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US20090148847A1 - Rapid magnetic flow assays - Google Patents

Rapid magnetic flow assays Download PDF

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Publication number
US20090148847A1
US20090148847A1 US12/203,779 US20377908A US2009148847A1 US 20090148847 A1 US20090148847 A1 US 20090148847A1 US 20377908 A US20377908 A US 20377908A US 2009148847 A1 US2009148847 A1 US 2009148847A1
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Prior art keywords
peptidyl
hapten
primer
paramagnetic
test pad
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Mark Kokoris
Melud Nabavi
Wayne L. Breidford
John Gerdes
Stephen Mordue
C. Frederick Battrell
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Revvity Health Sciences Inc
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Micronics Inc
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Assigned to MICRONICS, INC. reassignment MICRONICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KOKORIS, MARK, BREIDFORD, WAYNE L., MORDUE, STEPHEN, NABAVI, MELUD, BATTRELL, C. FREDERICK, GERDES, JOHN
Publication of US20090148847A1 publication Critical patent/US20090148847A1/en
Assigned to PERKINELMER HEALTH SCIENCES, INC. reassignment PERKINELMER HEALTH SCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MICRONICS, INC.
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Definitions

  • the present invention relates to the general fields of molecular biology and medical science, and more particularly to improved methods for nucleic acid and immunological bioassays.
  • the problem is one of diffusion kinetics and mass transfer.
  • amplicons are prepared by amplification of target nucleic acid sequences in the presence of forward and reverse primers conjugated with biotin and digoxigenin, respectively, for use in lateral flow assays.
  • the amplicons are bound to particles with streptavidin and agglutinate in the presence of antibody to digoxigenin.
  • bifunctional amplicon complexes are detected as trapped aggregates excluded from the fibrous matrix.
  • Other solids are interferences in the assay.
  • the avidin conjugates are wicked into a membrane and migrate until encountering a detection strip coated with a capture agent. Accumulation of dyed particles at the detection strip is detected.
  • the assays are generally dependent on flow rate in the materials, particle size and pore dimensions as well as laminar barriers to diffusion.
  • Magnetic beads are used to concentrate bioanalytes before or during assay (see for example US 2003/0032028). Beads have several advantages over arrays because beads have a higher specific surface area, move through the liquid sample matrix, and hence have more encounters per unit time with an assay target than the corresponding array.
  • use of magnetic microspheres is generally regarded as a concentration step, substituting for centrifugation or filtration.
  • Magnetic microbeads are also commonly used to position and contact analytes with reagents or solid substrates, as for example described in U.S. Pat. No. 5,660,990, U.S. Pat. No. 5,707,807, U.S. Pat. No. 6,815,160, 2002/0086443, 2002/0192676, 2003/0215825, 2004/0018611, 2004/01211364, 2005/0142582, and cumulative related citations representative of the prior art, all of which are incorporated here in full by reference. These examples show the breadth of the applications for microbeads. In US 2006/0292588, where magnetic control circuitry for bead washing is provided in an assay apparatus, time to assay endpoint is again the critical factor (FIG. 1 of US 2006/0292588, showing 5 hr to endpoint).
  • Magnetic beads have proven remarkably amenable to surface chemistry, and are routinely derivatized as assay reagents. Such chemistries include functional groups selected from carboxylate, amine, amide, hydrazide, anhydride, hydroxyl, sulfhydryl, chloromethyl, aldehyde, glycidyl (epoxy), and others. A broad range of applications exists.
  • ligand-tagged paramagnetic microbeads are readily extracted from a moving magnetic field by formation of molecular tethers with solid phase substrates coated with affinity ligand-binding molecules.
  • tagged paramagnetic beads can be affinity extracted from a moving magnetic field, not simply directed to or retained on a test pad by a stationary magnetic field.
  • a detectable endpoint for a bioassay can thus be achieved in one simple step wherein first a population of ligand-tagged paramagnetic microbeads is captured on an affinity-binding test pad as a magnetic field moves the bead complexes across the test pad, and second, as the magnetic field moves away, affinity tagged paramagnetic beads remain bound, but unbound paramagnetic beads are separated and pulled away to waste.
  • the magnetic force field has both a perpendicular force vector and a lateral force vector.
  • the paramagnetic beads are attracted to a surface or substrate by a magnetic force field emanating from the opposite side of the surface, and as the magnetic field moves laterally, the paramagnetic beads are dragged across the test pad while following the magnetic flux laterally.
  • Tagged magnetic beads so readily adhere to the test pad in this way that visual detection endpoints may be used. Although a visual endpoint is preferable for its simplicity, the invention is not to be construed as limited to such.
  • microfluidic devices are used in the embodiments of the examples reduced to practice herein, the invention again should not be construed as limited to such.
  • the method comprises the steps of:
  • peptidyl-conjugates to the 5′ tail of amplification primer sets are generally applicable in polymerase-dependent amplification protocols and are further robust, surprisingly retaining full antigenicity and binding integrity following amplification.
  • an immobilized antibody for example a monoclonal antibody, specific to a peptide-conjugated amplication primer will capture the products of amplification tagged with the primer.
  • a second primer tagged with a second affinity ligand rapid methods for forming target specific detection complexes are readily designed.
  • Peptidyl-conjugated oligonucleotides have not previously been used as primers in PCR amplification, or in other amplification protocols, or used as means for tagging and discriminating mixed PCR products in multiplex target detection protocols. These detection complexes thus serve essentially as means for interrogating a peptidyl-primer amplicon library.
  • this method has more breadth than prior art methods of tagging primers, which are limited to a few species of binding pairs, permitting simultaneous separation and detection of an essentially infinite number of amplicons by the step of tagging each amplicon with a unique peptide hapten (herein “peptidyl hapten”) and employing the corresponding antibody to capture and immobilize it.
  • the magnetic bead assay methods illustrated here are one embodiment of this discovery.
  • FIGS. 1-4 depict affinity-immobilized paramagnetic target molecule binding complexes as detection complexes. Shown are four “sandwich” detection complexes involving a paramagnetic target molecule binding complex and a test pad.
  • FIG. 5 is a pictograph describing (in panel 5 A) the use of a vectored moving magnetic field to sweep paramagnetic two-tailed amplicon complexes across and over two test pads while magnetically contacting them with two species of capture antibodies immobilized on the test pads, and (in panel 5 B) the resultant immuno-immobilized complexes on the test pad bearing an antibody specific to the paramagnetic target molecule complexes.
  • FIG. 6 is a pictograph describing (in panel 6 A) the use of a vectored moving magnetic field to sweep paramagnetic antigen:antibody complexes across and over a test pad while magnetically contacting them with capture antigen immobilized on the test pad, and (in panel 6 B) the resultant immuno-immobilized complexes on the test pad.
  • FIG. 7 is a pictograph describing (in panel 7 A) the use of a vectored moving magnetic field to sweep paramagnetic target antibody:antigen complexes across and over a test pad while magnetically contacting them with capture anti-antibodies immobilized on the test pad, and (in panel 7 B) the resultant immuno-immobilized complexes on the test pad.
  • FIG. 8 is a pictograph describing (in panel 8 A) the use of a vectored moving magnetic field to sweep paramagnetic target antibody:target antigen complexes across and over a test pad while magnetically contacting them with capture antibodies immobilized on the test pad, and (in panel 8 B) the resultant immuno-immobilized complexes on the test pad.
  • FIG. 9 is a sketch showing the use of the method in a microfluidic detection chamber for a multiplex assay.
  • the magnetic field can be used to sweep the paramagnetic target molecule complexes across and over multiple test pads, or to scrub the test pads back and forth with the complexes in order to form affinity-immobilized complexes.
  • FIG. 10 is a conceptual schematic of test pads and test pad arrays as may be useful in the method.
  • FIG. 11 is a flow chart depicting steps of a method for detection of affinity-immobilized amplicons.
  • FIG. 12 is a flow chart depicting steps of a method for detection of affinity-immobilized target antibody:antibody complexes with antigen.
  • FIG. 13 is a flow chart depicting steps of a method for detection of affinity-immobilized target antibody:antigen complexes with anti-antibody.
  • FIG. 14 is a flow chart depicting steps of a method for detection of affinity-immobilized target antigen:antibody complexes with complementary antibody.
  • FIG. 15 is a reproduction of a photograph of parallel detection chambers in a microfluidic card treated by the inventive method.
  • FIG. 16 pictographically depicts an affinity-immobilized molecular detection complex with complex paramagnetic microbead tethered to a solid phase by a two-tailed amplicon complex.
  • Test samples include, for example: blood, serum, plasma, buffy coat, saliva, wound exudates, pus, lung and other respiratory aspirates, nasal aspirates and washes, sinus drainage, bronchial lavage fluids, sputum, medial and inner ear aspirates, cyst aspirates, cerebral spinal fluid, stool, diarrhoeal fluid, urine, tears, mammary secretions, ovarian contents, ascites fluid, mucous, gastric fluid, gastrointestinal contents, urethral discharge, synovial fluid, peritoneal fluid, meconium, vaginal fluid or discharge, amniotic fluid, semen, penile discharge, or the like may be tested.
