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WO2024149910A2 - Extraction automatisée d'échantillons d'acides nucléiques à partir d'une pluralité d'échantillons - Google Patents

Extraction automatisée d'échantillons d'acides nucléiques à partir d'une pluralité d'échantillons Download PDF

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Publication number
WO2024149910A2
WO2024149910A2 PCT/EP2024/062942 EP2024062942W WO2024149910A2 WO 2024149910 A2 WO2024149910 A2 WO 2024149910A2 EP 2024062942 W EP2024062942 W EP 2024062942W WO 2024149910 A2 WO2024149910 A2 WO 2024149910A2
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WO
WIPO (PCT)
Prior art keywords
matrix
receptacles
sample
samples
nucleic acid
Prior art date
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PCT/EP2024/062942
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English (en)
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WO2024149910A3 (fr
Inventor
Navid ASLANI
Jay BULLEN
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Untap Ltd
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Untap Ltd
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Filing date
Publication date
Priority claimed from GBGB2306923.0A external-priority patent/GB202306923D0/en
Priority claimed from GBGB2314626.9A external-priority patent/GB202314626D0/en
Application filed by Untap Ltd filed Critical Untap Ltd
Priority to EP24723920.5A priority Critical patent/EP4611937A2/fr
Priority to AU2024208619A priority patent/AU2024208619A1/en
Publication of WO2024149910A2 publication Critical patent/WO2024149910A2/fr
Publication of WO2024149910A3 publication Critical patent/WO2024149910A3/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped

Definitions

  • the present disclosure relates to devices and methods for automated extraction of nucleic acid samples.
  • PCR-based techniques such as quantitative PCR (qPCR), or reverse transcription qPCR (RT-qPCR) enable highly accurate and sensitive detection of nucleic acids in samples of interest.
  • qPCR quantitative PCR
  • RT-qPCR reverse transcription qPCR
  • the volumes of the samples and or reagents provided during the extraction of nucleic acids by the devices for automated extraction of nucleic acids will change with wear and tear of the components involved.
  • Pre-packaged and pre-measured reagents provide a non-cost-effective alternative, which additionally creates an additional need for the device to comprise means for waste-management, further increasing the cost and footprint of such a device, while also increasing the engineering complexity of the device by introducing the need to provide means for releasing the reagents from the packaging. This further limits the utility of the devices
  • SUBSTITUTE SHEET (RULE 26) for automated extraction of nucleic acids known in the art, e.g., for continuous monitoring of biomarkers in environmental samples.
  • a skilled person could consider the use of precise liquid handlers e.g. acoustic-based liquid handling (such as an Echo machine) to decrease the effect of wear and tear on the accuracy and precision of volume measurements.
  • acoustic liquid handlers are unlikely to be suitable for applications such as for example the extraction of nucleic acids for environmental monitoring of pathogens, due to the small orders of volumes of the liquids used in acoustic liquid handling.
  • acoustic liquid handlers are characterised by a considerable size and hardware cost and thus their potential use in the devices for automated extraction of nucleic acid is not desirable.
  • the invention provides the following aspects.
  • the invention provides a device for automated extraction of nucleic acid samples from a plurality of samples, comprising: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, and c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples; and ii) a multi-channel pump module, comprising: d) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples, and e) at least one pump configured to deliver the plurality of samples and/or the reagents for extraction of nucleic acid samples to and/or through the matrix of any receptacle of the plurality of receptacles; and wherein: the device comprises means for measuring the volume of ana sample
  • the invention provides a process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples, comprising the steps of a) receiving a sample of the plurality of samples in a receptacle of a plurality of receptacles, b) binding a nucleic acid to a matrix, c) washing the matrix, and d) eluting the nucleic acid from the matrix; wherein a volume of sample used in the process and/or a volume of any reagent of a plurality of reagents used in the process are controlled using image processing, and wherein image processing is used to measure the volume of the sample and/or the volume of any reagent of a plurality of reagents received in the receptacle.
  • the invention provides a device for automated extraction of nucleic acid samples from a plurality of samples, comprising: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples, and d) a regeneration unit configured to regenerate the matrix in any receptacle of the plurality of receptacles; and ii) a multi-channel pump module, comprising: e) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples and a plurality of reservoirs for storing reagents for regeneration of the matrix, and f) at least one pump configured to deliver the plurality of samples, the reagent
  • the invention provides a process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples using a plurality of matrices for nucleic acid binding, the process comprising a nucleic acid extraction step and a matrix regeneration step, wherein the nucleic acid extraction step and the matrix regeneration step are performed in-parallel.
