotatable) relative to the first fluidic system FS1.
  • a first rim may border aperture 110.
  • the first rim may be used to position a first gasket between the stationary unit 100 and movable (rotatable) unit SW, 200.
  • a second rim may border aperture 112. The second rim may be used to position a second gasket between the stationary unit 100 and movable (rotatable) unit DW, 600.
  • the first gasket may be arranged within a groove Grl arranged around aperture 110 and arranged adjacent to the first rim.
  • a further groove Gr2 may be arranged around aperture 112 and adjacent to the second rim, e.g. in order to hold and to position the second gasket.
  • the first fluidic system (stationary) FS1 may comprise four groups of “taps” that are arranged around aperture 110 in the following sequence and in a clockwise direction:
  • Border B may be arranged between taps of the third group and of the fourth group.
  • a tap at the border may be omitted or may be realized as well.
  • Taps 1 to 14 may have a circumferential offset between adjacent taps 1 to 7 of a second value. The second value may be half of the first value.
  • Taps 1 to 13 a fourth group e.g. connected to channels o to zl arranged on the left side of a border B between channels of the third group and of the fourth group.
  • Taps 1 to 13 may have a circumferential offset between adjacent taps 1 to 13 of the second value.
  • the second value may be half of the first value.
  • the taps may be formed as axial portions within the stationary unit 100. Moreover, holes within the first gasket and/or cylindrical portions of the first gasket may be parts of the taps. Alternatively, at least one continuous fluidic interface portion may be used comprising only one continuous elongated hole instead of several taps of one of the first group and/or of the second group.
  • the fluidic system FS1 may comprise a further channel z3 as well as channels CH10 and CH20.
  • the first fluidic system FS1 (stationary) may comprise e.g. three processing (reaction) chambers PCI to PC3, e.g. a lysing chamber (PCI), a magnetic separation chamber (MSC, PC2) and a master mix chamber (MMC, PC3).
  • PCI processing (reaction) chambers
  • MMC magnetic separation chamber
  • PC3 master mix chamber
  • the fluidic system FS1 may comprise a plurality of buffer chambers used for storage of fluids or of dry chemicals.
  • the following buffers/chambers may be provided within cartridge C: - Master mix buffer chamber, preferably for storage of a so called master mix MM.
  • the master mix MM may be stored as a dry powder.
  • the respective chamber may be named as a dry master mix buffer chamber DMC.
  • - Buffers (chambers) Bl to B6 e.g. used for storing liquids, e.g. in this sequence an aqueous solution (e.g. distilled water), isopropanol, a first washing solution, a second washing solution, ethanol and an elution (de-binding) buffer, etc.
  • aqueous solution e.g. distilled water
  • isopropanol e.g. distilled water
  • a first washing solution e.g. distilled water
  • second washing solution e.g. ethanol
  • ethanol and an elution (de-binding) buffer e.g. ethanol
  • sample tube ST may comprise a sample S, e.g. a sample S comprising the at least one substance under test.
  • the sample tube ST may also include a solution that may enhance or allow testability of the at least one substance, e.g. a buffer solution as mentioned above, e.g. comprising chaotropic salt(s) and/or surface-active substances.
  • the sample S may be inserted in the sample tube ST, e.g. by a practitioner, e.g. using a pipette or a tweezer. Thereafter, the sample tube ST may be closed, e.g.
  • a plunger of the test device D may be used to press the sample S out of the sample tube.
  • a distal pin may be used to open the sample tube ST and to connect the sample tube ST to the channel z3 fluidically.
  • a waste chamber W e.g. used to store waste liquid produced in the micro reactor that may be implemented in cartridge C and that may be taken into operation using machine or device D. However, especially buffer chambers that have been emptied may also be used as waste chambers during the operation of the micro reactor.
  • a reservoir R e.g. in order to store a portion of the sample, especially of a lysate of the sample, for further purposes, e.g. for performing further test and/or for research or scientific purposes.
  • a chamber 120 for storing of a drying powder e.g. a powder that makes sure that the master mix may be stored as a dry powder even if cartridge C may be stored in an environment having high or higher humidity.
  • An auxiliary chamber 122 of fluidic system FS1 may be used to dissolve the master mix MM during operation of the micro reactor implemented by cartridge C and device D.
  • the cartridge C may comprise less or more channel s/buffer than illustrated.
  • the switching wheel SW, 200 may be optionally, e.g. if the sample is prepared in another device or manually.
  • the disc wheel DW, 600 may be optional too. Thus, only one stationary chamber Cl may be used for testing.
  • a flexible hose H may be connected to two fluidic ports FP1 (first) and FP2 (second).
  • the flexible hose H may be pressed and released by a peristaltic pump.
  • pumps operating according to other pumping principles may be used.
  • the channel CH10 may connect first fluidic port FP1 and the first coupling portion channel CPCH1.
  • the channel CH20 may connect second fluidic port FP2 to the second coupling portion channel CPCH2.
  • Waste chamber W may also comprise an opening to the environment of cartridge C.
  • Biological filters may be used in openings 01 to 03 and at the opening of waste chamber W in order to protect the environment.
  • Opening 01 may be connected to a valve VI or may be part of a valve that may be operated by the machine/device D into which cartridge C may be inserted to perform at least one test or a plurality of tests.
  • At least one, an arbitrary selection of or all of the following fluidic sub-systems may be provided within the first fluidic system FS1, especially arranged in the clockwise direction:
  • the first coupling portion channel CPCH1 fluidically connected to taps 1 to 8 of the first group and e.g. to channel CH10, i.e. to first fluidic port FP1.
  • a channel z2 may be connected to the first coupling portion channel CPCH1 immediately below tap 1 of the first group of taps.
  • the channel CH10 may be fluidically connected to the first coupling portion channel CPCH1 immediately below tap 5 of the first group of taps or at another appropriate location thereby forming a first fluidic passage. Regardless of taps, there may be no further fluidic connection to the first coupling portion channel CPCH1.
  • the first coupling portion channel CPCH1 may comprise an intermediate volume IV, IV1 that may be arranged between the first fluidic passage and the taps connected to or arranged on the first coupling portion channel CPCH1.
  • one void tap position arranged between the first coupling portion channel CPCH1 and the second coupling portion channel CPCH2, e.g. between tap 8 of the first group of taps and tap 1 of the second group of taps.
  • the second coupling portion channel CPCH2 connected to taps 1 to 7 of the second group and to channel CH20, i.e. to second fluidic port FP2.
  • Channel CH20 may be fluidically connected to the second coupling portion channel CPCH2 immediately below or at tap 1 of the second group of taps or at another appropriate location thereby forming a second fluidic passage. Regardless of taps, there may be no further fluidic connection to the second coupling portion channel CPCH2.
  • the second coupling portion channel CPCH2 may comprise an intermediate volume IV, IV2 that may be arranged between the second fluidic passage and the taps connected to or arranged on the second coupling portion channel CPCH2.
  • Tap 1 of the third group of taps e.g.
  • auxiliary chamber 122 fluidically connected via the channel a to one passage of auxiliary chamber 122 and to chamber 120 (drying powder or gel, e.g. MiniPax (may be a trademark)).
  • the other passage of auxiliary chamber 122 may be fluidically connected to the upper portion of master mix chamber DMC.
  • the lower portion of master mix chamber DMC may be connected to channel b.
  • Channel b may be fluidically connected to tap 2 of the third group.
  • a fluidic loop may be established via channel c, a portion of the fluidic system on disc wheel DW, 600 and channel d as well as using other portions of fluidic systems FS1 and of the second fluidic system.
  • border B may be arranged between taps of the third group and taps of the fourth group,
  • Waste buffer W may comprises an opening, preferably at its upper end as mentioned already above.
  • - Tap 4 of the fourth group of taps may be unused but may be used for special purposes, e.g. for a further single connection to a buffer having a “venting” opening.
  • the channel to the upper portions of a buffer or of a reaction chambers comes first, i.e. before the channel to the lower portion of the respective buffer or reaction chamber, if the switching wheel SW, 200 may be rotated in a clockwise direction.
  • the channel to the lower portions of a buffer or of a reaction chambers come first, i.e. before the channel to the upper portion of the respective buffer or reaction chamber, if the switching wheel SW, 200 may be rotated in a clockwise direction. Therefore, at the border B, the channel n that may be connected to the lower portion of buffer chamber B4 may be adjacent to the channel o that may be connected to the lower portion of buffer chamber B3.
  • a channel z3 may fluidically connect the lower portion of sample tube holder STH, especially the lower portion of piercing pin PP, with a passage in processing (reaction) chamber PCI (e.g. lysis chamber).
  • processing (reaction) chamber PCI e.g. lysis chamber
  • the passage in the processing (reaction) chamber PCI may be arranged within a middle portion of the processing chamber PCI, e.g. between the lower portion and the upper portion of the processing chamber PCI.
  • Apertures 110 and 112 may extend through the first flat member and through the second flat member of the cartridge C.
  • two rigid flat members may be used for cartridge C.
  • at least one flat member may comprise or may consist of a flexible foil, e.g. the flat member may be flat on both sides, e.g. without grooves for channels and/or buffers and/or reaction chambers.
  • the switching unit/wheel SW, 200 may comprise a central channel CHI, e.g. double spirally wounded and four channels extending on its circumference. Switching unit/wheel SW, 200 may be used to connect the pump to several buffer chambers/processing chambers.
  • the (PCR) disc wheel DW, 600 may comprise the test chambers Cl to Cl 1, a calibration chamber CC, the short circuit CC as well as other components.
  • Rotatable, (PCR) disc wheel DW, 600 may be filled sequentially and automatically using channels c and d.
  • disc wheel DW, 600 may be used to perform a PCR test.
  • Disc wheel DW, 600 may not be rotated during performing the PCR test or another test, e.g. a test involving temperature cycling.
  • At least one temperature unit may be arranged at disc wheel DW, 600 during the test.
  • an optical unit e.g. comprising a radiation transfer unit may be arranged on disc wheel DW, 600 during the test, e.g.
  • a fluidic interface of disc wheel IFDW may allow fluidic communication between stationary unit 100 and chambers Cl to Cl 1.
  • the cartridge C may have an overall width Wil in the range of 10 cm (centimeter) to 20 cm.
  • the height Hel of the cartridge C may be in the range of 10 cm to 15 cm.
  • the thickness of the cartridge C may be in the range of 0.5 cm to 3 cm.
  • a maximum height He2 of processing chamber PC2 may be in the range of 4 cm to 1 cm or in the range of 3 cm to 2 cm, e.g. 2.5 cm.
  • the maximum width Wi2 of processing chamber PC2 may be in the range of 2 cm to 0.5 cm, e.g. 1 cm.
  • the maximum thickness of the inner chamber volume of processing chamber PC2 may be in the range of 0.3 to 1 cm, e.g. 0.5 cm.
  • the chamber volume may be in the range of 100 microliter to 800 microliter or in the range of 200 microliter to 600 microliter.