  • Assay from swabs or lavages representative of mucosal secretions and epithelia are acceptable, for example mucosal swabs of the throat, tonsils, gingival, nasal passages, vagina, urethra, rectum, lower colon, and eyes, as are homogenates, lysates and digests of tissue specimens of all sorts.
  • Mammalian cells are acceptable samples. Besides physiological fluids, samples of water, industrial discharges, food products, milk, air filtrates, and so forth are also test specimens. In some embodiments, test samples are placed directly in the device; in other embodiments, pre-analytical processing is contemplated.
  • Bioassay Target Molecule may include a nucleic acid, a protein, an antigen, an antibody, a carbohydrate, a cell component, a lipid, a receptor ligand, a small molecule such as a drug, and so forth.
  • Target nucleic acids include genes, portions of genes, regulatory sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, cDNA and may be single stranded, double stranded or triple stranded.
  • Some nucleic acid targets have polymorphisms, deletions and alternate splice sequences. Multiple target domains may exist in a single molecule, for example an immunogen may include multiple antigenic determinants.
  • An antibody includes variable regions, constant regions, and the Fc region, which is of value in immobilizing antibodies.
  • Pathogen an organism associated with an infection or infectious disease.
  • Pathogenic condition a condition of a mammalian host characterized by the absence of health, i.e., a disease, infirmity, morbidity, or a genetic trait associated with potential morbidity.
  • Target nucleic acid sequence refers to a nucleic acid sequence in a biosample that is to be amplified in the assay by a polymerase and detected.
  • the “target” molecule can be present as a “spike” or as an uncharacterized analyte in a sample, and may consist of DNA, cDNA, gDNA, RNA, mRNA, rRNA, or miRNA, either synthetic or native to an organism.
  • the “organism” is not limited to a mammal.
  • the target nucleic acid sequence is a template for synthesis of a complementary sequence during amplification. Genomic target sequences are denoted by a listing of the order of the bases, listed by convention from 5′ end to 3′ end.
  • Reporter refers to a biomolecule or modification of a biomolecule that can be detected by physical, chemical, electromagnetic and other related analytical techniques.
  • detectable reporters include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, dyed particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, enzymes linked to nucleic acid probes, and enzyme substrates. Reporters are used in bioassays as reagents, and are often covalently attached to another molecule, adsorbed on a solid phase, or bound by specific affinity binding.
  • Ligand any molecule for which there exists another molecule (i.e., an “antiligand” or ligand binding molecule) that binds with specific affinity to the ligand with stereochemical recognition or “fit” of some portion of the ligand by the ligand binding molecule.
  • Forces between ligand and binding molecule are typically Van der Waals, hydrogen bond, hydrophobic bond, and electrostatic bond.
  • Ligand binding is not typically covalent and is thus distinguished from “crosslinked” and “derivatized”.
  • the term “ligand” is reserved for binding moieties that are not “Peptidyl haptens”.
  • Peptidyl hapten refers to a subclass of haptens that is a peptide fragment. As used herein, peptidyl haptens, or “peptide haptens” are used with their complementary antibody to the peptide fragment as a means for capturing two-tailed amplicons on a solid phase.
  • Haptens are “molecular keys” in the Kekulean sense, that when bound to an immunogenic carrier and introduced into a vertebrate, will elicit formation of antibodies specific for the hapten or epitope. These molecular keys have stereochemical specificity, are generally exposed on the surface of the carrier, and are of lower molecular weight than the carrier.
  • Illustrative examples include small-molecule derivatives of native proteins, RNA loop-stem structures, a drug or steroid such as digoxigenin, the carbohydrate side-chains that decorate a mucopeptide, and short chain peptides or helices of non-native proteins such as diphtheria toxin or toxoid.
  • a dipeptide or a lipid when conjugated on a suitable immunogenic carrier, can produce an antibody response, and affinity-captured antibody specific to the dipeptide or lipid itself, not the immunogen, can be produced by absorbing out the non-specific antibodies in an antiserum or by preparing a monoclonal antibody by lymphocyte selection.
  • a hapten is not immunogenic of itself, it has very finely directed immunospecificity and is recognized by a very limited set of complementary antibodies.
  • short chain peptides are a preferred hapten for tagging amplicons as used to create peptidyl-amplicon libraries because of their robust chemistry, compatibility with enzymes as primer labels, and essentially infinite immunospecificity.
  • Capture agent or “affinity capture agent” is a generic term for a complementary partner in an affinity binding pair and is generally used to capture a ligand or hapten by binding it to a solid phase.
  • Affinity binding pairs include streptavidin:biotin, antibody:antigen, hapten:antibody, peptidyl hapten:antibody, and antigen:antibody, for example, and either member of the affinity binding pair may be the capture agent.
  • Test pad area is an area or zone occupied by an affinity capture agent.
  • the area is 3-dimensional at a nanomolecular level and is generally formed on the surface of a substrate in a liquid flow path.
  • the test pad is generally the site in the assay where the assay endpoint is observed or measured, and as such may be housed in a detection chamber with optical window.
  • Heterogeneous capture or immobilization refers use of affinity binding pairs to concentrate an analyte or detection complex on a solid phase surface, particle, or porous adsorbent material, generally so that the analyte can be detected, concentrated or purified.
  • Heterogeneous or solid phase capture may be achieved with capture agents such as immobilized antigen, antibody, avidin, nickel-NTA, lectin, or other ligand/receptor systems.
  • the molecular complex formed by heterogeneous capture is the “immobilized reporter complex” and may be the detection complex of a heterogeneous binding assay. Such complexes are stabilized by non-covalent and cooperative binding.
  • Amplification refers to a “template-dependent process” that results in an increase in the concentration of a nucleic acid sequence relative to its initial concentration.
  • a “template-dependent process” is a process that involves “template-dependent extension” of a “primer” molecule.
  • a “primer” molecule refers to a sequence of a nucleic acid that is complementary to a known portion of the target sequence.
  • a “template dependent extension” refers to nucleic acid synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the rules of complementary base pairing of the target nucleic acid and the primers.
  • Amplicon refers to a double stranded DNA product of a prior art amplification means, and includes double stranded DNA products formed from DNA and RNA templates.
  • Two-tailed Amplicon refers to a double stranded DNA product of a prior art amplification means in which tagged primer pairs are covalently incorporated, a first primer conjugated with one affinity tag, a second primer conjugated with a second affinity tag, the two tags being different.
  • the two-tailed amplicon functions as a “hetero-bifunctional” tether, and links a magnetic bead to a solid substrate.
  • Primer is a single-stranded polynucleotide or polynucleotide conjugate capable of acting as a point of initiation for template-directed DNA synthesis in the presence of a suitable polymerase and cofactors.
  • Primers are generally at least 7 nucleotides long and, more typically range from 10 to 30 nucleotides in length, or longer.
  • the term “primer pair” refers to a set of primers including a 5′ “forward” or “upstream” primer that hybridizes with the complement of the 5′ end of the DNA template to be amplified and a 3′ “reverse” or “downstream” primer that hybridizes with the 3′ end of the sequence to be amplified.
  • primer extension has 5′ and 3′ ends and that primer extension always occurs in the direction of 5′ to 3′. Therefore, chemical conjugation at or near the 5′ end does not block primer extension by a suitable polymerase.
  • Primers may be referred to as “first primer” and “second primer”, indicating a primer pair in which the identity of the “forward” and “reverse” primers is interchangeable. Additional primers may be used in nested amplification.
  • the first primer is a monospecific or class-specific oligonucleotide conjugated to a peptide hapten or epitope recognized by a specific antibody.
  • the second “primer” is an oligonucleotide conjugated to a hapten, to a biotin, a digoxin, a steroid, a polysaccharide, an antigen or fragment thereof, a folic acid, a phycoerythrin dye, a fluorophore, to an Fc fragment of an antibody, to a nickel chelator such as NTA, or to a lectin, 2,4-dinitrophenyl, and so forth, at or near the 5′ terminus.
  • Complementary refers to two single-stranded nucleic acid sequences that can hybridize to form a double helix.
  • the matching of base pairs in the double helix of two complementary strands is not necessarily absolute.
  • Selectivity of hybridization is a function of temperature of annealing, salt concentration, and solvent, and will generally occur under low stringency when there is as little as 55% identity over a stretch of at least 14-25 nucleotides. Stringency can be increased by methods well known in the art. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984).
  • a primer that is “perfectly complementary” has a sequence fully complementary across the entire length of the primer and has no mismatches.
  • a “mismatch” refers to a site at which the base in the primer and the base in the target nucleic acid with which it is aligned are not complementary.
  • Complementary refers to antibody:immunogen or antibody:hapten binding that is immunospecific.
  • Magnetic Microbead refers to a “nanoparticle”, “bead”, or “microsphere”, or by other terms as known in the art, having at least one dimension, such as apparent diameter or circumference, in the micron or nanometer range.
  • An upper range of such dimensions is 600 um, but typically apparent diameter is under 200 nm, and may be 1-50 um or 5-20 nm, while not limited to such.
  • Such particles may be composed of, contain cores of, or contain granular domains of, a paramagnetic or superparamagnetic material, such as the Fe 2 O 3 and Fe 3 O 4 ( ⁇ -Fe crystal type), ⁇ ′-FeCo, ⁇ -Cobalt, CoPt, CrPt 3 , SmCo 5 , Nickel and nickel alloys, Cu 2 MnAl, ⁇ -FeZr, Nd 2 Fe 14 B, NoTi, for example.