  • Figure 1 A process diagram for nucleic acid extraction using image processing to provide reproducible reagent volumes measured directly within the receptacle containing the membrane.
  • the diagram demonstrates an outline of exemplary the steps performed during the process of aspects of the invention.
  • Figure 2 A logic diagram for achieving reproducible, accurate and precise measurement of reagent volumes using image processing.
  • the diagram demonstrates an example of the feedback loop used in aspects of the invention.
  • Figure 3B shows the camera-image plane of the refraction-based liquid level detector, demonstrating the application of refraction techniques to improve the contrast when using image processing with a camera to measure reagent volumes within the receptacle used for nucleic acid extraction.
  • the liquid level is determined in the refracted image area.
  • Pixel brightness is measured in the area marked as “refracted image” in Figure 3B.
  • Figure 4 Exemplary test data showing a single receptacle with the membrane used 10 times to extract nucleic acids from positive and negative spiked samples. The process was entirely automated. A regeneration step consisting of a bleach wash followed by water rinse was performed between each nucleic acid extraction. The negative samples were used to evaluate residual nucleic acid (i.e. cross-contamination): the 2.5 log reduction in gene copy numbers (gene units per mL) indicates >99% removal of nucleic acid from the receptacle’s membrane. The input sample was 1 mL wastewater spiked with 50 uL of a SARS-CoV-2 RNA standard (enveloped in viral capsids, NIB SC).
  • SARS-CoV-2 RNA standard enveloped in viral capsids, NIB SC.
  • the lysis buffer, wash buffers and elution buffers were from a typical commercial kit.
  • the extracted nucleic acids were quantified using a commercial RT-qPCR assay on a commercial qPCR thermocycler instrument with an external calibration curve and included positive controls.
  • Figure 5 Exemplary nucleic acid extraction data collected using three membranes loaded simultaneously onto the carousel test data.
  • the data shows multiple receptacles loaded onto the carousel of the nucleic acid extraction module simultaneously and each receptacle re-used multiple times to extract nucleic acids from positive and negative spiked samples.
  • the process was entirely automated. A regeneration step consisting of a bleach wash followed by water rinse was performed between each nucleic acid extraction.
  • the negative samples were used to evaluate residual nucleic acid (i.e. cross-contamination): the 2-3 log reduction in gene copy numbers (gene units per mL) indicates up to 99.9% removal of nucleic acid from the receptacle’s membrane.
  • the input sample was 1 mL wastewater spiked with 50 uL of a SARS-CoV-2 RNA standard (enveloped in viral capsids, NIBSC).
  • the lysis buffer, wash buffers and elution buffers were from a typical commercial kit.
  • the extracted nucleic acids were quantified using a commercial RT-qPCR assay on a commercial qPCR thermocycler instrument with an external calibration curve and included positive controls.
  • Figure 6 An exemplary embodiment of the system of the invention wherein the at least one pump is a syringe pump in communication with a valve terminal.
  • the communication depicted in Figure 6 between the single pump and the valve terminal, the reagent reservoirs, the extraction unit, the regeneration unit and the in-line sample lysis module is configured to enable positive (air) pressure to be provided from the single pump and through the valve terminal.
  • the communication depicted in Figure 6 between the reagent reservoirs for storing reagents for nucleic acid extraction and the projections (e.g. pipette tips), between the regeneration reagent reservoirs, the in-line sample lysis module and the projections (e.g. pipette tips) is configured to enable the delivery of the reagents and sample to the receptacle.
  • Figure 7 An exemplary embodiment of the system of the invention wherein the at least one pump comprises a syringe pump in communication with a valve terminal and a plurality of metering pumps.
  • the communication between the single pump, the valve terminal and the extraction and regeneration units and the in-line sample lysis module is configured to enable positive (air) pressure to be provided from the single pump and through the valve terminal.
  • the communication between the reagent reservoirs for storing nucleic acid extraction reagents, the metering pumps and the projections (e.g. pipette tips); between the regeneration reagent reservoirs, the metering pumps and, the in-line sample lysis module and the projections (e.g. pipette tips) is configured to enable the delivery of the reagents and sample to the receptacle.
  • analyte is used to describe any liquid and/or suspension, such as a sample and/or a reagent.
  • the term "about” is used to modify, for example, the quantity of an ingredient in a composition, concentration, volume, process temperature, process time, yield, flow rate, pressure, and like values, and ranges thereof, employed in describing the examples of the disclosure.
  • the term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and like proximate considerations.