  • Processing chamber PC2 (MSC, magnetic separation chamber) may comprise a stirring element StB2 and/or magnetizable particles, e.g. beads Be.
  • Beads Be may be beads Be which are commercially available and/or which comprise e.g. a ferromagnetic material. Beads Be may have a core comprising a first material and at least one coating layer, e.g. a sorbent coating as mentioned above.
  • a stirring element e.g. a stirring bar StBl may be arranged within processing chamber PCI.
  • a third stirring element e.g. a stirring bar StB3 may be arranged within the third processing chamber PC3.
  • the stirring bars StBl to StB3 may be commercially available.
  • the stirring bars StBl to StB3 may be magnetizable, e.g. comprising a ferromagnetic material.
  • Stirring bars StBl to StB3 may be moved by three separate stirring units of a device D that may be used for performing a test. Alternatively, only one (single) stirring unit may be used for moving and/or rotation all three stirring bars StBl to StB3 in sequence. This is described in more detail below. This may reduce the number of components and/or the weight and/or the costs of device D considerably.
  • each processing chamber PCI to PC3 may be heated and/or cooled by a separate heating and/or cooling element.
  • a movable single temperature unit may be used to heat the processing chamber PCI to PC3 in sequence.
  • the single temperature unit may be coupled to the single stirring unit. This may reduce the number of components and/or the weight and/or the costs of device D further.
  • FIG. 2 illustrates a first - more general - embodiment of a three dimensional (3D) positioning unit PU 1 (or particle retaining unit PRU) for a magnetic field generation unit Magi .
  • Positioning unit PU1 may be comprised within an automatic test device D, PRD (particle retaining device) e.g. within a PCR (polymerase chain reaction) test device that may be able to perform, e.g. more than 20 tests automatically using one cartridge C.
  • PRD particle retaining device
  • PCR polymerase chain reaction
  • the three dimensions may relate to:
  • the 3D positioning unit PU1 may comprise:
  • An x-axis module XAM1 configured to implement a mechanical movement into both directions of the x-axis x
  • a y-axis module YAM1 configured to implement a mechanical movement into both directions of the y-axis y
  • a z-axis module ZAM1 configured to implement a mechanical movement into both directions of the z-axis z.
  • the x-axis module XAM1 may be the basis module that carries the other two axis modules YAM1 and ZAM1.
  • the x-axis module XAM1 may comprise:
  • a track TRxl that may extend in the x-direction and that may be e.g. mounted on a chassis or on a casing, e.g. on a mechanically stable casing of device D.
  • An x-direction frame FRxl or other appropriate support structure that may be slideable or otherwise movable in the x direction relative to track TRxl,
  • a coupling portion CPxl that may mechanically couple the track TRxl and the frame FRxl to enable the relative movement of the frame FRxl in x-direction relative to track TRxl.
  • Coupling portion CPxl may comprise a carriage/slider or other mechanical element that may be movable relative to track TRxl.
  • the track TRxl and the coupling portion CPxl may be comprised within an x-axis guiding unit, e.g. within a linear guiding unit LGU.
  • an x-axis guiding unit e.g. within a linear guiding unit LGU.
  • pivoting/rotation or curved movement may be used.
  • the x-axis module XAM1 may comprise further mechanical elements, e.g. a driving unit, especially a driving unit comprising an electrical motor and a force transmission unit, e.g. a spindle and a spindle nut or a tooth wheel and a tooth rack.
  • a driving unit is mentioned below for the device D as illustrated in figure 3, i.e. an electrical motor, e.g. a stepper motor.
  • the y-axis module YAM1 may be a secondary module that may be carried by the x-axis module XAM1 and that carries the z-axis module ZAM1.
  • the y-axis module YAM1 may comprise:
  • a track TRyl in y-direction that may extend in the x-direction and that may be e.g. mounted on the frame FRxl of the x-axis module XAM1 or that may be part of the frame FRxl .
  • a coupling portion CPyl (for the y-direction) that may mechanically couple the track TRyl and the frame FRyl in order to enable the relative movement of the frame FRyl in y- direction relative to track TRyl.
  • the coupling portion CPyl may comprise a carriage/slider or other mechanical element that may be movable relative to track TRyl and that carries the frame FRyl, e.g. a slider that is mounted only one-sided or alternatively a slider that is mounted at two sides.
  • the track TRyl and the coupling portion CPyl may be comprised within a guiding unit, e.g. within a linear guiding unit LGU.
  • a guiding unit e.g. within a linear guiding unit LGU.
  • pivoting/rotation or curved movement may be used.
  • the y-axis module YAM1 may comprise further mechanical elements, e.g. a y-axis driving unit, especially a driving unit comprising an electrical motor and a force transmission unit, e.g. a spindle and a spindle nut or a tooth wheel and a tooth rack.
  • a driving unit is mentioned below for the device D as illustrated in figure 3, i.e. an electrical motor, e.g. a stepper motor.
  • the z-axis module ZAM1 may be a tertiary module that may be carried by the x-axis module XAM1 and by the y-axis module YAM1.
  • the z-axis module ZAM1 may comprise:
  • a track TRzl in z-direction that may extend in the z-direction and that may be e.g. mounted on the frame FRyl of the y-axis module YAM1.
  • a z-direction frame FRzl or other support structure that may be slideable or otherwise movable relative to track TRzl.
  • a coupling portion CPzl (for z-direction) that may mechanically couple the track TRzl and the frame FRzl to enable the relative movement of the frame FRzl in z-direction relative to track TRzl.
  • the coupling portion CPzl may comprise a carriage/slider or other mechanical element that may be movable relative to track TRzl.
  • the track TRzl and the coupling portion CPzl may be comprised within a z-axis guiding unit, e.g. within a linear guiding unit LGU.
  • a z-axis guiding unit e.g. within a linear guiding unit LGU.
  • pivoting/rotation or curved movement may be used.
  • the z-axis module ZAM1 may comprise further mechanical elements, e.g. a driving unit, especially a driving unit comprising an electrical motor and a force transmission unit, e.g. spindle and spindle nut or tooth wheel and tooth rack.
  • a driving unit is mentioned below for the device D as illustrated in figure 3, i.e. an electrical motor, e.g. a stepper motor.
  • the magnetic field generation unit e.g. magnet unit Magi may be mounted on the z- axis module ZAM1 and may be movable into all three directions x, y and z using modules XAM1, YAM1 and ZAM1. This movement may allow at least one of the following:
  • a movement of the magnet unit Magi along the y-axis may be used for this purpose.
  • a movement of the magnet unit Magi along the y axis may be used for a vertical arrangement of at least two processing chambers and for changing the position of the magnet unit Magi relative to these at least two vertically “staggered” processing chambers (not illustrated).
  • the magnetic field generation unit e.g. magnet unit Magi may be rotatable relative to the frame FRzl. This rotation may allow stirring/mixing of fluids within processing (reaction chambers) of the cartridge C when inserted into device D, see e.g. processing chamber PCA and processing chamber PCB.
  • a rotatable permanent magnet see arrow Rot may be used for implementing the magnetic field generation unit, e.g. magnet unit Magi.
  • the permanent magnet may comprise neodymium.
  • a rotating magnet field electromagnettic field
  • the device D, PRD may comprise at least one receiving space, e.g. two receiving spaces RSA and RSB.
  • the receiving spaces RSA and RSB may be arranged at the same y position and at the same z position but on different x positions.
  • the first receiving space RSA may be configured to receive and/or to retain a first processing chamber PCA or a first reaction chamber RSA.
  • the second receiving space RSA may be configured to receive and/or to retain a second processing chamber PCB or a first reaction chamber RSA.
  • the processing may comprise changing of temperature, mixing, filling, removing a liquid etc.
  • the reaction may be a chemical or a biochemical reaction.
  • At least one or both of the processing chambers PCA, PCB may comprise a respective stirring bar StBA, StBB, e.g. comprising a magnetizable material, e.g. a material that becomes magnetic under the influence of an external magnetic field (e.g. disregarding the magnetic field of the earth).
  • At least one of the stirring bars (or other shaped element) StBA, StBB may be rotated using the magnet unit Magi, preferably both stirring bars StBA, StBB in sequential time order.
  • At least one or both of the processing chambers PCA, PCB may comprise magnetizable beads Be.
  • the magnetizable beads Be may be magnetized using magnetic unit Magi .
  • device D may comprise a control unit CUI, e.g. comprising a microprocessor, a microcontroller or an electronic circuitry comprising a finite state machine that does not execute program instructions but that changes its internal operation states depending on electrical inputs and/or depending on internal signal, e.g. expiry of internal timers.
  • the microprocessor may be configured to execute program instructions of a program that may be stored in an electronic memory, e.g. in a volatile storing memory (RAM, random access memory) or in a non-volatile storing memory (ROM, read only memory) and variants thereof, e.g. Flash EEPROM (electrically erasable programmable ROM)). Examples for a higher programming language are mentioned in this application, e.g. with regard to mixing instructions.
  • the instructions of the higher programming language may be translated, interpreted, etc. into instructions that may be executed by the microprocessor or by the microcontroller as is known to the person skilled in the art.
  • the control unit CUI may e.g. control driving elements of the axis modules XAM1, YAM1 and ZAM1, e.g. electrical stepper motors.
  • the control unit CUI may be a specific control unit (dedicated control unit) of the device D.
  • the control unit CUI may be a central control unit of the device D, PRD.
  • the device D, PRD may comprise an electronic camera Caml, e.g. a CCD (charge coupled device) type camera or a CMOS (complementary metal (or highly doped semiconductor material) oxide semiconductor) type camera.
  • the camera Caml may be used to take pictures of at least one processing chamber PCA, PCB, e.g. for regulatory purposes.
  • camera Caml may be able to take pictures of other parts of cartridge C, e.g. o switching wheel SW, 200 or of disc wheel DW, 600.
  • Pump P may be any appropriate pump, e.g. an axial pump (main fluid (e.g. liquid) flow may be directed in the axial direction of the pump, e.g. in the direction of a rotation axis of a rotating part of the pump), a radial pump (main fluid (e.g. liquid) flow may be directed radially with regard to an axial direction of the pump) or a diagonal pump (main fluid (e.g. liquid) flow may be directed diagonally to an axial direction of the pump) or other type of mechanical pump or of a pump using other physical pumping principles.
  • main fluid e.g. liquid
  • main fluid e.g. liquid
  • pump P may be a roller pump that may be also known as a peristaltic pump.
  • pump P may be used to pump liquids through the fluidic system, e.g. fluidic system FS1 as mentioned above, of the cartridge C or of another appropriate analysis carrier.
  • - z-axis module directly (e.g. without another intermediated axis module between) positioned on y-axis module that may be arranged on x-axis module, e.g. as illustrated in figure 2, ZAM1 on YAM1 that may be arranged on XAM1,
  • - z-axis module directly arranged on x-axis module that may be arranged on y-axis module.