  • the Ferrites defined as ferrimagnetic or ceramic compound materials consisting of various mixtures of iron oxides such as Hematite (Fe 2 O 3 ) or Magnetite (Fe 3 O 4 ) and iron oxides in alloys with other metals.
  • These materials as used generally are particles having dimensions smaller than a magnetic domain, and may be formed into particles, beads or microspheres with binders such as latex polymers (generically), silica, as is generally well known and inclusive of such materials as are commercially available.
  • nanoparticles of Fe 3 O 4 with diameters in the 50 nm-100 um range are commercially available for magnetic bioseparations. These particles are “superparamagnetic”, meaning that they are attracted to a magnetic field but retain no residual magnetism after the field is removed. Therefore, suspended superparamagnetic particles tagged to the biomaterial of interest can be removed from a matrix using a magnetic field, but they do not agglomerate (i.e., they stay suspended) after removal of the field. Also of interest are nickel and cobalt microbeads. These beads may be reactive with peptides containing histidine.
  • Paramagnetic beads have the property that they align themselves along magnetic flux lines and are attracted from areas of lower magnetic flux density to areas of higher magnetic flux density.
  • magnetic microbeads may be composite materials. Such beads may further contain other micro- or nanoparticles agglomerated with a binder. Composites with RF-tags, QDots, up-converting fluorophores, colloid sols and clays, and the like are contemplated for use in the present invention.
  • a magnetic bead need not be formed entirely of a magnetic material, but may instead comprise both magnetic and non-magnetic materials.
  • Microbeads may themselves be colloidal and have chromogenic properties, or may be combined with other colloidal metal particles with chromogenic properties. Mixed suspensions of differently modified microbeads may be used.
  • Microbeads are by no means simply commodities. They may be modified with surface active agents such as detergents to control their rheological properties, as in ferrofluids.
  • the surface of microbeads may be modified by adsorption or covalent attachment of bioactive molecules, including immunoaffinity agents, antibodies, enzymes, dyes, fluorescent dyes, fluorescent quenchers, oligomers, peptide nucleomers, and the like, and more specifically by coating with streptavidin or single stranded DNA oligomers, for example.
  • microbeads of interest herein are comprised of at least one paramagnetic element therein, as would be readily recognized by those skilled in the prior arts.
  • Suitable matrices for microbeads include polystyrene, divinylbenzene, polyvinyltoluene, polyester, polyurethane, with optional functional groups selected from SO3, COOH, NH2, Glycidyl (COC), OH, Cl, Tosyl, aldehyde, and sulfhydryl. Particles often range from 0.3 to 5 um or larger. Latex particles of 100 nm, and 1, 5, 20, 50 or 100 um are commercially available in bulk. Silica may be used as a matrix or as a capsule. Derivatized silane includes OH, NH2, COOH and more. Particles often range from 0.5 to 3 um. Dextran may also be used as a matrix.
  • Particles often range from 20-50 nm.
  • Polysaccharide may also be used with silane as silica fortified microbeads of particle size around 250 nm.
  • Agarose and cellulose matrices include particles in the range of 1-10 um, and may be activated for introduction of functional groups.
  • Protein particles such as of gelatin and albumin, have long been used for magnetic microspheres. These are readily activated for amine, carboxyl, hydroxyl and sulfhydryl linkages with ligands or tags. Liposomes are somewhat more refractory to chemical derivatization, but have been used to make magnetic particles.
  • Naked iron oxide, and other paramagnetic metal particles are also known, and may be derivatized by adding sulfhydryl groups or chelators. These particles often have sizes of 5 to 300 nm. Certain types of particle populations are known to be uniform in size; in others the heterogeneity may be controlled or selected.
  • microbeads may be readily prepared.
  • carboxyl-modified microbeads containing ⁇ 20-60% magnetite are made by dispersing a (magnetite)/styrene/divinylbenzene ferrofluid mixture in water, and emulsion-polymerizing the monomers to trap the magnetite in a polymer matrix of microbeads of ⁇ 1 ⁇ m diameter. The magnetite is thus dispersed throughout the solid beads.
  • Other prior art means for synthesizing and modifying microbeads are commonly known.
  • Suitable microbeads for practicing the present invention may also be purchased from vendors such as Bang's Laboratories, Inc. (Fishers Ind.) and Polysciences, Inc (Warrington Pa.), as well as numerous suppliers of specialty modified microbeads such as Bioscience Beads (West Warwick R.I.). Tradenames of such beads, again not as a comprehensive recitation, include Estapor® SuperParaMagnetic Microspheres, COMPELTM Uniform Magnetic Microspheres, Dynabeads® V MyOneTM Microspheres, and the like. Cobalt paramagnetic microbeads are sold as Dynabead's MyOne TALON. BioMag Plus microbeads from Polysciences have an irregular shape, and thus more surface area for affinity chemistry.
  • Populations—of microbeads are generally used to assay populations of assay targets.
  • a population as used herein refers to a set of members sharing some common element or property.
  • a population of beads may be similar in that the beads share a common tag, such as an avidin coat, or a barcode.
  • a population of nucleic acids comprising an assay target may simply share a target nucleic acid sequence, or may contain a common tag.
  • a population of antibodies may share a common specificity. And so forth.
  • Paramagnetic and Superparamagnetic are taken as functionally synonymous for the present purposes. These materials when fabricated as microbeads, have the property of responding to an external magnetic field when present, but dissipating any residual magnetism immediately upon release of the external magnetic field, and are thus easily resuspended and remain monodisperse, but when placed in proximity to a magnetic field, clump tightly, the process being fully reversible by simply removing the magnetic field.
  • Magnetic Force Field is the volume defined by the magnetic flux lines between two poles of a magnet or two faces of a coil. Electromagnets and driving circuitry can be used to generate magnetic fields and localized magnetic fields. Permanent magnets may also be used. Preferred permanent magnetic materials include NdFeB (Neodymium-Iron-Boron Nd 2 Fe 14 B), Ferrite (Strontium or Barium Ferrite), AlNiCo (Aluminum-Nickel-Cobalt), and SmCo (Samarium Cobalt).
  • NdFeB Neodymium-Iron-Boron Nd 2 Fe 14 B
  • Ferrite Strontium or Barium Ferrite
  • AlNiCo AlNiCo
  • AlNiCo AlNiCo
  • SmCo Scamarium Cobalt
  • a localized magnetic field is a magnetic field that substantially exists in the volume between the poles of two magnets, and may be attractive or repulsive.
  • Robustness refers to the relative tolerance of an assay format to variability in execution, to materials substitutions, and to interferences, over a range of assay conditions. Robustness generally increases with the strength of the detection signal generated by a positive result. Robustness negatively correlates with the difficulty and complexity of the assay.
  • Sensitivity refers to the lower limit of detection of an assay where a negative can no longer be reliably distinguished from a positive.
  • Assay endpoint “Endpoint” or “datapoint” is used here as shorthand for a “result” from either qualitative or quantitative assays, and may refer to both stable endpoints where a constant plateau or level of reactant is attained, and to rate reactions, where the rate of appearance or disappearance of a reactant or product as a function of time (i.e., the slope) is the datapoint.
  • Detection of a “molecular detection complex”, also termed an “immobilized reporter complex”, may constitute an assay endpoint.
  • Microfluidic cartridge a “device”, “card”, or “chip” with fluidic structures and internal channels having microfluidic dimensions. These fluidic structures may include chambers, valves, vents, vias, pumps, inlets, nipples, and detection means, for example.
  • microfluidic channels are fluid passages having at least one internal cross-sectional dimension that is less than about 500 ⁇ m and typically between about 0.1 ⁇ m and about 500 ⁇ m, but we extend the upper limit of the range to 600 um because the macroscopic character of the bead suspensions used here have a dramatic effect on the microfluidic flow regime, particularly as it relates to restrictions in the fluid path.
  • microfluidic channels are fluid passages having at least one internal cross-sectional dimension that is less than 600 um.
  • the microfluidic flow regime is characterized by Poiseuille or “laminar” flow.
  • the particle volume fraction ( ⁇ ) and ratio of channel diameter to particle diameter (D/d) has a measurable effect on flow characteristics.
  • Microfluidic cartridges may be fabricated from various materials using techniques such as laser stenciling, embossing, stamping, injection molding, masking, etching, and three-dimensional soft lithography. Laminated microfluidic cartridges are further fabricated with adhesive interlayers or by thermal adhesiveless bonding techniques, such by pressure treatment of oriented polypropylene. The microarchitecture of laminated and molded microfluidic cartridges can differ.
  • Lateral flow Assay refers to a class of conventional assays wherein particle aggregation, agglutination or binding is detected by applying a particle-containing fluid to a fibrous layer such as a permeable membrane and observing the chromatographic properties as the particles and particle aggregates move into and through the material. Penetration of clumps of particles is impeded, whereas free particles penetrate between the fibers. Similarly, free particles may accumulate as clumps in zones of the fibrous layer treated with affinity binding agents.
  • the devices and methods described here are not lateral flow assays.