  • matrix or “matrix for nucleic acid binding” is used with reference to any matrix suitable for binding of nucleic acid binding.
  • Suitable matrices include e.g., silica matrices (such as silica membranes), zeolite or glass fibre matrices. Paper (or cellulose-based) matrices are also known in the art.
  • nozzle refers to an apparatus for docking onto a receptacle for supplying positive air pressure to the receptacle.
  • nozzle is not to be interpreted as limited a cylindrical or round spout at the end of a pipe, hose, or tube used to control a jet of gas or liquid.
  • the term “reagent” is used to describe any substance which is used in a process such as nucleic acid extraction and/or matrix regeneration.
  • the term “reagent” includes lysis buffers, wash buffers, elution buffers, water, hypochlorites (such as sodium hypochlorite also commonly referred to as “bleach”) and any other substances known in the art to be used in the processes of nucleic acid extraction and/or matrix regeneration.
  • Reagents for nucleic acid extraction are readily available from commercial vendors and a skilled person will recognize that a plurality of such reagents from commercially available kits could be comprised by the devices and methods of the invention.
  • matrix regeneration refers to the process of washing, cleaning or otherwise regenerating a matrix that has been used in a nucleic acid extraction process such that it can be used again for further nucleic acid binding according the processes disclosed herein.
  • Regeneration solutions also referred to herein as “cleaning solutions” for regenerating the matrix may comprise reagents such as bleach (referred to herein as a chemical regeneration reagent), water, phosphate buffer solution (PBS) or borate buffers.
  • a device for automated extraction of nucleic acid samples from a plurality of samples comprises: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, and c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples; and ii) a multi-channel pump module, comprising: d) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples, and e) at least one pump configured to deliver the plurality of samples and/or the reagents for extraction of nucleic acid samples to and/or through the matrix of any receptacle of the plurality of receptacles; and wherein: the device comprises means for measuring a volume of an ana
  • the at least one pump is a single pump. In some embodiments, the at least one pump is a plurality of pumps.
  • the extraction unit comprises a plurality of tubes configured to deliver the sample and/or the reagents to the matrix.
  • the volume measurement provided by the means for measuring the volume of the analyte is used to control the at least one pump.
  • the volume measurement is used to control the at least one pump using a closed loop feedback system.
  • the control of the at least one pump is performed continuously.
  • the control of the plurality of the pumps using the volume measurement allows for the adjustment of pump activity to deliver an accurate and precise volume of the sample and/or reagent, without the need for servicing the device and manually recalibrating the pumps.
  • the control of the plurality of the pumps using the volume measurement allows for the adjustment of pump activity to deliver the sample and/or reagent through the matrix.
  • the volume of the sample and/or reagent delivered by the at least one pump would be expected to change over time due to wear and tear of the components of the device, resulting in a deterioration of both accuracy and precision of the device.
  • Measuring the volume directly in the receptacle overcomes this problem.
  • the measurement of volumes directly in the receptacle allows to overcome the issues caused by the “dead-volumes” resulting from the volume of the tubing and other comprised by the device, which would have to be carefully considered and accounted for, should the volume measurement in the receptacle not be used.
  • the expansion and compression of air additionally creates a “non-linearity” of pressure differences in the syringe pump, further limiting the accuracy and precision when no volume measurements in the receptacle are performed.
  • a plurality of embodiments can be employed to provide means for measuring the volume of analyte in any receptacle of the plurality of receptacles.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a weight-measurement system.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a load cell.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a sound-based system, optionally an ultrasound-based system.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a camera system.
  • the camera system can be positioned in sufficiently close proximity to the plurality of tubes configured to deliver the sample and/or the reagents to the matrix, in order to enable the volume of sample and/or reagents in the receptacle to be measured, and to enable the volume of sample and/or reagents delivered to the matrix to be controlled.
  • the camera system comprises a refraction target, which provides increased contrast between the background of the image and the meniscus of the liquid (analyte) in the receptacle.
  • the refraction target facilitates the measurements of the volumes directly in the receptacle.
  • the refraction target is a vertical, dark colour slip placed horizontally behind the receptacle, in the camera-receptacle-refraction target axis.
  • An exemplary set up of the camera system is provided in Figure 3A.
  • the camera system does not comprise a refraction target.
  • a basic level (or line) detection is used to determine the position of the meniscus of the liquid in the sample receptacle and thus determine the volume of the liquid in the sample receptacle.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles further comprises means for measuring the flow rate of the reagents and/or the sample through the device such that the volume of the analyte in the receptacle can be determined from the flow rate measurements.