  • a two dimensional (2D) or three dimensional (3D) movable robot arm may be used.
  • other movable mechanical stages may be used, e.g. two dimensional (2D) stages or three dimensional stages (3D), i.e. orthogonal multi axis systems.
  • a particle retaining device D, PRD for processing nucleic acid molecules NMA using particles Be comprising:
  • At least one receiving space RS2 for a fluidic system FS, FS1 comprising at least one particle retaining portion PRP, e.g. processing chamber PCA, PCB,
  • At least one particle retaining unit PU1, PRU that may be configured to apply an external magnetic field to the particle retaining portion PRP e.g. processing chamber PCA, PCB in order to provide retaining forces to the particles Be within the particle retaining portion PRP which retain the particles Be within the particle retaining portion PRP,
  • a fluid transport unit FTU, P that may be configured to transport fluid through the particle retaining portion PRP, e.g. processing chamber PCA, PCB,
  • a control unit CUI, CU2 that may be configured to control the at least one particle retaining unit PU1, PRU and the fluid transport unit P, FTU in a fluid transport operation state of the device D, PRD such that the at least one particle retaining unit PRU retains the particles, e.g. beads Be within the at least one particle retaining portion PRP when the fluid transport unit FTU transports a fluid through the particle retaining portion PRP.
  • Figure 3 illustrates a second - more specific - embodiment of a positioning unit PU2, PRU (particle retaining unit) for a magnetic field generation unit Mag2.
  • Device D, PRD (particle retaining device) as illustrated in figure 3 may comprise the same units that are described above for device D, PRD as illustrated in figure 2.
  • reference is made to the description of figure 2, e.g. for:
  • An x-axis module XAM2 that may be similar to x-axis module XAM1 and that may have the same function(s),
  • a y-axis module YAM2 that may be similar to y-axis module YAM1 and that may have the same function(s),
  • a z-axis module ZAM2 that may be similar to z-axis module ZAM1 and that may have the same function(s).
  • the device D, PRD may comprise a main chassis (not illustrated) that may be surrounded by a casing of device D, PRD (also not illustrated).
  • a sub chassis CHA2 may be mounted onto the main chassis.
  • the (sub) chassis CHA2 may comprise an essentially U shaped frame as illustrated in figure 3, e.g. comprising two sidewall which extend into x direction and comprising a base plate that extends also into the x direction.
  • the x-axis module XAM2 may be mounted onto the chassis CHA2 as is described in more detail below.
  • the x-axis module XAM2 may comprise:
  • the linear driving unit LDUx in x direction may comprise:
  • - A (driving) motor Mx e.g. a stepper motor, that may be mounted on the chassis CHA2 or on the main chassis or on another appropriate part of device D, PRD, and/or
  • a spindle SPIx connected to a shaft of the motor Mx and extending in the same axial direction (x direction) as the shaft of motor Mx, and/or
  • the linear driving unit LDUx may form a first part of a coupling portion CPx that corresponds to coupling portion CPxl.
  • the spindle nut may be configured to interact mechanically with the spindle SPIx, especially with a screw thread of the spindle SPIx.
  • the spindle SPIx may comprise a trapezoid screw thread or another appropriate type of screw thread.
  • the linear guiding unit LGUx in x direction may comprise:
  • a slide SLx (or carriage) that may be arranged slideable within linear guiding track
  • the guiding track GTx may be mounted onto chassis CHA2 and may extend in the x direction.
  • the slide SLx may carry the x-direction frame FRx2 or may be integrally formed with this frame FRx2.
  • the part of the slide SLx that may be arranged within guiding track GTx may have the shape of a “swallow tail” or “dove tail” or other appropriate shape.
  • the guide track GTx may have a shape in a cross section which shape may be complementary to the shape of the side SLx that may be arranged within guiding track GTx.
  • a T-shaped portion of slide SLx may be arranged within guiding track GTx enabling movement of slide SLx in x-direction but preventing movement of slide SLx in y-direction and in z-direction.
  • another type of linear guiding unit LGUx may be used, e.g. similar to the linear guiding system LGUz in z direction that may be mentioned below.
  • the linear guiding unit LGUx may form a second part of coupling portion CPx.
  • other types of coupling may be used as well, e.g. using a one part coupling portion CPx that comprises only a linear driving unit configured to fulfill the functions of driving and of guiding.
  • the x-direction frame FRx2 may extend into the y direction from slide SLx.
  • the frame FRx2 may have four columns that extend from slide SLx into the y direction away from chassis CHA2. Two columns may be arranged at a front part of slide SLx, e.g. closer to the viewer in the illustration of figure 3 compared to the two other columns of frame FRx2 which may be arranged at a rear part of slide SLx.
  • a left track and a right track may be formed by frame FRx2 which tracks are configured to guide frame FRy2 that holds tooth rack TR of the y-axis module YAM2 as described in more detail below.
  • x-axis module XAM2 may be configured to position frame FRx2 and finally magnetic unit Mag2 therewith at an x position depending on control signals of control unit CU2 which may be similar to control unit CU 1.
  • a rotation of the shaft of the motor Mx may be transmitted to a rotation of spindle SPIx.
  • Rotation of spindle SPIx may be converted by the spindle nut within frame FRx2 into a linear movement of slide SLx within guiding track GTx into the x direction, e.g. to the viewer of figure 3 and/or to a front part of chassis CHA2 or away from the viewer of figure 3 and/or to a rear part of chassis CHA2.
  • the y-axis module YAM2 may comprise:
  • the linear driving unit LDUy in y direction may comprise:
  • a motor My e.g. stepper motor that may be mounted on frame FRx2, e.g. on the two rear part columns of frame FRx2, and/or
  • a toothed wheel TW that may be mounted on a shaft of motor My, and/or
  • a tooth rack TR that may be configured to interact with toothed wheel TW.
  • the tooth rack TR may be arranged slideable between the two left columns of frame FRx2 or other type of support.
  • a part of frame FRy2 may be guided slideable within the two right columns of frame FRx2, e.g. in order to provide a second guiding track that may stabilize linear movement of frame FRy2 relative to frame FRx2.
  • the linear driving unit LDUy may form a first part of a coupling portion CPy that corresponds to coupling portion CPyl.
  • the linear guiding unit LGUy in y direction may comprise:
  • At least one guiding track e.g. formed by the column of frame FRx2, and/or
  • An optional second guiding track e.g. formed by at least one other column of frame FRx2, and/or
  • a slide or carriage e.g. formed by lower parts of frame FRy2 and/or by tooth rack TR which may be connected to frame Fry2.
  • this slide may be one-sided mounted and/or supported.
  • the guiding tracks formed by the respective columns of frame FRx2 may extend into the y direction.
  • the frame FRy2 may be configured to be movable, e.g. to slide into y direction within the guiding tracks formed by the respective columns of frame FRx2.
  • movement of frame FRy2 relative to frame FRx2 into the x direction or into the z direction may be prevented by linear guiding unit LGUy.
  • the linear guiding unit LGUy may form a second part of coupling portion CPy.
  • other types of coupling may be used as well, e.g. using a one part coupling portion CPy that comprises only a linear driving unit configured to fulfill the functions of driving and of guiding.
  • the y-direction frame FRy2 may comprise a lower part that interacts with the columns of the frame FRx2 and an upper part that forms of guiding track GTz which extends into the z direction.
  • the lower part of frame FRy2 may carry tooth rack TR, e.g. may be connected to tooth rack TR.
  • tooth rack TR may be formed integrally with frame FRy2.
  • the lower part of frame FRy2 may be arranged telescopically within the columns of frame FRx2 thereby enabling upwards or downwards movement of the frame FRy2 depending on the rotation direction of the shaft of motor My that interacts via the tooth wheel TW with toot rack TR.
  • y-axis module YAM2 may be configured to position frame FRy2 and finally magnetic unit Mag2 therewith at a y position depending on control signals of control unit CU2 which may be similar to control unit CU 1.
  • a rotation of the shaft of the motor My may be converted by the tooth wheel TW and by the tooth rack into a linear upwards or into a linear downwards movement of frame FRy2 within the guiding tracks that are formed by the at least one column of frame FRx2.
  • the z-axis module ZAM2 may comprise:
  • the linear driving unit LDUz in z direction may comprise:
  • a motor Mz e.g. a stepper motor or other electrical motor, and/or
  • the linear driving unit LDUz may form a first part of a coupling portion CPz that corresponds to coupling portion CPzl.
  • the motor Mz may be mounted onto frame FRy2.
  • the spindle SPIz may be mounted onto a shaft of the motor Mz and may extend into the same axial direction as the shaft of the motor Mz, e.g. into the z-direction.
  • the spindle nut may be arranged within the frame FRz2.
  • the spindle nut may be configured to interact mechanically with the spindle SPIz, especially with a screw thread of the spindle SPIz.
  • the screw thread of the spindle SPIz may have an appropriate cross section, e.g. trapezoid.
  • the linear guiding unit LGUz in z direction may comprise:
  • a guiding track GTz that may extend into the z direction, and/or
  • a slide SLz (carriage) that may be arranged movable on guiding track GTz.
  • the linear guiding unit LGUz may form a second part of coupling portion CPz.
  • other types of coupling may be used as well, e.g. using a one part coupling portion CPz that comprises only a linear driving unit configured to fulfill the functions of driving and of guiding.
  • the guiding track GTz may have two tranversally extending protrusions that may extend into the x direction, e.g. over the whole length (z-direction) of guiding track GTz.
  • a lower part of slide SLz may be attached around the protrusions in order to allow movement in the z direction but to block movement into the x direction and into the y direction.
  • another type of linear guiding unit LGUz may be used, e.g. similar to the linear guiding system LGUx in x direction that is mentioned above.
  • the z-direction frame FRz2 may be mechanically connected to slide SLz or may be formed integrally with slide SLz.
  • Frame FRz2 may be used to mount a magnetic field generation unit, e.g. magnet unit Mag2 as mentioned below.
  • z-axis module ZAM2 may be configured to position the frame FRz2 and finally magnetic unit Mag2 therewith at a z position depending on control signals of control unit CU2 which may be similar to control unit CU 1.
  • a rotation of the shaft of the motor Mz may be transmitted to a rotation of spindle SPIz.
  • Rotation of spindle SPIz may be converted by the spindle nut within frame FRz2 into a linear movement of the slide SLz onto guiding track GTz into the z direction, e.g. to the left of figure 3 and/or to the right, e.g. as seen by a viewer of figure 3.
  • the magnet unit Mag2 may be similar to magnet unit Magi mentioned above.
  • a rotatable permanent magnet e.g. comprising neodymium or other super magnetic material
  • a rotating magnet field (electromagnetic field) unit may be used, e.g. an electromagnet.
  • Other possibilities are rotary magnet fields as known from the stator part of stepper motors or other electrical motors.
  • a motor Mmag e.g. a high speed motor may be mounted on frame FRz2.