  • Means for extracting refers to various cited elements of a device, such as a solid substrate, filter, filter plug, bead bed, frit, or column, for capturing target nucleic acids from a biological sample, and includes the cumulative knowledge in the art cited herein by reference to a few examples.
  • a means for polymerizing may refer to various species of molecular machinery described as polymerases and their cofactors and substrates, for example reverse transcriptases and TAQ polymerase, and includes the cumulative knowledge of enzymology cited herein by reference to a few examples.
  • thermocycling Means for Amplifying: Include thermocycling and isothermal means.
  • the first thermocycling technique was the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al. Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), and in Innis et al., (“PCR Protocols”, Academic Press, Inc., San Diego Calif., 1990).
  • Polymerase chain reaction methodologies are well known in the art. Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of a target sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the template to form reaction products, excess primers will bind to the template and to the reaction products and the process is repeated. By adding fluorescent intercalating agents, PCR products can be detected in real time.
  • a DNA polymerase e.g., Taq polymerase
  • LAMP loop-mediated isothermal amplification of DNA
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation (Walker et al. Nucleic Acids Research, 1992:1691-1696).
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • CPR cyclic probe reaction
  • a probe having 3′ and 5′ sequences of non-specific DNA and a middle sequence of specific RNA is hybridised to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • RT-PCR reverse transcription polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ Replicase may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • primers are used in a PCR-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labelled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989 , Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al., 1989 , Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315.
  • NASBA the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerisation.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • T7 or SP6 an isothermal cyclic reaction
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5′ to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of E. coli DNA polymerase D, resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridisation of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include “RACE” and “one-sided PCR” (Frohman, M. A., In: “PCR Protocols: A Guide to Methods and Applications”, Academic Press, N.Y., 1990; Ohara et al., 1989 , Proc. Natl. Acad. Sci. U.S.A., 86: 5673-567).
  • Means for detecting refers to an apparatus for displaying an endpoint, i.e., the result of an assay, and may include a detection channel and test pads, and a means for evaluation of a detection endpoint. Detection endpoints are evaluated by an observer visually in a test field, or by a machine equipped with a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode, nephlometer, photon counter, voltmeter, ammeter, pH meter, capacitative sensor, radio-frequency transmitter, magnetoresistometer, or Hall-effect device.
  • Magnetic particles, beads and microspheres having impregnated color or having a higher diffraction index may be used to facilitate visual or machine-enhanced detection of an assay endpoint.
  • Magnifying lenses in the cover plate, optical filters, colored fluids and labeling may be used to improve detection and interpretation of assay results.
  • Means for detection of magnetic particles, beads and microspheres may also include embedded or coated “labels” or “tags” such as, but not limited to, dyes such as chromophores and fluorophores, for example Texas Red; radio frequency tags, plasmon resonance, spintronic, radiolabel, Raman scattering, chemoluminescence, or inductive moment as are known in the prior art.
  • QDots such as CdSe coated with ZnS, decorated on magnetic beads, or amalgamations of QDots and paramagnetic Fe 3 O 4 microparticles, optionally in a sol gel microparticulate matrix or prepared in a reverse emulsion, are a convenient method of improving the sensitivity of an assay of the present invention, thereby permitting smaller test pads and larger arrays. Fluorescence quenching detection endpoints are also anticipated.
  • a variety of substrate and product chromophores associated with enzyme-linked immunoassays are also well known in the art and provide a means for amplifying a detection signal so as to improve the sensitivity of the assay. Detection systems are optionally qualitative, quantitative or semi-quantitative. Visual detection is preferred for its simplicity, however detection means can involve visual detection, machine detection, manual detection or automated detection.
  • FIGS. 1 through 4 illustrate examples of amplicon capture on immobilized antibody, target antibody capture on immobilized antigen, target antibody capture on immobilized capture agent, and target antigen capture on immobilized antibody, respectively.
  • the detection complex of FIG. 1 depicts a paramagnetic bead 1 immobilized on test pad 2 .
  • the tether between bead and antibody 3 is formed by a two-tailed amplicon 4 , in this case with first primer 5 tagged with peptidyl hapten 6 and second primer 7 tagged with biotin 8 , for illustration.
  • the paramagnetic beads are coated with bound avidin 9 .
  • the immobilized antibody on the test pad 1 has captured a “two-tailed amplicon” ( 4 ), i.e., an amplicon with peptidyl-oligomer-tagged primer at a first end and biotin-tagged primer at the opposite end.
  • These two-tailed amplicons are synthesized during an amplification step by providing reagent primer sets in which biotin has been used to tag a second primer and peptide hapten the first primer by conventional chemistries.
  • the biotin tagged amplicon is captured by the avidin-coated microbead, and the reporter bead complex in turn is then immobilized on the test pad.
  • the two-tailed amplicon thus serves as a heterobifunctional tether.
  • a sufficient number of immobilized beads, as present in a few microliters of reagent result in a distinct visual coloration of the test pad. Biotin is only one such ligand useful in constructing these unique molecular detection complexes with magnetic beads
  • composite magnetic beads can be prepared with materials such as QDots, fluorophores, dyes, enzymes, RFIDs, and so forth, so as to be readily detectable by alternative detection means when immobilized on the respective test pads. Detection can involve visual detection, machine detection, manual detection or automated detection. Methods for preparation of hapten-tagged primers are also readily extracted from the prior art.
  • a “positive detection complex” results when an amplicon becomes tethered to a test pad as shown in FIG. 1 .
  • Those test pads to which magnetic beads are tethered indicate a positive result for one specific two-tailed species of hapten-tagged amplicon.
  • Those test pads to which no magnetic beads are tethered indicate a negative result for the respective hapten-tagged amplicon. Because the species of hapten are known and are assigned to a particular forward or reverse primer conjugate, the detection event can be interpreted as positive detection of the particular nucleic acid sequence corresponding to the target nucleic acid sequence under investigation.
  • peptidyl-haptens peptide epitopes
  • large libraries of peptidyl-hapten-tagged amplicons can be prepared by amplification, and interrogated by the magnetic bead methods described here. Methods using the much more limited prior art toolbox of non-peptide ligands as haptens or binding agents are not so robust.
  • biotin:avidin affinity binding pair is one of many ligand binding pairs that might be chosen for affinity binding.
  • Others include nickel:nickel binding complexes, as may be suitable to nickel-bearing microbeads. Or digoxin and digoxigenin and complementary antibodies, or the antibody Fc fragment and Protein A or Protein G.
  • Antibody-coated microbeads may also be used to capture peptidyl hapten-tagged second primers (i.e., a unique peptidyl hapten on both primers), and so forth.
  • FIG. 2 we see a second detection complex, again taking advantage of a bifunctional tether.
  • Paramagnetic bead 20 is immobilized on test pad 21 coated with antigen.
  • the tether between bead and antigen 22 is formed by an antibody 23 .
  • the paramagnetic beads are coated with bound anti-antibody 24 .
  • Species specific anti-antibodies are useful for this method, as are also Protein A and Protein G.
  • Paramagnetic bead 30 is immobilized on test pad 31 coated with an anti-antibody 32 .
  • the tether between bead 30 and antibody 32 is again an antibody 33 , but the beads are now coated with antigen 34 .
  • Species specific anti-antibodies are useful for this method.
  • antigen is the target molecule of the assay.
  • Paramagnetic bead 40 is coupled to test pad 41 coated with antibody 42 specific for the antigen.
  • the tether between bead 40 and antibody 42 is now an antigen 43 .
  • the beads are coated with an antibody 44 specific for the antigen.
  • the antibodies are not necessarily identical, one antibody may be a hybridoma antibody, the other a polyvalent antibody, and the antibodies may bind to different recognition sites on a macromolecular antigen, or share a common binding site where multiple binding sites are present, for example when the antigen is a viral particle.
  • FIG. 5 a key is used to describe the elements of the step.
  • paramagnetic bead amplicon binding complexes on the left are in the process of being “swept” or “dragged” onto, through and across two patches of immobilized antibody illustrating the test pads. It can be appreciated that at a molecular level the test pads are not two-dimensional layers, but are in fact 3-dimensional surfaces coated with a layer of bound and unstirred water. The cone of the magnetic force field (long arrow) is moving parallel to the plane of the test pad, but the magnetic flux lines are experienced by paramagnetic particles as being directed downwardly (short arrow).
  • the magnetic force is thus seen to have two vectors, one directed “downwardly” and the other “laterally” (relative to the plane of the test pad).
  • Paramagnetic particles are attracted to the magnet from which the magnetic force field emanates, and the magnet is positioned under the plane of the test pad and is moving from left to right.
  • the “J-hook” at the base of the amplicons represents a peptidyl hapten that is recognized by one of the test pads, which are coated with different antibodies (left vs. right test pad).
  • the magnetic force field has moved past the test pads, “sweeping” or “dragging” with it unbound paramagnetic particles while—surprisingly—paramagnetic bead complexes bearing the amplicon have been captured and extracted from the magnetic field, and are seen in the panel to be immunoimmobilized on the test pad coated with capture antibody specific for the J-hook of the peptidyl hapten.
  • the bead complexes are dragged through the mat of antibody and liquid crystalline water bound to the test pad.
  • the magnetic force pulls the complexes down into close approach and full contact with the antibody mat, contacting antibody and hapten.