  • the extraction unit comprises a nozzle configured to force the sample and/or the reagents through the matrix.
  • a positive pressure is provided on the analyte in the receptacle.
  • the positive pressure provided on the analyte is from +lkPa to +100 kPa.
  • the nozzle of the extraction unit can form an airtight seal with any receptacle of the plurality of receptacles. The airtight seal enables for the pressure in the receptacle to be increased such that the analyte can be forced through the matrix.
  • the extraction unit comprises a linear actuator (e.g. an electric or pneumatic actuator) for docking the nozzle to the receptacle.
  • the device comprises a valve for releasing and/or maintaining positive pressure, optionally a solenoid valve.
  • the valve can be used to control the flow rate of the analyte through the matrix, by decreasing the air pressure in the receptacle and thus stopping the analyte from moving through the matrix.
  • the nozzle is further configured to measure the air pressure in the receptacle.
  • the air pressure measurements provided by the nozzle are used together with the volume measurements to control the activity of the pumps.
  • the air pressure measurements are used to determine the presence of a leftover, undesired sample and/or reagent on the matrix.
  • the presence of the leftover, undesired sample and/or reagent on the matrix is determined using a backpressure measurement.
  • the backpressure measurement is used to adjust the number of wash cycles required to remove the leftover, undesired sample and/or reagent from the matrix.
  • the backpressure measurements are used to adjust a time for which the positive pressure is provided to force the sample and/or the reagents through the matrix, without the leftover, undesired sample and/or reagent staying on the matrix.
  • the device comprises an in-line sample lysis module for lysis of any sample of the plurality of samples.
  • the samples are lysed prior to delivery to the device.
  • the samples are lysed after delivery to the device.
  • Methods for lysis of samples for nucleic acid extraction are well known in the art.
  • the in-line sample lysis module comprises a mechanical vibration device for mixing of the sample with a lysis buffer.
  • the in-line sample lysis module comprises a load cell for measuring the volumes of the sample and the lysis buffer that are mixed together.
  • the lysis can be performed directly in the receptacle, by mixing of the sample and the lysis buffer in the receptacle.
  • the in-line sample lysis module comprises a magnetic stirrer.
  • the device comprises a waste reservoir for collection of waste liquid.
  • the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.
  • the reagents for the extraction can be selected from the plurality of reagents well-known in the art. Examples of the reagents for nucleic acid extractions are described in for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012).
  • the matrix is a silica matrix (optionally a silica membrane), a glass fibre matrix or a zeolite. In preferred embodiments the matrix is a silica membrane.
  • the plurality of receptacles is a plurality of columns. In some embodiments, the plurality of receptacles is a plurality of spin columns. In preferred embodiments, the plurality of receptacles is a plurality of minispin columns. Minispin columns comprising silica membranes for nucleic acid extractions are well known in the art, available commercially and cost- effective.
  • the number of receptacles in the plurality of receptacles can vary depending on the desired sample-processing capacity of the device.
  • the plurality of receptacles is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 receptacles.
  • the means for housing the plurality of receptacles is a cassette.
  • the means for housing the plurality of receptacles is a carousel.
  • the means for housing the plurality of receptacles can be a conveyor belt.
  • tubing used to deliver the nucleic acid sample is manufactured form an inert material, which nucleic acid molecules will not stick to.
  • the carousel is configured to move in a single plane (through rotation). In other embodiments, the carousel is configured to move in two planes, the planes being perpendicular to each other.
  • the at least one pump comprises at least one peristaltic pump.
  • the at least one pump comprises at least one syringe pump.
  • the at least one pump comprises at least one diaphragm pump.
  • the at least one pump comprises at least one metering pump.
  • the at least one pump is at least one pump in communication with a valve terminal.
  • the at least one pump comprises at least one pump in communication with a valve terminal.
  • the valve terminal comprises a plurality of solenoids.
  • the positive air pressure provided on the analyte in the receptacle may be generated by any suitable means for generating positive air pressure.
  • the positive air pressure is generated by the at least one pump.
  • the positive air pressure provided on the analyte in the receptacle is generated by the at least one syringe pump.
  • the positive air pressure provided on the analyte in the receptacle is generated by the at least diaphragm pump.
  • the positive air pressure provided on the analyte is provided by means for storing compressed air, for example by a compressed air tank.
  • the device of the first aspect of the invention is further configured to regenerate any matrix of the plurality of matrices to enable more than one extraction from any matrix of the plurality of matrices.
  • the regeneration of any matrix of the plurality of matrices further increases the sample-processing capacity of the device.