  • the magnet unit Mag2 may be mechanically coupled, e.g. fixedly attached to the shaft of the motor Mmag.
  • the motor Mmag may be a stepper motor or another appropriate motor.
  • the shaft of motor Mmag may extend into the z direction.
  • the magnet unit Mag2 may correspond to magnet unit Magi and may comprise:
  • At least one magnetic north pole N preferably only one magnetic north pole N, and/or
  • At least one magnetic south pole S preferably only one magnetic south pole S.
  • the magnet unit Mag2 may be configured to interact, e.g. to magnetize, to move, to rotate, etc. with the at least one stirring bar StB2 that is arranged in processing chamber PC2 or with another stirring bar that is arranged in another processing chamber, e.g. PCI, PC3, etc.
  • the magnet unit Mag2 may be configured to interact with magnetizable beads directly or indirectly via an optional stirring bar, e.g. stirring bar StB2, e.g. to magnetize the beads, to hold the beads or to move the beads Be.
  • no auxiliary (stirring) element may be used and the magnet unit Mag2 may interact only with the particles, e.g. beads within the respective processing chamber or other part of the fluidic system FS, e.g. a portion of a channel a to z3.
  • the positioning unit PU2 may be configured to perform the functions that are described in more detail below, e.g. performing static mixing/ stirring and/or performing dynamic mixing and/or stirring and/or enabling collection of beads Be and/or enabling or promoting binding of molecules to the beads, e.g. of nucleic acid molecules NAM and/or enabling or promoting de-binding or release of molecules from the beads Be.
  • An embodiment of the electrical connection of motors Mx, My, Mz and of motor Mmag to a control unit CU2 is described in the following. Other connection schemes are possible as well.
  • Control unit CU2 may be similar to control unit CUI mentioned above.
  • a first printed circuit board PCB1 mounted on the frame FRx2, e.g. on the right side of the frame FRx2 according to the view illustrated in figure 3, and/or
  • a second printed circuit board PCB2 mounted on the frame FRy2, e.g. on the right side of the frame FRy2 according to the view illustrated in figure 3, and/or
  • a third printed circuit board PCB3 mounted on frame FRz2, e.g. on the top of the frame FRz2.
  • the following flexible connections may be used in order to connect the printed circuit boards PCB or other carriers for carrying electrical connections to each other and/or to the control unit CU2 thereby allowing mechanical movement of the frames FRxl, FRy2 and FRz2 relative to each other, e.g. by using appropriated flexible “loops” of the connections as is illustrated in figure 3 :
  • the first flexible flat cable FFC1 may be connected to the control unit CU2 at one of its ends.
  • the other end of the first flexible flat cable FFC1 may be connected to the first printed circuit board PCB1, e.g. using a connector CONI, e.g. releasable.
  • a first loop of the first flexible flat cable FFC1 may enable movement of frame FRx2 away from control unit CU2 by a predetermined distance, e.g. in the range of 5 cm (centimeter) to 20 cm.
  • the second flexible flat cable FFC2 may connect the first printed circuit board PCB1 and the second printed circuit board PCB2 electrically.
  • a second flexible loop of the second flexible flat cable FFC2 may allow movement of frame FRy2 relative to frame FRx2 into the y direction, e.g. by a distance within a range of 5 cm (centimeter) to 20 cm.
  • the third flexible flat cable FFC3 may connect the second printed circuit board PCB2 and the third printed circuit board PCB3 electrically.
  • a third flexible loop of the third flexible flat cable FFC3 may allow movement of frame FRz2 relative to frame FRy2 into the z direction, e.g. by a distance within a range of 5 cm (centimeter) to 20 cm.
  • the motor Mx may be connected to the control unit CU2 by an electrical connection (bundle) that is not illustrated in figure 3.
  • This connection may not comprise a loop, e.g. additional cable length since the motor Mx is arranged stationary to chassis CHA2 and also relative to the control unit CU2.
  • this connection may comprise a releasable connector CONO (not illustrated) or may not comprise a releasable connector CONO.
  • the following further releasable electrical connectors CON may be used to connect other electrical drive units of positioning module PU2 to the control unit CU2:
  • An electrical connector CONMag e.g. releasable for motor Mmag that may carry the magnet as mentioned above.
  • the second electrical connector CON2 may be arranged on the first printed circuit board PCB1.
  • the second electrical connector CON2 may comprise a socket or a plug.
  • the respective counterpart may be connected mechanically and electrically to connector CON2 in order to implement an electrical connection to the motor My, especially a releasable electrical and mechanical connection.
  • the third electrical connection CON3 may be arranged on the second printed circuit board PCB2.
  • the third electrical connector CON3 may comprise a socket or a plug.
  • the respective counterpart may be connected mechanically and electrically to connector CON3 in order to implement an electrical connection to the motor Mz, especially a releasable electrical and mechanical connection.
  • the electrical connection CONMag may be arranged on the third printed circuit board PCB3.
  • the electrical connector CONMag may comprise a socket or a plug.
  • the respective counterpart may be connected mechanically and electrically to connector CONMag in order to implement an electrical connection to the motor Mmag.
  • the releasable connect! on/conenctors may ease assembling of unit PU2, PRU considerably.
  • no releasable connectors CONI, CON2, CON3 or CONMag or only a part of these connectors CON is used.
  • the motors My, Mz and/or Mmag may be electrically connected fixedly to the respective printed circuits boards PCB1, PCB2, PCB3 or other carrier for electrical connections.
  • no printed circuit boards or other carrier for electrical connections or only a part of the printed circuit boards PCB1, PCB2, PCB3 are used.
  • a cable harness may be used instead of the printed circuit boards.
  • control unit CU2 may control all four motors Mx, My, Mz and Mmag via the electrical connections mentioned above, see the two arrows which point away from control unit CU2 in figure 3.
  • control unit CU2 or another control unit of device D may control a pump P, e.g. a peristaltic pump that comprises a flexible hose H.
  • hose H may be arranged on the cartridge C that comprises also at least one processing chamber, e.g. the processing chamber PC2 within the processing chamber retaining space RS2.
  • device D may comprise a camera Cam2 that may be similar to camera Caml mentioned above.
  • Camera Cam2 may be controlled by control unit CU2 or by another control unit of device D.
  • PRU Positioning unit PU2, PRU may be identified by a type shield TS that may be arranged on the right wall of chassis CHA2.
  • Type shield TS may be used for regulatory purposes or for other purposes.
  • An optional sensor module SM may be mounted on frame FRy2, e.g. on the left side.
  • the sensor module SM may carry at least one light barrier or another appropriate positioning sensor in order to allow to position frame FRx2 relative to chassis CHA2 or to another chassis or casing of device D, PRD.
  • a protrusion (not illustrated) may be arranged on chassis CHA2 or on another appropriate element of device D. As soon as the light barrier or other sensor reaches the protrusion a corresponding detection signal is generated by the sensor and transmitted to the control unit CU2, thereby enabling exact positioning and/or calibration of positioning for the x direction.
  • Similar optional sensor modules may be used on the frame FRy2 and/or on the frame FRz2 in order to allow positioning and/or calibration of positioning in y direction and/or in z direction.
  • Corresponding protrusions may be arranged on the frame FRx2 for the y direction and/or on the frame FRy2 for the z direction.
  • PRD the at least one particle retaining unit PU2, PRU may comprise at least one of, at least two of or all three of
  • z-axis module ZAM1, ZAM2 that may be configured to move the magnetic field along a z-axis transversally towards or away from the particle retaining portion PRP, and/or
  • YAM1 - a y-axis module YAM1, YAM2 that may be configured to move the magnetic field along a y-axis laterally relative to the particle retaining portion PRP, wherein the y-axis is arranged perpendicular or about perpendicular relative to the z-axis, e.g. in the direction of gravity,
  • an x-axis module XAM1, XAM2 that may be configured to move the magnetic field along an x-axis laterally relative to the particle retaining portion PRP, wherein the x-axis is arranged perpendicular or about perpendicular relative to the z-axis and/or to the y-axis, e.g. perpendicular to the direction of gravity.
  • Figure 4 illustrates an arrangement of a processing chamber, e.g. processing chamber PC2 and of a magnetic field generation unit, e.g. comprising or consisting of a permanent magnet, especially magnet unit Mag2 during a dynamic mixing (stirring) operation state/mode in a first high magnetic flux density operation mode of a device D.
  • Device D may be used for enriching nucleic acid molecules NAM using optional magnetizable beads Be or other particles.
  • the magnet unit Mag2 may rotate Rot around a rotation axis RA, e.g. a longitudinal axis of the shaft of motor Mmag as described above.
  • the rotation axis RA may extend in z direction.
  • a distance Dil between the magnet unit Mag2 and processing chamber PC2 may be low, e.g. within the range of 1 mm (millimeter) to 5 mm, especially 2 mm.
  • Stirring bar, e.g. StB2 and optional magnetizable beads may be magnetized by the high magnetic field of magnet unit Mmag that is arranged closely to the processing chamber, e.g. PC2.
  • only beads Be may be magnetized, e.g. using a magnetic stirring bar StB2.
  • the magnet unit Mag2 may rotate around rotation axis RA, see rotation Rot.
  • the speed of rotation may be in the range of e.g. 1500 rpm to 2500 rpm (rounds per minute), especially 1900 rpm.
  • the magnetized stirring bar, e.g. StB2 will follow the rotation of the magnet unit, e.g. Mmag.
  • the magnetizable beads Be may be arranged on the magnetized stirring bar StB2, especially at the start of mixing, see e.g. description of figures 11 to 15 further below.
  • an additional movement M of the magnet unit e.g. Mmag may be implemented, e.g. using positioning unit PU1, PRU or positioning unit PU2, PRU as mentioned above.
  • An example of a movement M is described below in more detail with reference to figure 6.
  • Simple movements may be used alternatively, e.g. only an up or down movement, e.g. in y direction, a combined upwards movement followed by a downwards movement or vice versa.
  • Another example for a simple movement may be a left or right movement, e.g. in x direction, or a combined left movement/right movement.
  • the additional movement of magnet unit Mmag may be continuously or stepwise or in another appropriate way.
  • Dynamic mixing/ stirring may allow excellent mixing, e.g. involving particles, e.g. beads within the whole chamber volume of processing chamber PC2.
  • Example la dynamic mixing/ stirring (e.g. mixing of lysis and isopropanol): Before step S57 perform optional steps S51 to S56 as mentioned below, i.e. collect beads after filling in lysate, isopropanol and chaotropic salt, e.g. in solution. Steps S51 to S56 are described in more detail below, see e.g. figure 9, positions Pl to end position P6a.
  • the instructions that are mentioned in this application are instructions for a simulation program that is used to simulate the method steps performed in the real testing device, e.g. in the device D, PRD.
  • the instructions are written in xml language (extended markup language).