  • the magnetic force (which is a weak force) is not strong enough to rip the hapten from the primer, or even to rip the antibody from the test pad, but instead releases the bead complex, which remains immobilized on the test pad.
  • the process of removing unbound paramagnetic material from the test pads after immunocapture could also be accomplished by repositioning the source of the magnetic field above the test pad.
  • Paramagnetic beads will always move from a field of less dense magnetic flux lines to a field of more dense magnetic flux lines.
  • capture is accomplished by sweeping the beads from outside to inside the test pad area
  • removal of unbound material is accomplished by sweeping the beads from inside to outside the test pad area, without reference to particular geometries.
  • the magnetic field may also serve to remove the unbound material to waste.
  • Immobilization is specific.
  • the peptide hapten is recognized only by the complementary antibody of the right test pad, not the left, and the bead complexes are therefore immobilized only on the right test pad.
  • Detection of the immunoimmobilized bead complexes is thus a positive detection event and indicates here the presence of the target amplicon.
  • Detection of the immobilized complexes can be as simple as a visual estimate of the color of the test pad before and after binding, or a comparison with positive and negative control test pads.
  • Paramagnetic beads typically have a distinct color or can be suitably dyed. More complex detection means may also be used.
  • paramagnetic bead:antibody binding complexes on the left are in the process of being “swept” or “dragged” onto, through and across a patch of immobilized antibody on the test pad.
  • the bead antibody complexes consist of a mixture of target antibody (black body) and nonspecific antibodies (black tips), all of which have been bound to the beads by an anti-antibody, for example in the case of an assay for antigen-specific IgG immunoglobins in human serum, a mouse or hybridoma anti-human IgG antibody.
  • the test pad is coated with adsorbed antigen specific for the antibody targeted in the assay.
  • the magnetic force field (long arrow) is moving parallel to the plane of the test pad, but is experienced by paramagnetic particles as being directed downwardly (short arrow).
  • the lateral vector corresponding to movement of the magnetic field from left to right
  • the perpendicular vector corresponding to the magnetic flux lines which are not shown.
  • Paramagnetic particles are attracted to the magnet from which the magnetic flux lines emanate, and the magnet is positioned beneath the plane of the test pad and is moving from left to right. The paramagnetic particles will follow the motion of the magnetic force field, and are pulled against the test pad while being dragged from left to right.
  • the magnetic force field has moved past the test pads, “sweeping” or “dragging” with it nonspecific antibody and unbound paramagnetic particles while—surprisingly—paramagnetic bead complexes bearing target antibody specific for the antigen have been captured and extracted from the magnetic field, and are seen in the panel to be immunoimmobilized on the test pad.
  • paramagnetic bead:antibody binding complexes on the left are in the process of being “swept” or “dragged” onto, through and across immobilized antibody on the test pad.
  • the microbeads are coated with antigen complementary or specific for the target antibody of the assay.
  • the test pad is coated with an anti-antibody, for example in the case of an assay for antigen-specific IgG immunoglobins in human serum, a mouse or hybridoma anti-human IgG antibody.
  • the magnetic force field (long arrow) is moving parallel to the plane of the test pad, but is experienced by paramagnetic particles as having a downward vector (short arrow).
  • Paramagnetic microbeads are attracted to the magnet from which the magnetic force field emanates, and the magnet is positioned beneath the plane of the test pad and is moving from left to right. The paramagnetic beads are thus pulled down on the test pad, in close contact with the capture agent, while simultaneously transversing the test pad from left to right.
  • the magnetic force field has moved past the test pads, “sweeping” or “dragging” with it unbound paramagnetic particles while—surprisingly—paramagnetic bead complexes bearing target antibody have been captured and immunoextracted from the magnetic field.
  • paramagnetic bead:antigen complexes on the left are in the process of being “swept” or “dragged” onto, through and across immobilized antibody on the test pad.
  • the beads are coated with antibody complementary for the target antigen of the assay.
  • the test pad is coated with an anti-antigen antibody.
  • the magnetic force field (long arrow) is moving parallel to the plane of the test pad, but is experienced by paramagnetic particles as being directed with a downward vector component (short arrow).
  • Paramagnetic particles are attracted to the magnet from which the magnetic force field emanates, and the magnet is positioned beneath the plane of the test pad and is moving from left to right. The paramagnetic particles will follow the motion of the magnetic force field, and are pulled up against the test pad while being dragged from left to right.
  • the magnetic force field has moved past the test pads, “sweeping” or “dragging” with it unbound paramagnetic particles while—surprisingly—paramagnetic bead complexes bearing target antigen have been captured and immunoextracted from the magnetic field.
  • the invention is a way of solving a critical problem of bioassays, that of facilitating the close approach of target and target capture agent by dislodging the boundary or unstirred layer of water at the surface of the capture layer.
  • this barrier is a critical barrier in affinity binding.
  • this problem has been overcome by extending incubation time or by convective close approach (for example as in the wicking effect of lateral flow) followed by diffusion and capture.
  • unbound paramagnetic complexes are first brought into contact with a capture surface or substrate under the direction of a magnetic force field and are then extracted from the magnetic field, while unbound paramagnetic substrates are dragged away from the capture surface or substrate by the continued lateral motion of the magnetic field.
  • the magnetic force field thus has two vectors, one directed “downwardly” (relative to the plane of the capture surface or test pad) and the other “laterally” (again relative to the plane of the capture surface or test pad).
  • the downward vector penetrates the unstirred water layer around the capture molecule, and draws the target molecule into the required close approach or “close encounter” where affinity binding can occur.
  • the lateral vector is through the unstirred water layer, and again draws the target molecules into the required close approach to capture molecules, but further serves to differentiate bound and unbound material. Unbound paramagnetic molecular complexes remain with the moving magnetic field and continue their lateral path. Bound paramagnetic materials are immobilized at the site of capture and are not dislodged by the continuing lateral vector of the magnetic force field.
  • the magnetic force field is manipulated by moving its source (a permanent magnet or electromagnet) laterally across or through the plane of the test pad, and may be disengaged by withdrawing the magnet or turning off current to the electromagnet).
  • a permanent magnet or electromagnet a permanent magnet or electromagnet
  • FIG. 9 illustrated is a simple device for conducting the method. It can be seen that the lateral motion of the magnetic force field is optionally bidirectional, here shown with a net motion in a fluid path of detection chamber 60 from upstream 61 (Paramagnetic Complexes In) to downstream (Waste Out).
  • the source of the magnetic field is again “underneath” (or “behind”) the test pad. This draws the target into close approach with the binding sites, and facilitates the detection step.
  • the path of the magnetic field must necessarily contactingly traverse one or more test pads 62 coated with affinity capture agent 63 .
  • a viewing window 64 permits detection of the bound complexes after the moving magnetic field has passed, thus simplifying detection.
  • the magnetic field is further useful in directing unbound paramagnetic materials to waste.
  • a multiplex detection device is shown, having a plurality of test pads.
  • test pads are treated to immobilize a capture agent prior to assembly of the device, for example, on polystyrene, by plasma treatment of plastic areas delimited by a mask, followed by application of the capture agent and drying. Spotting of capture agent, for example with a laserjet printer, can eliminate the need for masking test pads. If necessary, test pads are “blocked” with blocking agents to prevent non-specific adsorption of target molecules prior to sealing the detection chamber or channel.
  • Test pads are a feature of the detection step of the method described herein.
  • Test pads 70 and 71 constitute for example a negative and positive detection field for an assay and may be used as a pair.
  • Test pad array 72 is a vertical stack of banded or striped test pads in the form of an array, not unlike that shown in FIG. 9 .
  • Test pad array 73 is a rectangular array of individual test squares, each treated with a unique capture agent.
  • Test pad 74 is circular and is adapted to inkjet printing.
  • Test pad 75 is treated with a gradient of a capture agent so as to display a readily interpretable semi-quantitative endpoint.
  • Test pads have in common a test field bounded by an edge inside of which a bioactive capture agent is immobilized.
  • the capture agent may be a protein such as an antibody, an anti-antibody, an anti peptidyl hapten antibody, Protein A, Protein G, or antigen, or a non-protein such as an aptimer, a carbohydrate antigen, a mucopolysaccharide, a binding protein such as folic acid binding protein or an avidin, or a nucleotide oligomer.
  • Capture agents may also include denatured viral antigens and microbial antigens in general and cellular components or whole cells in general.
  • test pads are not necessarily impermeable substrates, and may be porous or fibrous in character.
  • the microbead fluid path in the magnetic field may be across or through the test pad area, as in from side-to-side or from front-to-back.
  • the test pad architecture, at a molecular level, is inherently three-dimensional, although it may be represented as a two-dimensional plane.
  • Solid substrates for test pads include olefin or other thermoplastic materials such as polystyrene, polycarbonate, polypropylene, polyethylene terephthalate, polyether sulfone, polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, and polyamides and also inorganic materials such as glass.
  • Certain fibrous or porous supports such as nitrocellulose, nylon, hydrogel, and polyethylene may also be applied as test pads, and may be pretreated with capture agent for ease of assembly. To enhance binding of capture agents, crosslinked proteins are sometimes employed. Drying also promotes irreversible binding of the capture agent.