  • PRD especially in control unit CUI, CU2 or in another control unit of device D, PRD there may be stored corresponding program instructions in order to perform in reality the functions that are simulated by the simulation program.
  • the program instructions of the simulation program may have equivalent program instructions sored in a memory of device D, PRD.
  • similar instructions may be used to program device D, PRD e.g. using other naming of the variables, using another sequence of the variables, using more variables/parameters and/or using less variables/parameters per instruction.
  • A30MixProgramExecute may specify a call of a dynamic mixing program or a subroutine that implements the function of dynamic mixing. “A30” may be the name of device D, PRD e.g. a trademark name.
  • Chamber “MagneticSepChamber”: may specify the processing chamber for mixing or stirring, e.g. processing chamber PC2, e.g. magnetic separation chamber or another processing or reaction chamber, e.g. of the analysis carrier, especially of the cartridge C.
  • RotSpeed “1900”: may specify the rotation speed of a magnetic unit, e.g. of magnetic unit Magi or Mag2.
  • the rotation speed may be similar to the rotation speed of at least one stirring bar within the processing/reaction chamber.
  • the rotation speed is high, e.g. 1900 rpm or may have a value within a range as specified in the introductory part of the description.
  • Base may specify the type of the mixing program, e.g. “basic” or a more enhanced mixing program type.
  • An example for the type “basic” may be as described below with reference to figure 6.
  • Count “3”: may specify the number of mixing cycles, e.g. how often the mixing program is performed, e.g. three times as in the example la. This may specify the duration of mixing implicitly or indirectly.
  • Bottom YPos “0”: may specify a reference point for a mixing “rectangle” or other mixing region.
  • the mixing region may be covered by movements and/or by rotations of the stirring bar or other mixing element or only by the external magnet if no auxiliary (mixing) element is used. Another reference point may be used as well, e.g. instead of the bottom, the top or a comer of the region.
  • the mixing region may be a rectangle (2D, two dimensional) or a square (2D) or another appropriate region, e.g. a circle (2D), an ellipse (2D), a cuboid (3D, three dimensional), a cube (3D), a sphere (3D) or an ellipsoid (3D).
  • Z “2”: may specify the distance between the magnetic unit, e.g. Magi, Mag2 and the processing chamber, e.g. PC2. This distance may be measured from the outer surface of processing chamber to an outer surface of the magnetic unit, e.g. Magi, Mag2. Alternatively, other appropriate distances may be specified.
  • the z value of 2 mm (millimeter) may be used in the example la.
  • the duration of mixing may not be specified explicitly. However, the duration may be specified in an alternative embodiment explicitly. See embodiment for a possible moving path of the magnetic unit during dynamic mixing as illustrated in figure 6 and as described in more detail below.
  • S59 optionally promote release of particles, see below.
  • Binding may follow, e.g. steps S62 to S65, e.g. comprising a waiting loop including pumping, e.g. within the range of 30 s (seconds) to 60 s, or within other ranges as mentioned above, no picture may be taken, optionally a picture of the binding process may be taken during binding.
  • Example lb dynamic mixing/stirring (e.g. for washing 1 step (washing 1 solution)):
  • the beads Be may still be attached on stirring element/bar, e.g. stirring bar StBB. If not, optional steps similar or identical to steps S51 to S56 may be performed to collect the beads Be on the stirring element, e.g. on StBB.
  • S97 optionally promote release, see below.
  • S98 optional, depending of the implementation, stop of the rotation of the stirring bar and/or of the rotating magnet.
  • Binding may follow, e.g. steps S99 to S107 comprising e.g. waiting loop including pumping and taking of picture.
  • Collection of the beads Be may follow, e.g. steps S108 to SI 12 as mentioned below, figure 9, position 1 Pla to P6b.
  • Example 1c dynamic mixing/stirring (e.g. washing 2 step (e.g. using washing 2 solution):
  • Beads Be may still be on stirring element/bar, e.g. StB2. If not, optional steps similar or identical to steps S51 to S56 may be performed to collect the beads Be.
  • SI 30 optional static mixing, see below.
  • Binding may follow, e.g. steps S133 to S136 (e.g. using a waiting loop and not taking of picture or optionally taking a picture of the binding process).
  • Collection of the beads Be may follow, e.g. step S137 to S141 as mentioned below, figure 9, positions Pl to P6c.
  • Example Id dynamic mixing/stirring (e.g. ethanol washing):
  • the beads Be may still be on stirring element/bar, e.g. stirring bar StBB. If not, optional steps similar or identical to steps S51 to S56 may be performed in order to collect the beads Be on the stirring element, e.g. on stirring bar StBB.
  • SI 60 optional static mixing, see below.
  • SI 62 optional, depending of the implementation, separate step in order to stop the rotation of e.g. the stirring bar.
  • Binding may follow, e.g. steps SI 63 to SI 66 at least one waiting loop and not taking of a picture or optionally taking picture of the binding process. )
  • Collection of the beads Be may follow, e.g. steps SI 67 to SI 83 as mentioned below, see e.g. figure 10, positions PIO to P27.
  • Example le dynamic mixing/ stirring (e.g. in preparation of de-binding):
  • step S256 to S260 collect the beads Be in step S256 to S260 as mentioned below, see figure 8, example 3f. If beads Be are still attached on the stirring element, these steps may not be performed. Optional releasing of the beads Be may be possible as well.
  • S263 optionally promote release, see below.
  • De-binding may follow, e.g. steps S265 and S266 comprising at least one waiting loop, e.g. not including pumping and not including taking of a picture of the processing chamber and/or of other parts of the cartridge C or of another analysis carrier.
  • Collection of the beads Be may follow in step S276 to S281 as mentioned below, e.g. from lower portion of chamber to upper portion, see figure 8, example 3g.
  • PRD the control unit CU may be configured to control the at least one particle retaining unit PRU such that in a dynamic mixing state of the device D:
  • the magnetic field may enable to transmit a mixing force to the particles Be and/or to a magnetizable element MME, preferably a stirring element StB A, StBB arranged within the particle retaining portion PRP, PC2, etc.
  • the magnetic field may be rotated with a rotation speed above 1500 rpm, and that at least one particle retaining unit PRU may be moved in order to move the magnetic field in addition to the rotation continuously laterally within the particle retaining portion PRP,
  • the dynamic mixing state may be performed before the fluid transport operation state of the device in order to mix a fluid within the particle retaining portion PRP.
  • Figure 5 illustrates an arrangement of a magnetic field generation unit e.g. comprising or consisting of a permanent magnet, e.g. Magi or Mag2 relative to a processing chamber, e.g. processing chamber PC2 during a static mixing (stirring may be used equivalently) operation state/mode in the first high magnetic flux density operation mode, e.g. of a device D, PRD for enriching nucleic acid molecules (NAM) using e.g. magnetizable beads or other appropriate particles.
  • a magnetic field generation unit e.g. comprising or consisting of a permanent magnet, e.g. Magi or Mag2 relative to a processing chamber, e.g. processing chamber PC2 during a static mixing (stirring may be used equivalently) operation state/mode in the first high magnetic flux density operation mode, e.g. of a device D, PRD for enriching nucleic acid molecules (NAM) using e.g. magnetizable beads or other appropriate particles.
  • NAM nucleic acid molecules
  • Example 2a e.g. if lysate, isopropanol and chaotropic salt are within magnetic separation chamber (MSC), processing chamber PC2):
  • the instruction A30MixerSetPosAndSpeed may be used to program static mixing using a higher-level program language. Alternatively, the same function may be programmed using a programming language that is closer to the instruction set of a microprocessor or of a microcontroller. Static mixing may be performed on a position that is specified in the instruction(s). More parameters than specified below may be used. Alternatively, less parameters than specified below may be used. The parameters may be named different but may still have the same function:
  • Chamber "MagneticSepChamber”: see description mentioned above for dynamic mixing.
  • static mixing may be performed in the processing chamber PC2 of the cartridge C or in another chamber, e.g. processing chamber PCI or PC3.
  • X "0": may specify the x position for static mixing, e.g. position “0”.
  • the Cartesian coordinate system as illustrated in figures 9 and 10 may be used.
  • Y "8": may specify the y position for static mixing, e.g. position “8” in the example.
  • the Cartesian coordinate system as illustrated in figures 9 and 10 may be used.
  • the distance Dil between the stirring element and the outer surface of the wall of the processing chamber, e.g. PC2 may be 2 mm (millimeters).
  • RotFeedback "On”: see the feature rotation feedback as mentioned above, e.g. using mixing sound or other detection principles.
  • Comment "": an optional comment may be provided.
  • the duration parameter is set to 3 seconds.
  • the rotation speed may be increased from 1900 rpm to 2000 rpm.
  • the z distance may be increased from 2 mm to 15 mm. This may promote release of particles from the stirring element. Release of the particles from the stirring element may be promoted by this optional step.
  • the duration parameter is set to 0 seconds.
  • the rotation speed may be decreased from 2000 rpm to 0 rpm, e.g. rotation may be switched off or a low rotation speed may be selected, e.g. below 10 rpm.
  • Static mixing may be performed in processing chamber PC2 (magnetic separation chamber) using rotation speed of 1900 rpm for 5 minutes.
  • the moving speed is set to 10 mm per second.
  • the position for static mixing (stirring) may be 0, 6 (x, y).
  • the distance Di 1 may be low, e.g. 2 mm.
  • the magnetic field density B may be high within processing chamber PC2.
  • the duration parameter is set to 1 second.
  • the rotation speed may be increased from 1900 rpm to 2000 rpm for 1 second.
  • the moving speed may be increased from 10 mm per second to 25 mm per second. This may be relevant for movement in z direction. No movement in x direction and/or y direction may be performed.
  • the z distance may be increased from 2 mm to 15 mm. Release of the particles from the stirring element may be promoted by this optional step.
  • the duration parameter is set to 0 seconds.
  • the rotation speed may be decreased from 2000 rpm to 0 rpm, e.g. rotation may be switched off or a low rotation speed may be selected, e.g. below 10 rpm.
  • the duration parameter is set to 5 seconds.
  • Static mixing may be performed in processing chamber PC2 (magnetic separation chamber) using rotation speed of 1900 rpm for 5 seconds.
  • the moving speed is set to 10 mm per second.
  • the position for static mixing (stirring) may be 0, 6 (x, y).
  • the distance Dil may be low, e.g. 2 mm.
  • the magnetic field density B may be high within processing chamber PC2.
  • the duration parameter is set to 3 seconds.
  • the rotation speed may be increased from 1900 rpm to 2000 rpm.
  • the moving speed may be increased from 10 mm per second to 25 mm per second. This may be relevant for movement in z direction. No movement in x direction and/or y direction may be performed.
  • the z distance may be increased from 2 mm to 15 mm. Release of the particles from the stirring element may be promoted by this optional step.
  • the duration parameter is set to 0 seconds.
  • the rotation speed may be decreased from 2000 rpm to 0 rpm, e.g. rotation speed may be switched off or a low rotation speed may be selected, e.g. below 10 rpm.
  • Example 2d (e.g. during ethanol washing within magnetic separation chamber (MSC), PC2)):
  • the duration parameter is set to 5 seconds.
  • Static mixing is performed in processing chamber PC2 (magnetic separation chamber) using rotation speed of e.g. 1900 rpm for 5 seconds.
  • the moving speed is set to 10 mm per second.
  • the position for static mixing (stirring) may be 0, 7 (x, y).
  • the distance Dil may be low, e.g. 2 mm.
  • the magnetic field density B may be high within processing chamber PC2.
  • the duration parameter is set to 2 seconds.
  • the rotation speed may be increased from 1900 rpm to 2000 rpm.
  • the moving speed may be increased from 10 mm per second to 25 mm per second. This may be relevant for movement in z direction. No movement in x direction and/or y direction may be performed.
  • the z distance may be increased from 2 mm to 15 mm. Release of the particles from the stirring element may be promoted by this optional step.
  • the duration parameter is set to 0 seconds.
  • the rotation speed may be decreased from 2000 rpm to 0 rpm, e.g. rotation may be switched off or a low rotation speed may be selected, e.g. below 10 rpm.
  • Static mixing may be performed in processing chamber PC2 (magnetic separation chamber) for 2 seconds using a rotation speed of 1900 rpm.
  • the moving speed may be set to 10 mm per second.
  • the position for static mixing (stirring) may be 0, 2 (x, y).
  • the distance Dil may be low, e.g. 2 mm.
  • the magnetic field density B may be high within processing chamber PC2.
  • the rotation speed may be increased from 1900 rpm to 2000 rpm.
  • the duration parameter is set to 3 seconds.
  • the moving speed may be increased from 10 mm per second to 25 mm per second. This may be relevant for movement in z direction. No movement in x direction and/or y direction may be performed.
  • the z distance may be increased from 2 mm to 15 mm. Release of the particles from the stirring element may be promoted by this optional step.
  • the duration parameter is set to 0 seconds.
  • the moving speed is decreased slightly.
  • the rotation speed may be decreased from 2000 rpm to 0 rpm, e.g. rotation may be switched off or a low rotation speed may be selected, e.g. below 10 rpm.
  • the position z is set to z equal to 15 mm.
  • the control unit CU may be configured to control the at least one particle retaining unit PRU such that in a static mixing state of the device D:
  • the magnetic field may enable to transmit a mixing force to the particles, e.g. beads Be and/or to a magnetizable element MME, e.g. StBB arranged within the particle retaining portion PRP, PC2, etc.,
  • the at least one particle retaining unit PRU may be positioned at a first lateral static position relative to the particle retaining portion PRP that enables to transmit a mixing force to the particles and/or to a magnetizable element MME arranged within the particle retaining portion PRP,
  • the magnetic field may be rotated at the first static position with a rotation speed above (or of at least) 1500 rpm or above (or of at least) 1500 rpm for at least 0.5 seconds for at least 0.5 s or 1 second, and
  • the at least one particle retaining unit PRU may be positioned thereafter at a second lateral static position that is different from the first lateral static position, wherein the second lateral static position enables to transmit a mixing force to the particles and/or to a magnetizable element MME, preferably a stirring element StBA, StBB arranged within the particle retaining portion PRP,
  • the magnetic field may be rotated at the second lateral static position with a rotation speed above 1000 rpm or above 1500 rpm for at least 0.5 s or for at least 1 second,
  • the static mixing state may be performed before the fluid transport operation state of the device in order to mix a fluid within the particle retaining portion PRP.
  • the rotation speed of the magnetic field may be controlled by the control unit CU, CUI, CU2 to such a value that centrifugal forces and/or shear forces within the particle retaining portion PRP are generated to the particles Be that result in release of the particles Be form at least one, from at least two of or from all of:
  • a force mitigation element or other auxiliary element that is arranged within the particle retaining portion PRP preferably a stirring element StBA, StBB.
  • Figure 6 illustrates a possible mixing path during the dynamic mixing mode/ operation state.
  • Figure 4 illustrates the configuration during dynamic mixing along a cross section through the processing chamber, e.g. a processing chamber PC or processing chamber PC2.
  • the movement M may follow various predefined paths.
  • An example of a movement M is described in the following.
  • the processing chamber PC (or a reaction chamber), e.g. PC2 may have a predetermined inner volume defined by an inner surface of the wall of the processing chamber PC.
  • a mixing region MR may be defined for dynamic mixing, e.g. a rectangular (2D) or a cuboid region (3D).
  • the mixing region MR may be smaller than the chamber volume and may be specified by a reference position and by:
  • a height e.g. H6, and/or
  • a width e.g. W6, and/or
  • the movement path M may be performed such that the center point of the stirring element, e.g. the rotation axis thereof remains within the mixing region MR.
  • the movement path may be specified such that the ends of the stirring element, e.g. stirring bar StBB remain within the mixing region MR during mixing.
  • the movement path M may comprise:
  • a first movement Ml e.g. a straight downwards movement in y direction within mixing region MR, and/or
  • a second movement path M2 e.g. a straight upwards movement in y direction within mixing region MR, and/or
  • a third movement path M3 e.g. along a first circle or ellipsoid in anticlockwise direction within mixing region MR, e.g. combined movement in x direction and in y direction within mixing region MR, and/or
  • a fourth movement path M4 e.g. along a second circle or ellipse in anticlockwise direction within mixing region MR, e.g. combined movement in x direction and in y direction within mixing region MR.
  • Figure 7 illustrates the arrangement of the magnetic field generation unit relative to the processing chamber during a preparation step for the collection of particles (e.g. beads).
  • particles e.g. beads
  • the same arrangement of the magnetic field generation unit relative to the processing chamber, e.g. PC2 may be used during a binding step of the nucleic acid molecules NAM to the beads Be or during or a de-binding (release) step of the nucleic acid molecules NAM from the beads Be.
  • a distance Di2 between the outer surface of the wall of the processing chamber, e.g. PC2 and the stirring element may be large, e.g. more than 10 mm or more than 13 mm, e.g. 15 mm.
  • the magnetic field density B within the processing chamber PC2 may be low, e.g. not sufficient to magnetize and/or to attract the stirring element, e.g. stirring bar StB2 to the magnetic unit. Therefore, the stirring element may sink to the bottom of the processing chamber, e.g. PC2.
  • the magnetic field density B within the processing chamber PC2 may be low, e.g. not sufficiently to magnetize the magnetizable beads Be. Therefore, the beads Be may not be attracted to the stirring bar and/or may not attract each other.
  • the beads Be may swim or float within the liquid that is filled within the processing chamber, e.g. PC2.
  • the liquid may be a binding buffer (bb) solution, a non-binding buffer (non bb) solution or another appropriate solution.
  • a first method step that may use the arrangement illustrated in figure 7 is the release of the beads Be.
  • steps S59, S97, S131, S161, S263 are mentioned in steps S59, S97, S131, S161, S263 as mentioned above.
  • a second method step that may use the arrangement illustrated in figure 7 is the binding of NAMs to the beads.
  • NAMs nucleic acid molecules binding buffer
  • steps S60, S98, SI 32, SI 62, S264 as mentioned above.
  • Binding of NAMs to the beads Be is described with reference to figure 12 in detail below.
  • the solution e.g. a NAMbb (nucleic acid molecules binding buffer) may come to rest, e.g. there may be no additional external mixing or stirring. Binding may take place based on Brownian motion of molecules within the NAMbb in order to enhance binding.
  • the distances between the NAMs and the beads should be as short as possible.
  • a temperature value within the processing chamber, e.g. PC2 (PRP, particle retaining portion) above room temperature may be used to further enhance binding.
  • the processing chamber, e.g. PC2 may be heated accordingly using a heating element, e.g. a Peltier element or other appropriate heating element (e.g. resistive heating).
  • the temperature may be in the range of 25 °C (degree Celsius) to 37 °C. However, lower temperatures or higher temperatures may be used as well for binding.
  • a third method step that may use the arrangement illustrated in figure 7 is the de-binding (release) of NAMs from the beads Be.
  • An example for de-binding of NAMs from the beads Be is mentioned in step S264 as mentioned above.
  • NAMs de-binding (release) of NAMs from the beads is described with reference to figure 14 in detail below.
  • the solution e.g. a non-NAMbb (nucleic acid molecules non-binding buffer) may come to rest, e.g. there may be no additional external mixing or stirring. Binding may take place based on Brownian motion of molecules within the non NAMbb in order to enhance release.
  • the distances between the molecules of the non NAMbb and the particles, e.g. beads should be as short as possible. This may be reached by excellent mixing/stirring in order to have a homogeneous dispersion of particles (e.g. beads) within the non NAMbb.
  • a temperature value within the processing chamber, e.g. PC2 (PRP, particle retaining portion) above room temperature may be used to further enhance binding.
  • the temperature may be in the range of 20 °C (degree Celsius) to 60 °C.
  • lower temperatures or higher temperatures may be used as well for de-binding (release of the NAMs from the particles, e.g. from the beads.
  • the processing chamber, e.g. PC2 may be heated accordingly using a heating element, e.g. a Peltier element or other appropriate heating element (e.g. resistive heating).
  • the control unit CU, CUI, CU2 may be configured to control the at least one particle retaining unit PRU, e.g. PC2 such that in a first particle release state of the device D, PRD:
  • the value of the magnetic flux density B e.g. the maximum value within the particle retaining portion PRP may be decreased from a value that retains the particles, e.g. beads Be within the particle retaining portion PRP, preferably at a wall thereof and/or at a magnetizable element MME to a value at which the particles Be are released from the wall of the particle retaining portion PRP and/or from a magnetizable element MME, preferably a stirring element StBA, StBB arranged within the particle retaining portion PRP.
  • the magnetic field is not rotated or only rotated with a rotation speed of at most 20 rpm.
  • Figure 8 illustrates the arrangement of the magnetic field generation unit relative to the processing chamber, e.g. PC2 during a second high flux density operation mode used for the collection of particles, e.g. of beads Be.
  • a distance Dil between the outer surface of the wall of the processing chamber, e.g. PC2 and the stirring element may be low, e.g. lower than 5 mm or lower than 3 mm, e.g. 2 mm.
  • the magnetic field density B within the processing chamber PC2 may be high, e.g. sufficient to magnetize and/or to attract the stirring element, e.g. stirring bar StB2 to the magnetic unit, e.g. Magi or Mag2. Therefore, the stirring element may be hold within the processing chamber, e.g. PC2.
  • the magnetic field density B within the processing chamber PC2 may be high, e.g. sufficient to magnetize the magnetizable beads Be. Therefore, the beads Be may be attracted to the stirring bar, e.g.
  • the beads Be may not swim or float within the liquid that is filled within the processing chamber, e.g. PC2 after all beads Be have been collected to the stirring element, e.g. stirring bar StB2, StBB, etc.