  • a preferred method for pretreating plastic prior to adsorbing the capture agent is low pressure gas plasma treatment. Exposure of the surface to pure oxygen or nitrogen produces an activated hydroxylated and carboxylated substrate layer or an activated aminated and nitroxidated layer, respectively. Argon may also be used.
  • polystyrene plastic is used as the substrate for immobilizing capture agent. Masking, followed by gas plasma treatment is used to activate designated areas as test pads. The capture agent is applied, dried in place, and the mask is removed. When antibody is used as the capture agent, application by hand or with an automated printer is followed by drying and blocking. Other capture agents may require modified protocols as are known in the art.
  • an assay for a target nucleic acid sequence becomes the steps of first preparing the sample for amplification of the target sequence, amplifying the target by a PCR or related isothermal protocol whereby tagged primers are incorporated into the product amplicon, then binding those tagged “two-tailed” amplicons on paramagnetic beads coated with an affinity binding agent, and magnetically sweeping or dragging the beads into close contact with a test pad area with immobilized capture antibody so as to form immunoimmobilized paramagnetic complexes of test pad:capture antibody:amplicon with hapten tag of first primer and ligand tag of second primer:binding agent and paramagnetic bead (i.e., the detection complex), before sweeping from the test pad any un-immobilized paramagnetic material. And finally a step for detecting any molecular detection complexes on the test pad.
  • Preparation of a sample may involve lysing cells to release the target nucleic acids, removing interferences such as hemoglobin from a blood lysate by selective adsorption and elution of the nucleic acids from a glass solid phase, and dissolution of the nucleic acids with a suitable buffer for a polymerase. Also required in some applications are preliminary steps for reverse transcription, as when mRNA contains the target sequences and must be converted to duplex DNA before amplification.
  • multiplex or nested primer sets may be used.
  • the method uses a second primer with tag suitable for complexation with an affinity binding agent on the paramagnetic beads, and often this a biotin tag as illustrated in FIG. 1 .
  • the method uses a first primer with tag suitable for immunoaffinity immobilization of the formed amplicon:bead complex on the surface of the test pad.
  • affinity binding agents may be used in each phase of formation of the detection complex.
  • capture antibodies for second phase immobilization is the specificity of antibody:peptidyl hapten binding, which permits design of protocols for simultaneous assay of multiple target nucleic acid sequences.
  • Immuno-immobilization of target analyte with antibody capture agent is a preferred embodiment, but the invention is not limited to such.
  • the next step is to use a magnetic field to localize and contact the analyte complexes with the test pad so that the immunoimmobilized detection complexes can be formed.
  • the magnetic field is moved and optionally modulated to perform this. Lateral motion of the magnetic field sweeps or drags the bead complexes onto the test pad, through the unstirred layer and the 3-dimensional network of bound capture antibody, and finally across the test pad, where unbound paramagnetic material is carried off the test pad and away with the lateral motion of the magnetic force. This step promotes binding interactions without the need for multi-minute incubations.
  • the double-stranded, two-tailed amplicon bound by avidin:biotin on one end (for example) and antibody:peptidyl hapten on the other (for example), is sufficiently strong to selectively tether the paramagnetic bead to the test pad and resist delocalization by the moving magnetic force field. It can be said that the capture antibody “extracts” the amplicon:bead complexes from the moving magnetic field. Sufficient numbers of bound bead complexes are readily identified and form a positive result by visual endpoint. A visual detection step is illustrated.
  • the primer set is essentially a first assay reagent, and may be prepared and placed in an assay device or kit, optionally in dried form, at any time prior to performing the assay.
  • the beads are essentially a second assay reagent, and may be sensitized with the desired binding agent, and optionally dried in place prior to the assay.
  • Test pads are prepared in advance of the assay itself and may be rehydrated prior to use or rehydrated by the test sample in performance of the assay. Drying promotes irreversible binding of the capture agent to the test pad substrate. Reagents for sample preparation and amplification may also be prepared separately.
  • the sample is first processed to prepare a liquid fraction, which might be serum or plasma, a paracellular fluid, saliva, or other biological sample. Generally any solid fraction of the sample is separated from the aqueous liquid fraction.
  • a liquid fraction which might be serum or plasma, a paracellular fluid, saliva, or other biological sample.
  • any solid fraction of the sample is separated from the aqueous liquid fraction.
  • interferences are then adsorbed and any antibody:target antigen complexes in the biological sample are disrupted so as to release the analytical target.
  • the target antibody in free solution is then bound by paramagnetic beads coated with an anti-antibody.
  • This method is of use, for example, when a particular class of target antibody is of interest, as in distinguishing acute, convalescent, and chronic stages of infection, or when all antibody in the sample is to be interrogated for specificity to a plurality of antigens.
  • the bead:antibody:antibody:antigen tether is sufficiently strong to selectively anchor the paramagnetic bead to the test pad and resist disruption by the magnetic field. Sufficient numbers of bound bead complexes are readily identified and form a visually positive detection endpoint.
  • the detection complex is formed of test pad:antigen:target antibody:affinity bound paramagnetic bead.
  • the detection complex may contain an enzyme, for example, and may be further developed for detection by enzymatic assay.
  • the common step in all these assays is to simultaneously use a magnetic field a) to localize and contact the analyte:bead complexes with the test pad so that the immobilized detection complex can be formed and further b) to separate bound and unbound paramagnetic bead complexes.
  • the magnetic field is moved and optionally modulated to perform this. Lateral motion of the magnetic field sweeps or drags the bead complexes onto the test pad, through the unstirred layer and the 3-dimensional network of bound capture antigen, and finally across the test pad, where unbound paramagnetic material is carried off the test pad and away with the lateral motion of the magnetic force.
  • Paramagnetic bead complexes bearing target antibody remain behind, immuno-immobilized on complementary, irreversibly adsorbed antigen on the test pad.
  • bifunctional or “two-tailed” tether confers assay specificity.
  • nonspecific antibody may be bound to the paramagnetic beads, but would not be captured by the antigen on the test pads, so no false-positive detection complex will form.
  • Anti-antibodies directed at the Fc fragment of the target antibody are preferable for this assay so that the variable regions of the target antibody arms are free to recognize and bind to the bound antigen.
  • the beads are essentially a first assay reagent, and may be sensitized with the desired binding agent, and optionally dried in place prior to the assay.
  • Test pads are prepared in advance of the assay itself and may be rehydrated prior to use or rehydrated by the test sample in performance of the assay. Reagents for sample preparation may also be prepared separately.
  • the method differs from that of FIG. 12 essentially by the polarity of the tether.
  • the solid fraction of the sample is separated from the aqueous liquid fraction. If needed, interferences are then adsorbed and any antibody:target antigen complexes in the biological sample are disrupted so as to release the analytical target.
  • the target antibody in free solution is then bound by paramagnetic beads coated with complementary antigen, forming immunospecific antibody:antigen complexes on the bead (also termed a “reporter:analyte complex”.
  • the next step is common to all these assays and involves the simultaneous use a magnetic field to a) localize and contact the analyte:bead complexes with the test pad so that the immobilized detection complex can be formed and b) to separate bound and unbound paramagnetic bead complexes. Lateral motion of the magnetic field sweeps or drags the bead complexes onto the test pad, through the unstirred layer with a downward vector on the paramagnetic beads, penetrating the 3-dimensional network of bound capture anti-antibody on the test pad, and finally across the test pad, whereupon unbound paramagnetic material is carried away with the lateral motion of the magnetic force. Paramagnetic bead complexes bearing target antibody remain trapped by immunoimmobilization on adsorbed anti-antibody on the test pad.
  • the bead:antigen:antibody:antibody tether is sufficiently strong to selectively anchor the paramagnetic bead to the test pad and resist disruption by the magnetic field. Sufficient numbers of bound bead complexes are readily identified and form a positive visual detection endpoint.
  • the detection complex is formed of test pad:antibody:target antibody:affinity bound paramagnetic bead.
  • the detection endpoint may be further developed to amplify the detection sensitivity, for example by excitation of a fluorophore.
  • the bifunctional tether confers assay specificity.
  • the capture anti-antibody on the test pad in panel 13 B will likely capture a broad spectrum of antibodies in the sample, but only those immunocomplexed by paramagnetic beads will result in a positive assay.
  • the bifunctional specificity of the tether ensures assay specificity.
  • the paramagnetic bead reagent is a mixture of beads coated with either a first antigen and beads coated with a second antigen, an immunoimmobilized positive assay endpoint will form if antibodies to either antigen are present in the sample.
  • the identity of individual antibodies to particular antigens is, however, obtained with the method of FIG. 12 , even in a multiplexed bead format.
  • the beads are essentially a first assay reagent, and may be sensitized with the desired binding agent, and optionally dried in place prior to the assay.
  • Test pads are prepared in advance of the assay itself and may be rehydrated prior to use or rehydrated by the test sample in performance of the assay. Reagents for sample preparation may also be prepared separately.
  • individual antigens in a biological test sample may be identified by the method of FIG. 14 .
  • FIG. 14 we see the same principles illustrated to assay for a target antigen.
  • the sample is first processed to prepare a liquid fraction containing a solution or suspension of the target antigen.
  • interferences are adsorbed and any antibody:target antigen complexes in the biological sample are disrupted so as to release the analytical target.