  • the liquid may be a binding buffer (bb) solution, a non-binding buffer (non bb) solution or another appropriate solution.
  • No rotation of the magnet unit e.g. Magi or Mag2 may be used during usage of the arrangement illustrated in figure 8 for the collection of particles.
  • a slow rotation speed may be used, e.g. a rotation speed below 10 rpm (rounds per minute).
  • Example 3a (I: collection of beads Be before mixing of lysate, isopropanol and chaotropic salt, see also figure 9):
  • the meaning of the instruction as well as the meaning of the parameters has been described above.
  • the duration parameter is set to 0 seconds, e.g. if the position has been reached the next step follows immediately, e.g. without further waiting time.
  • the z distance may be increased to distance Di2 equal to 15 mm using a moving speed of e.g. 20 mm per minute. No rotation is used.
  • the position of the magnetic unit may be 0, 2 (x, y). No rotation feedback may be used as there is essentially no rotation.
  • This step S51 may be a preparation step for the preparation of the collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the meaning of the instruction as well as the meaning of the parameters has been described above.
  • the duration parameter is set to 1 second, e.g. if the position has been reached the next step follows e.g. after a waiting time of 1 second.
  • the z distance may be decreased to distance Dil equal to 2 mm using a moving speed of e.g. 20 mm per second. No rotation may be used.
  • the position of the magnetic unit may still be 0, 2 (x, y). No rotation feedback may be used.
  • This step S52 may result in the collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the duration parameter may still be set to 1 second.
  • the z distance may be hold at distance Dil equal to 2 mm.
  • a moving speed of e.g. 5 mm per second may be used to move the magnetic unit to the next collection position.
  • a slow rotation speed may be used, e.g. 5 rpm, especially in order to reduce the mean path length of the particles to the stirring element/bar.
  • the rotation speed may be such slow that no centrifugal forces and/or shearing forces occur. Thus, all collected particles may remain on the stirring element during particle collection using low rotation speeds.
  • the position of the magnetic unit may be changed to -2, 12 (x, y). No rotation feedback may be used. This step S53 may result in further collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the duration parameter may still be set to 1 second.
  • the z distance may be hold at distance Dil equal to 2 mm.
  • a moving speed of e.g. 5 mm per second may be used to move the magnetic unit to the next collection position.
  • No rotation may be used, e.g. the rotation speed may be set to 0 rpm again.
  • the position of the magnetic unit may be changed to 0, 13 (x, y). No rotation feedback may be used.
  • This step S54 may result in further collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the duration parameter may still be set to 1 second.
  • the z distance may be hold at distance Dil equal to 2 mm.
  • a moving speed of e.g. 5 mm per second may be used to move the magnetic unit to the next collection position.
  • No rotation may be used, e.g. the rotation speed may be still set to 0 rpm.
  • the position of the magnetic unit may be changed to 2, 12 (x, y). No rotation feedback may be used.
  • This step S55 may result in further collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the duration parameter may still be set to 1 second.
  • the z distance may be hold at distance Dil equal to 2 mm.
  • a moving speed of e.g. 5 mm per second may be used to move the magnetic unit to the next collection position.
  • No rotation may be used, e.g. the rotation speed may still be set to 0 rpm.
  • the position of the magnetic unit may be changed to 0, 9 (x, y). No rotation feedback may be used.
  • This step S56 may result in further collection of beads Be to the stirring element, e.g. stirring bar StB2. Collection of particles above a fluid level LI may be used to collect all particles or at least the majority of particles, e.g. more than 90 percent or more than 95 percent of the particles.
  • Example 3b (II: collection of beads Be after mixing of lysate, isopropanol and chaotropic salt, see also figure 9):
  • the duration parameter may still be set to 1 second.
  • the z distance may be hold at distance Dil equal to 2 mm.
  • a moving speed of e.g. 5 mm per second may be used to move the magnetic unit to the next collection position.
  • No rotation may be used, e.g. the rotation speed may be set to 0 rpm.
  • the position of the magnetic unit may be changed to 0, 6 (x, y).
  • No rotation feedback may be used.
  • This step S73 may result in further collection of beads Be to the stirring element, e.g. stirring bar StB2.
  • the collection of beads takes place in a lower portion of processing chamber PC2 compared with step S72 or with step S56, e.g. because of a lower fluid level L2.
  • the fluid level L2 may be below fluid level LI, e.g. with regard to the direction of gravity. Collection of particles above the fluid level L2 may be used to collect all particles or at least the majority of particles, e.g. more than 90 percent or more than 95 percent of the particles.
  • Example 3 c (III: collection of beads Be after mixing/washing in washing solution 1, see also figure 9):
  • the duration parameter may be set to 2 seconds.
  • the collection of beads may take place in a lower portion of processing chamber PC2 compared with step S56, e.g. because of a lower fluid level L2. Collection of particles above the fluid level L2 may be used to collect all particles or at least the majority of particles, e.g. more than 90 percent or more than 95 percent of the particles.
  • Example 3d (IV: collection of beads Be after mixing/washing in washing solution 2, see also figure 9):
  • the duration parameter may be set to 2 seconds.
  • the collection of beads may take place in a lower portion of processing chamber PC2 compared with step S56, e.g. because of a lower fluid level L3 that is lower than fluid level L2. Collection of particles above the fluid level L3 may be used to collect all particles or at least the majority of particles, e.g. more than 90 percent or more than 95 percent of the particles.
  • Example 3e (V: collection of beads Be after mixing/washing in ethanol, see also figure 10):
  • Example 3f (VI: collection of beads Be before elution (de-binding/release)):
  • Example 3g (VII: collection of beads Be after elution (de-binding/release)):
  • the control unit CU may be configured to control the at least one particle retaining unit PRU such that in a particle collecting operation state of the device D, RPD:
  • the at least one particle retaining unit PRU, PU1, PU2 may be arranged adjacent to the particle retaining portion PRP, e.g. PC2,
  • the magnetic field may apply a magnetic flux density B within the particle retaining portion PRP that is strong enough to generate retaining forces that retain at least a majority of the particles e.g. more than 90 percent by number within the particle retaining portion PRP,
  • the magnetic field may not be rotated or may only be rotated with a rotation speed of at most 20 rpm
  • the particle collecting operation step may be performed before the fluid transport operation state of the device in order to retain at least a majority of the particles within the particle retaining portion PRP, preferably at at least one wall of the particle retaining portion PRP and/or at a magnetizable element MME, e.g. StBB arranged within the particle retaining portion PRP.
  • a magnetizable element MME e.g. StBB arranged within the particle retaining portion PRP.
  • the control unit CU, CUI, CU2 may be configured to control the at least one particle retaining unit PRU such that in the particle collecting operation state of the device D:
  • the at least one particle retaining unit PRU may be positioned at a first lateral position relative to the particle retaining portion PRP in order to collect a first part of the majority of the particles,
  • the at least one particle retaining unit PRU may be positioned at a second lateral position relative to the particle retaining portion PRP in order to collect a second part of the majority of the particles Be, wherein the second lateral position is different from the first lateral position.
  • At least three positions within the particle retaining portion PRP may be used in order to collect the majority of the particles, e.g. beads Be or all of the particles.
  • the control unit CU, CUI, CU2 may be configured to control the at least one particle retaining unit PRU such that in the particle collecting operation state the particles, e.g. beads Be are collected within the particle retaining portion PRP from below to above with regard to gravity in the normal operation mode of the device D PRD.
  • the control unit CU, CUI, CU2 may be configured to control the at least one particle retaining unit PRU such that in the particle collecting operation state the particles, e.g. beads Be are collected within the particle retaining portion PRP from above to below with regard to gravity in the normal operation mode of the device D, PRD.
  • Figure 9 illustrates four variants I to IV (e.g. examples 3a to 3d as mentioned above) of a first embodiment of positions, e.g. stopping positions of a stirring element during the second high flux density operation mode, e.g. collection of particles, especially of beads Be:
  • Variant III e.g. example 3c as mentioned above, e.g. collection of beads Be after mixing/washing in washing solution 1: positions during a third sequence of positions Pl to P5 and then P6b. See steps SI 08 to SI 12 as mentioned above.
  • figure 9 indicates different filling levels LI to L3 of processing chamber PC2.
  • Filling level LI is the highest level.
  • Filling level L2 is a medium filling level located between filling level LI and filling level L3.
  • Filling level L3 is the lowest filling level.
  • the filling level, e.g. LI to L3 may be relevant to specify an end position of the particle collection, e.g. below the filling level, at half the filling level etc. Collection of particle above the respective filling level, e.g. LI to L3 may be used to collect also particles that are arranged above the respective filling level, e.g. due to the previous stirring at high rotation speeds catapulting particles into all directions.
  • Figure 10 illustrates a second embodiment (fifth variant V, e.g. example 3e as mentioned above) of positions, e.g. stopping positions of a stirring element during the second high flux density operation mode, e.g. collection of particles, especially of beads Be.
  • first variant V e.g. example 3e as mentioned above
  • second high flux density operation mode e.g. collection of particles, especially of beads Be.
  • Position Pl 1 to P25 may be used for collecting the beads during a sequence of positions step as mentioned above, see e.g. steps S167 to S183.
  • collection of beads Be may be started in a lower portion of the processing chamber, e.g. PC2. Thereafter, beads Be may be collected in an upper portion of the processing chamber, e.g. PC2. Thereafter, beads Be may be collected in a medium portion of the processing chamber, e.g. PC2. Finally, beads Be may be collected again in the lower portion.
  • the collecting scheme that is illustrated in figure 10 may result in complete or almost complete collection of particles.
  • Figures 11 to 15 illustrate method/processing steps that may be performed in order to purify NMAs before performing a further test, e.g. a PCR test.
  • the devices D, PRU described above may be used to perform the purification, see e.g. figures 1 to 10.
  • Figure 11 illustrates a mixing step, e.g. a mixing step performed in processing chamber PC2.
  • the processing chamber PC2 may comprise:
  • NAMbb A nucleic acid molecule binding buffer solution (NAMbb), e.g. as specified in the introductory part of the present document.
  • NAM1 to NAM3 e.g. DNA or RNA molecules
  • - Magnetizable particels or other appropriate particles e.g. beads Bel to Be5, preferably coated with a sorbent material (e.g. facilitating binding of NMA to the surface of the sorbent material) as specified in the introductory part of the present document, and/or
  • Molecule Mol to Mo5 forming “debris” that has to be removed during purifying of the NMAs.
  • Molecule Mol to Mo5 may be e.g. remains of a cell wall or of other cell components that are different from nucleic acid molecules or may be particles of non-cell pathogens that are different from nucleic acid molecules.
  • an external high magnetic flux density B may be provided to processing chamber PC2, especially to stirring element, e.g. stirring bar StB2 and to magnetizable particles, e.g. beads Bel to Be2.
  • the external filed intensity B may have a magnetic north pole N and a magnetic south pole S.