  • the target antigen in free solution is then bound by paramagnetic beads coated with an antibody complementary for the antigen. Multiple antigens may be targeted simultaneously. This method is of use, for example, when a sample is suspected of carrying an enteric pathogen, a virus, or a marker released from malignant cells.
  • the common step in all these assays is use a magnetic field to a) localize and contact the analyte:bead complexes with the test pad so that the immobilized detection complex can be formed and to b) separate bound and unbound paramagnetic bead complexes. Essentially this is done simultaneously, thus speeding the assay and eliminating multi-minute incubations for the binding interaction.
  • the step for magnetic sweeping is comprised of applying a magnetic force to said paramagnetic bead reagent, wherein said magnetic force comprises generally lateral and generally perpendicular force vectors generated by a moving magnetic force field comprising flux lines extending from less dense to more dense. Because paramagnetic beads move from areas of less dense magnetic flux to areas of more dense magnetic flux, the magnetic force pulls the beads onto and into the arms of the capture agent. Because the magnetic field is moving laterally, the magnetic force sweeps or pulls the beads laterally over and across the test pad, separating bound and unbound materials as it goes. Rates of motion (linear velocity) for the magnetic sweep have been in the range of 25 to 100 mm/min (up to about 0.2 cm/sec). This step can be performed manually, or can be performed with an automated or semi-automated apparatus.
  • FIG. 15 illustrates a result of the assay.
  • Seven vertically elongate detection chambers are placed side by side on a microfluidic cartridge under an optical window. Within each detection chamber are seven test pads stacked vertically. Each test pad is about 0.5 ⁇ 2 mm in size.
  • Paramagnetic microbead reporter complexes are added to the detection chamber via a sample port and the beads are drawn up the detection chamber by a magnetic field originating from a magnet behind the cartridge. This magnetic field serves to a) draw the reporter complexes to the site of immobilization and b) remove unbound material.
  • the result is a striking rust colored band where reporter complexes are bound to the corresponding antibody on a test pad. Seven amplicons were used in preparation of this test cartridge, and seven corresponding antibody test pads were prepared in each detection chamber. The result thus appears as a “stairstep” from left to right.
  • the method as a rapid bioassay protocol comprising a step of moving a magnetic force field from outside to inside a test pad area so as to sweep a paramagnetic bead reagent in a fluid into close contact with an affinity capture agent in said test pad area, and thereby affinity capturing or extracting any bioassay target molecule bound to the paramagnetic bead reagent from the magnetic force field in the form of an immobilized paramagnetic microbead complex; and upon forming the immobilized paramagnetic bead complex (i.e., the detection complex), then moving the magnetic force field from inside to outside the test pad area so as to sweep from the test pad area any paramagnetic bead reagent not formed as immobilized paramagnetic complex, before detecting the detection complex, although it should be clear that, simplicity of description aside, the sweeping step in fact simultaneously integrates multiple simultaneous acts of formation of immobilized bead complexes and parallel acts of separation of not immobilized materials.
  • the tether is sufficiently strong to selectively anchor the paramagnetic bead to the test pad while resisting the separating force of the magnetic field.
  • the detection complex comprises bead:antibody:antigen:antibody:test pad, and may be further developed to increase assay sensitivity, for example by exciting an RFID tag or a fluorophore embedded in the bead matrix.
  • the bead thus acts as a reporter group itself, or as a complex with accessory reporter groups.
  • FIG. 14 which is copacetic with FIGS. 4 and 8 , specific antibody binds the target antigen, such as a drug or other small molecule, to the bead, and the target antigen:bead binding complex is bound to the test pad again by another antibody specific to the target analyte. Specificity and robustness is also demonstrated in FIG. 15 .
  • target antigen such as a drug or other small molecule
  • the beads are essentially a first assay reagent, and may be sensitized with the desired binding agent, and optionally dried in place prior to the assay.
  • Test pads are prepared in advance of the assay itself, are advantageously dried in place, and may be rehydrated prior to use or rehydrated by the test sample in performance of the assay. Reagents for sample preparation may also be prepared separately before use.
  • the size of magnetic beads preferred in the assay are about 0.01 to 50 microns, more preferably 0.5 to 10 microns, and most preferentially 0.8 to 2.8 microns, mean diameter. Homogeneously sized beads are preferred. Suitable beads may be obtained from Dynal Invitrogen (Carlsbad Calif.), Agencourt Bioscience Corp (Beverly Mass.), Bang's Laboratories, Inc. (Fishers Ind.), Polysciences, Inc (Warrington Pa.), Bioscience Beads (West Warwick R.I.), Bruker Daltonics (Nashville Tenn.) and AGOWA (Berlin Del.), for example.
  • the magnetic beads may be in the form of a ferrofluid, taken broadly.
  • the method serves as a sort of magnetic fluidized bed reactor for extraction of affinity captured beads and separation out of nonspecifically labeled beads, reagents and assay materials.
  • the magnet itself can be moved. Movement can be manual or powered with a stepper motor, servo motor, voice coil or with a spring-loaded mechanism and an x-z or y-z carriage can be constructed and automated.
  • the test pad may be moved relative to the magnetic field by similar means.
  • electromagnets are used in place of permanent magnets
  • c) an array of electromagnets can be actuated in sequence to redirect the magnetic field. It is possible to build a solid state system where a series of electromagnets are used to move the beads in a chamber.
  • the methods of the inventions should not be construed as being limited to a microfluidic device. Adaption to laminar flow, lateral flow, capillary, dipstick, multiwell plate, and test tube formats is also contemplated.
  • a stepper motor is used to move a rare earth magnet (neodymium) in an undercarriage mounted in close proximity to the detection chamber of a microfluidic device.
  • Simple software commands are used to move the undercarriage along y-axis of the detection chamber (see FIG. 9 ).
  • the speed of translation is adjustable and the carriage may be lowered in the z-axis to weaken the magnetic field in the chamber. Because paramagnetic beads line up on magnetic flux lines and are attracted to areas of higher magnetic flux, they cannot be repelled by the magnet and the orientation of the poles of the magnet is reversible.
  • the preferred apparatus accepts a microfluidic cartridge with detection chamber or “microchannel” configured in the body, the microchannel comprising a fluid path with axis of flow and with upper and lower aspects.
  • test pad or solid phase element which comprises an affinity capture agent for the analyte or for an analyte binding complex.
  • a means is provided for introducing a population of paramagnetic microbeads in a fluid into the microchannel, generally by assembling the cartridge with dehydrated beads inside and then rehydrating the beads in test sample fluid so that the beads complex target analyte.
  • the means for moving a magnetic force field comprises a subassembly external to said microfluidic cartridge, said subassembly with moveable carriage with track upon which said carriage is mounted, said track mounted in a plane parallel to said axis of flow, said carriage further comprising a first magnet, the subassembly further configured to move the magnet along said track, first bringing the magnetic force field into proximity to said test pad and then distancing the magnetic force field from said test pad element.
  • Neodynium (NdFeB) magnets obtained from K&J Magnetics (Jamison Pa.) were found to be suitable. Magnets designated D38, D40, and D44 were used. These magnets are cylindrical with poles on the long axis and have a Curie temperature of about 300° C. (maximum operating temperature of 80° C.). The magnets are Grade N52 neodynium and have a surface field strength of 4600 to 5000 Gauss. It should be recalled that magnetic field force is inversely proportionate to the 4 th power of the distance. Proximity to the test pad is in the range of 0.2 to 1.2 mm for these particular magnets. The diameter of the magnets range from to about 5 to 10 mm at the poles. For reference, the test pads themselves are about 0.5 mm ⁇ 2 mm, with the long axis perpendicular to the traverse of the magnet.
  • Magnets with a triangular cross-section and poles on two facets may also be used. These magnets have a sharply focused flux density above the apex of the facets.
  • Another aspect of the invention is use of peptidyl primer tagged amplicons in assays for nucleic acids.
  • a number of methods are now available for manufacture of specific peptide epitopes attached to oligonucleotide probes or primers (see C.-H. Tung and S. Stein, Bioconjugate Chem., 2000, 11, 605-618; E. Vives and B. Lebleu, Tetrahedron Lett., 1997, 38, 1183-1186; R. Eritja, A. Pons, M. Escarcellar, E. Giralt, and F. Albericio, Tetrahedron Lett., 1991, 47, 4113-4120; J. P. Bongartz, A. M. Aubertin, P. G.
  • the peptidyl hapten conjugated primers of this method are satisfactorily synthesized by the above chemistries and others.
  • primers of this class are compatible with PCR methods and with molecular biological nucleic acid amplifications in general.
  • the amplification product with peptide-tagged primer-labelled amplicons is first captured by an affinity capture agent specific for a ligand on the second primer of the amplification primer set and bound to a magnetic microbead.
  • the amplicon-bead complex is then interacted with peptidyl hapten-specific antibodies on the testpad and only those bead complexes with the peptide :amplicon molecular complex are captured by the testpad.
  • This method permits screening of peptidyl-amplicon libraries by heterogeneous binding assays using magnetic bead technology.
  • a molecular detection complex comprising a two-tailed amplicon with first end and second end, said first end comprising a first primer covalently conjugated with a peptidyl hapten, and said second end comprising a second primer covalently conjugated with a ligand, said first end further comprising a ligand-bound ligand binding agent-coated reporter group, and said second end further comprising a peptidyl hapten bound anti-peptidyl hapten antibody immobilized on a solid phase.
  • FIG. 16 shows a molecular detection complex with two-tailed amplicon as tether, as in FIG. 1 , but here not involving biotin, and utilizing a more complex magnetic microbead than that described in FIG. 1 .
  • the magnetic microbead 161 contains inclusion bodies or patches 162 of QDot, Texas Red, phycoerythrin, or other fluor, covalently attached or immobilized in the magnetic microbead matrix, here a latex binder with embedded particles of a ferrofluid.
  • the microbead further comprises adsorbed antibody 169 . This is the reporter group.
  • the tether consists of amplicon 164 with first primer 165 and peptidyl hapten tag 166 . Primer conjugates are incorporated into the amplicon during amplification.
  • Anti-peptidyl hapten antibody 166 immobilizes the tether to solid substrate 163 , which may be another bead or a fiber or a test pad.
  • the second primer 167 is conjugated with digoxigenin (for example). And the antibody in the reporter group is specific for digoxigenin.
  • the reporter group is thus immobilized in a complex comprising at least 5 non-covalent bonds—bead:antibody; antibody:ligand; DNA:DNA; peptide:antibody; and antibody:solid phase.
  • the assay method described here permits the tether and reporter group to be extracted with high specificity and robustness from a moving magnetic field in which the paramagnetic beads are carried.
  • soluble reporter groups and fluorophore dyed latex beads may be used with the two-tailed amplicons of the present invention.
  • bead libraries can be synthesized for analysis of mixed populations of two-tailed amplicons or of two-tailed amplicon libraries, and the resulting affinity binding complexes with pairs of beads tethered by the two-tailed amplicons can then be sorted or assayed using dual excitation fluorometry, a sort of liquid microarray.
  • the reporter group is a fluorophore of one emission frequency and the barcoded latex bead is selected from those of the prior art.
  • Reverse primers were first prepared and HPLC purified. Peptides were derivatized with n-terminal hydrazine before use. Oligonucleotides were treated with succinimidyl 4-formylbenzoate in formamide and then reacted with the hydrazine derivatized peptides to form hapten-tagged primers.
  • peptide epitopes were selected based on the availability of complementary antibodies. Alternate peptide conjugation chemistries may also be used. Forward primers were all conjugated with biotin.
  • Monodisperse streptavidin-coated magnetic beads (MyOne Streptavidin Cl Dynabeads) were purchased from Dynal, Carlsbad Calif. and washed and resuspended in 0.9 ⁇ PBS, 30 mg/mL BSA and 1% TritonX100 with 5% (v/v) of a solution of 80 mM MgCl 2 , 0.24% TritonX100, 1% BSA, in 0.5M TRIS pH 8 before use.
  • a microfluidic device was built from stencil-cut laminates and contained multiple detection chambers of the form illustrated in FIG. 9 .
  • Each detection chamber was formed with an inlet port and an outlet port fluidically connected to the detection chamber by microfluidic channels. Sufficient detection chambers were built for the experiment.
  • test pads in the detection chamber were masked and plasma treated with oxygen gas.
  • Peptidyl hapten-specific antibodies (Research Diagnostics, Flanders N.J.) and negative control solution were spotted on the test pads, 1 uL per pad, and dried in place under vacuum.
  • Each detection chamber contained one test pad corresponding to each primer set and a negative control.
  • the fully assembled device was treated with blocking/wash solution consisting of 0.9 ⁇ PBS, 30 mg/mL BSA and 1% TritonX100 to passivate untreated plastic surfaces. The blocking solution was removed before use and the chambers were dried.
  • PCR was performed with the prepared primer sets (above) for 35 cycles.
  • Platinum Quantitative RT-PCR Thermoscript One-Step System reagents were used for the amplification. Successful amplification was confirmed by 5% agarose gel electrophoresis. Amplicon 10 uL was then resuspended with 5 uL of beads (above) in about 20 uL of buffer containing 10 mM MgCl 2 , 0.5% BSA, 0.1% TritonX100 and 5 mM TRIS Buffer pH 8 and the bulk of this solution was loaded into a detection chamber. Each amplicon product corresponded to a single primer set and was loaded into a separate detection chamber.
  • the beads were first captured with a magnet positioned on the bottom of the detection chamber and the excess solution was removed. The magnet was then used to smear the bead paste onto, through and across the test pads, and the mixture was then allowed to incubate 1 min. With the magnet positioned on the bottom of the well, the well was gradually filled with blocking solution. The magnet was moved along the flow of the buffer, creating a bead front on the bottom layer of the detection chamber. The magnet was then shifted to the top of the detection chamber, lifting unbound beads out of the test pad areas. The unbound material could be resuspended in flowing buffer and rinsed to waste. The test pads were then rinsed with 1 volume of fresh buffer.
  • Bright orange test pad “stripes” were immediately visible and were determined to correctly reflect specificity of binding of the hapten-tagged amplicon to the test pad containing the complementary antibody. Because the detection chambers were aligned in parallel when constructed, a stairstep pattern was evident after all the amplicon bead mixtures were processed because each tagged amplicon was bound by only one test pad in each detection chamber.
  • PCR amplification was performed in a microfluidic device as follows:
  • Lysis Buffer, Wash Reagent, Elution Buffer, and Rehydration Buffer were aliquoted into sealed blister packs in designated chambers of the device.
  • the device was then fully assembled and placed in a pneumatic controller with variable temperature TEC heating blocks positioned under the PCR fluidics and thermal interface assembly.
  • An on-board sanitary bellows pump was used to pull sample through a pre-filter consisting of a depth filter element, made of polypropylene for example, supported on a laser-cut plastic ribs. A valve was then used to close the sample port.
  • the crude filtrate was then mixed with lysis buffer and drawn through a glass fiber filter to trap nucleic acids, and the filter retentate was rinsed thoroughly with ethanol. All rinses were sequestered in an onboard waste receptacle which vents through a 0.45 micron hydrophobic membrane filter.
  • the nucleic acids on the glass fiber membrane were then eluted with elution buffer and ported into the reaction channel containing primers, dNTPs, polymerase, magnesium, buffer and surface active agents in dehydrated form.
  • the reaction mixture in a volume of about 50 uL, was then heated to 95° C. in the PCR fluidics and thermal interface assembly for about 10 sec to effect denaturation of double stranded sequences and secondary structure in the sample. Heating and cooling was supplied by external Peltier chips mounted on suitable heat sinks and PID controlled within a 1° C. range from setpoint. Immediately thereafter, the temperature was returned to about 60° C. for a first round of annealing and extension, which was continued for about 20 sec. Thermocycling was repeated for 40 cycles over an 18 min period.
  • the amplicon products were moved to a mag mix chamber for mixing streptavidin-labelled magnetic beads (Dynal, MyOne Streptavidin C1) which had been rehydrated in Rehydration Buffer. This mixture was incubated with gentle mixing and then transferred to a MagnaFlow chamber. Optionally the reaction mix can be rinsed to remove unreacted hapten-conjugated primer while holding the magnetic beads in place.
  • the coated beads with putative target amplicon were brought into contact with the capture antibody test pads or array in the detection chamber, and unbound beads were moved away from the test pads with a moving magnetic field and sent to waste. Primers and non-specific amplicons were rinsed from the chamber with an excess of rehydration buffer, which again was discarded into on-board waste.
  • Forward primers for this example were conjugated with biotin.
  • Reverse primers were conjugated with peptide haptens for which antibodies were available (Research Diagnostics, Flanders N.J.). Covalent attachment of the haptens was at the 5′ terminus of the oligomer. Peptides were activated at the amino terminus for coupling.
  • a respiratory panel containing biotinylated and peptidyl hapten-tagged primer pairs is designed.
  • the primers are synthesized and then deposited in separate amplification channels or chambers of a device. Following the procedure of Example 2, throat swab washings are analyzed. A mini-bead impact mill is used to prepare the sample prior to analysis. A result is displayed in the detection chamber.
  • the product is packaged as a kit.
  • a sexually transmitted disease panel containing biotinylated and peptidyl hapten-tagged primer pairs is designed and the primers are synthesized.
  • the primers are then deposited in separate amplification channels or chambers of a device.
  • vaginal swab washings are analyzed.
  • a detection endpoint is displayed in the detection chamber.
  • the product is packaged as a kit.
  • An oncogene panel containing biotinylated and peptidyl hapten-tagged primer pairs is designed and the primers are synthesized. The primers are then deposited in a common amplification channel or chamber. Following PCR amplification, the amplification products are detected in a detection station. The product is packaged as a kit.

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US20090148933A1 (en) 2009-06-11
WO2007106579A3 (fr) 2008-02-28
WO2007106580A2 (fr) 2007-09-20
WO2007106579A2 (fr) 2007-09-20
EP2007905B1 (fr) 2012-08-22
US8772017B2 (en) 2014-07-08
EP2007905A2 (fr) 2008-12-31
ES2393758T3 (es) 2012-12-27
US8222023B2 (en) 2012-07-17
US20120329142A1 (en) 2012-12-27
WO2007106580A3 (fr) 2008-03-13
JP5254949B2 (ja) 2013-08-07

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