  • the external filed intensity B may magnetize stirring bar StB2 and beads Bel to Be2 which may comprise ferromagnetic materials.
  • magnetized stirring bar StB2 aligns with the external magnetic field B, e.g. magnetic north pole N of StB2 to magnetic south pole S of magnetic unit Magi, Mag2 and magnetic south pole S of StB2 to magnetic north pole N of magnetic unit Magi, Mag2. If the external magnetic field B is rotated, magnetized stirring bar StB2 will follow this rotation, thereby mixing the NMAbb solution, e.g. in order to create a dispersion in which the NAMs and the “debris” molecules are evenly distributed, e.g. thereby dissolving any clots or lumps.
  • a magnetic north pole N and a magnetic south pole S are formed in each of the beads Bel to Be5. Therefore, the magnetized beads Bel to Be5 are attracted to the magnetized stirring bar StB2, e.g. with their magnetic south poles S to the magnetic north pole N of the stirring bar StB2, see figure 11 and with their north poles N to the magnetic south pole S of the stirring bar StB2 (not illustrated). Moderate or no rotation may be performed until the beads Bel to Be5, e.g. all beads are attached to stirring bar StB2. Thereafter, rotation speed may be increased to enable enhanced mixing of NAMbb.
  • Figure 12 illustrates a binding step that may follow after the mixing step as illustrated in figure 11.
  • no external magnetic field B may be applied to processing chamber PC2. This may result in vanishing of the magnetization of stirring bar StB2 as well in vanishing of the magnetization of beads Bel to Be5.
  • stirring bar StB2 may be used or only a slow rotation during the binding step.
  • the magnetizable beads which are not magnetized anymore may detach from stirring bar StB2 and may also be distributed evenly in the dispersion, e.g. at the end of stirring when the strength of the magnetic field is reduced, e.g. step by step or more abruptly.
  • the binding step may take several seconds, e.g. a time duration within the range of 10 s (seconds) to 2 min (minutes), or in the range of 10 s to 1 min, e.g. 30 s.
  • the NAMs may bind to the surface of the beads Bel to Be5 under the influence of the NAMbb, e.g. NAM1 to bead Bel or to another bead, NAM2 to bead Be2 or to another bead, NAM3 to bead Be3 or to another bead, etc. It is of course possible that several NAMs bind to the surface of one bead Bel to Be5 or that there are beads that are left empty, i.e. with no NAM bind to it.
  • the molecules Mol to Mo5 may not bind to the beads Bel to Be5 under the influence of the NAMbb. Thus, the molecules Mol to Mo5 (debris) may remain within the fluid for later removal.
  • Figure 13 illustrates a particle collecting step whereby nucleic acid molecules (NAM) are attached to the particles (e.g. beads Be, Bel to Be5).
  • the particle collection step may be performed after the binding step as illustrated in figure 12.
  • Particle collection may be implemented by applying the external B field to processing chamber PC2, thereby magnetizing stirring bar StB2 and the beads Bel to Be5 again.
  • the magnetized beads Be are again attracted to the magnetized stirring bar StB2, e.g. using the collection steps as mentioned above, see e.g. figure 9.
  • the NAMs e.g. NAM1 to NAM3 that are attached to the beads Bel to Be3 will also be attached to the stirring bar StB2.
  • the molecules Mol to Mo5 which may not be bound to the beads Bel to Be5 may remain in the NAMbb and may therefore not be attached to the stirring bar StB2.
  • NAMbb together with the debris molecules Mol to Mo5 out of processing chamber PC2, e.g. using a pump P of device D, PRD.
  • the pump P may be a peristaltic pump actuating on hose H.
  • the stirring bar StB2 may be hold by the external magnetic field B in its position within processing chamber PC2 thereby holding also the beads Bel to Be5 and the attached NMAs within processing chamber PC2.
  • Collection of the beads Be may be performed as described above with reference to figures 9 and 10.
  • a non-NAMbb non nucleic acid molecule binding buffer (solution), nucleic acid molecule non-binding buffer (solution)
  • processing chamber PC2 e.g. using a pump P of device D, PRD.
  • the beads Be and the attached NAMs may be attracted to magnetized stirring bar StB2 during of non-NAMbb thereby preventing that the beads Bel to Be5 and the NAMs are flushed out of processing chamber PC2.
  • an external magnetic field may be applied and no or only moderate rotation may be used during exchange of the buffer solution/washing solution(s).
  • An optional mixing step may follow in order to disperse the beads Bel to Be5 evenly or homogenous within the non-NAMbb, thereby preparing the de-binding step that is illustrated in figure 14.
  • An external magnetic field may be used for mixing as well as rotation of the external magnetic field that is transferred to rotation of stirring bar StB2.
  • Figure 14 illustrates a de-binding (release) step that may be performed after the step that is illustrated in figure 13 and that is described above in more detail.
  • the NAMs may be released from the surface of the beads Bel to Be5, especially from the surface of the sorbent coating on the beads Bel to Be5.
  • the NAMs, e.g. NAM1 to NAM3 may be released into the non-NAMbb (solution).
  • the releasing step may be performed without the application of an external B field to processing chamber PC2 and also without performing rotation.
  • the de-binding step may take several minutes, e.g. a time in the range of 10s (seconds) to 5 min (minutes) or in the range of 20 s to 4 min or in the range of 30 s to 3 min.
  • Figure 15 illustrates a particle collecting step whereby no nucleic acid molecules (NAM) are attached to the particles (e.g. beads).
  • the particle collection step may be performed after the de-binding step as illustrated in figure 14.
  • An external magnetic field B high magnetic flux density
  • An external magnetic field B high magnetic flux density
  • Attraction of the beads Bel to Be5 to the stirring bar StB2 may be enhanced by a collection movement of stirring bar StB2, e.g. as mentioned above for steps S256 to S260
  • the NAMs remain within the non-NAMbb (solution) and are not attracted to or collected on stirring bar StB2.
  • the NAMs may be removed during a following pumping step together with non NAMbb out of the processing chamber, e.g. PC2 or another appropriate particle retaining portion of the fluidic system FS, FS1 of the cartridge C.
  • a method for enriching nucleic acid molecules may comprise the steps of:
  • composition which comprises the following components: a sample material comprising nucleic acid molecules, a nucleic acid molecule binding buffer, magnetizable beads which are capable of binding nucleic acid molecules, and a magnetizable stirring bar,
  • a method for isolating nucleic acid molecules may comprise the steps of:
  • the method for isolating nucleic acid molecules comprises the steps of:
  • a particle retaining system PRS may comprise a device D, PRD according to any one of the embodiments mentioned above and an analysis carrier C,
  • the analysis carrier e.g. cartridge C may comprise the fluidic system FS,
  • the particle retaining portion PRP may comprise at least one magnetizable element MME that preferably generates an induced magnetic field only under the influence of the magnetic field and/or that does not generate an induced magnetic field in the absence of the magnetic field, and/or
  • the fluidic system FS, FS1 preferably the particle retaining portion PRP comprises the particles, e.g. beads Be, and/or
  • beads Be are magnetizable particles which preferably generate an induced magnetic field only under the influence of the magnetic field and/or which do not generate an induced magnetic field in the absence of the magnetic field.
  • the at least one magnetizable element MME may be configured to fulfill at least one, at least two or all of the following function(s):
  • a usage of the device D, PRD according to any one of the preceding embodiments for enriching or other processing of nucleic acid molecules NAM within a solution is provided, preferably a usage of the device D, PRD to perform a method according to any one of the embodiments mentioned above.
  • Shielding of the permanent magnet may be used instead or in addition to the movement in the z-direction, e.g. away from the particle retaining portion PRP (chamber, e.g. PC2).
  • the design of the fluorescent probes may use FRET (Forster (Foerster) resonance energy transfer) principles or non-FRET principles.
  • FRET Form (Foerster) resonance energy transfer
  • the probes may be destroyed by Taq DNA polymerase in the course of the PCR, separating donor and acceptor fluorophores.
  • the disclosed cartridge and/or micro reactor may be used not only for PCR but also for other purposes, e.g. isothermal amplification.
  • pathogens may be subject to the analysis of at least one substance comprised therein (e.g. nucleic acid molecule such as DNA or RNA molecule):
  • IAI Intelligent- Abdominal Infection
  • the amplification products of the genes or genetic regions representative for the above-mentioned pathogen (bacterial) species may be detected with the radiation transfer unit (RU).
  • the detection is carried out via a color marking such as fluorescence marking.
  • the bacteria-specific nucleic acid molecule such as DNA or RNA molecule to be detected is labelled with a detectable dye such as fluorescence marker/probe like TaqMan probe.
  • a TaqMan probe may consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3 ’-end or vice versa.
  • fluorophores e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET
  • quenchers e.g. tetramethylrhodamine, acronym: TAMRA
  • the quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Forster resonance energy transfer (FRET).
  • TaqMan probes are designed such that they anneal within a nucleic acid such as DNA region amplified by a specific set of primers.
  • TaqMan probes can be conjugated to a minor groove binder (MGB) moiety, dihydrocyclopyrroloindole tripeptide (DPI3), in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature (T m ) due to increased stabilization of van der Waals forces.
  • MGB minor groove binder
  • DPI3 dihydrocyclopyrroloindole tripeptide
  • the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus, relieving the quenching effect and allowing fluorescence of the fluorophore.
  • fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of nucleic acid such as DNA template present in the PCR. This signal is then detected with the radiation transfer unit (RU).
  • a packet of assays/tests/probes may be used for a respective cartridge, e.g. for respiratory diseases.
  • the number of tests e.g. corresponding to the number of probes comprised within the cartridge
  • the number of tests may be within the range of 10 to 40 or in the range of 15 to 35 tests.
  • tests for the following viruses may be performed within a respiratory disease test package: Adenovirus, at least one Coronavirus (SARS CoV, MERS CoV, Cov 19, etc.), Influenza A (Orthomyxovirus), Parainfluenza (Krupps, Paramyxovirus), Respiratory Syncytial Virus (RSV) (Paramyxovirus), Rhinovirus (common cold, Picomavirus).
  • Adenovirus at least one Coronavirus
  • MERS CoV, Cov 19, etc. Influenza A
  • Parainfluenza Krupps, Paramyxovirus
  • Respiratory Syncytial Virus RSV
  • Rhinovirus common cold, Picomavirus
  • test packages may be used as well, e.g. for the detection of:

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Abstract

La présente invention concerne des procédés d'enrichissement et d'isolement de molécules d'acide nucléique à l'aide, par exemple, de particules magnétisables. De plus, la présente invention concerne un dispositif de traitement de molécules d'acide nucléique à l'aide, par exemple, de particules magnétisables.
PCT/EP2024/069813 2023-07-13 2024-07-12 Dispositif et procédés d'enrichissement et d'isolement de molécules d'acide nucléique Pending WO2025012429A2 (fr)

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