  • the device of the first aspect of is further characterized in that: i) the sample module further comprises a regeneration unit configured to regenerate the matrix in any receptacle of the plurality of receptacles, ii) the multi-channel module further comprises a plurality of reservoirs for storing reagents for regeneration of the matrix, iii) the at least one pump is further configured to deliver the reagents for regeneration of the matrix to and/or through the matrix of any receptacle of the plurality of samples, and: the multi-channel pump module and the sample module are further connected to enable regeneration of any matrix of the plurality of receptacles, and the means for housing the plurality of receptacles is further configured such that any of the plurality of the receptacles can be positioned for delivery of the reagents from the regeneration unit.
  • the extraction unit and the regeneration unit are part of the same unit. In other embodiments, the extraction unit and the regeneration unit are separate. In some embodiments, the regeneration unit comprises a plurality of tubes configured to deliver the reagents to the matrix.
  • the regeneration unit comprises a nozzle which is identical or similar to the nozzle of the extraction unit. In some embodiments, the regeneration unit comprises a nozzle configured to force the sample and/or the reagents through the matrix. To force the sample and/or the reagent through the matrix a positive pressure is provided on the analyte in the receptacle. In some embodiments, the positive pressure provided on the analyte is from +lkPa to +100 kPa. In some embodiments, the device comprises a valve for releasing and/or maintaining positive pressure, optionally a solenoid valve.
  • the valve can be used to control the flow rate of the analyte through the matrix, by decreasing the air pressure in the receptacle and thus stopping the analyte from moving through the matrix.
  • the nozzle of the regeneration unit can form an airtight seal with any receptacle of the plurality of receptacles. The airtight seal enables for the pressure in the receptacle to be increased such that the analyte can be forced through the matrix.
  • the nozzle is further configured to measure the air pressure in the receptacle.
  • the air pressure measurements provided by the nozzle are used together with the volume measurements to control the activity of the pumps.
  • the air pressure measurements are used to determine the presence of a leftover, undesired sample and/or reagent on the matrix.
  • the presence of the leftover, undesired sample and/or reagent on the matrix is determined using a backpressure measurement.
  • the backpressure measurement is used to adjust the number of wash cycles required to remove the leftover, undesired sample and/or reagent from the matrix.
  • the backpressure measurements are used to adjust a time for which the positive pressure is provided to force the sample and/or the reagents through the matrix, without the leftover, undesired sample and/or reagent staying on the matrix.
  • the regeneration unit comprises a linear actuator (e.g. an electric or pneumatic actuator) for docking the nozzle to the receptacle.
  • the reagents for regeneration of the matrix for nucleic acid binding are known in the art.
  • the reagents for regeneration of the matrix for nucleic acid binding can comprise any reagents for chemical or non-chemical regeneration of the matrix for nucleic acid binding known in the art.
  • the reagents for chemical regeneration of the matrix comprise a hypochlorite.
  • the reagents for chemical regeneration of the matrix comprise sodium hypochlorite, preferably 0.7% sodium hypochlorite.
  • the regeneration reagent is any suitable cleaning reagent such as bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IPA) and DNAZapTM PCR DNA.
  • the regeneration reagent is selected from bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IPA) and DNAZapTM PCR DNA Degradation reagent.
  • a cleaning solution for regenerating the matrix for nucleic acid binding does not comprise bleach-based reagents.
  • a cleaning solution comprises water, PBS and/or borate buffers.
  • the regeneration of the matrix is achieved by washing the membrane resulting in removal of the nucleic acid bound to the matrix.
  • the plurality of tubes of the device comprises projections (e.g. pipette tips), narrowing towards the end of the projections such that fine droplets can be created when a liquid (e.g. the samples or reagents) is delivered through the plurality of tubes.
  • projections e.g. pipette tips
  • the device of the invention comprises at least one actuator.
  • the device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 actuators.
  • at least one actuator is at least one electric actuator.
  • the at least one actuator is at least one pneumatic actuator.
  • the device comprises at least one linear actuator and at least one pneumatic actuator.
  • the device comprises an actuator configured to lower the end of the tube for delivery of any of the reagents and/or sample into the receptacle during delivery. Such lowering allows to prevent excessive splashing during delivery into the receptacle.
  • the device comprises a suction system.
  • the suction system comprises a suction pump, a waste reservoir for collection of waste liquid and a linear actuator configured to dock the waste reservoir of the suction system to the receptacle.
  • the waste reservoir is docked to the receptacle using a funnel.
  • the docking of the waste reservoir of the suction system to the sample receptacle is not air-tight.
  • the use of the suction system during operation of the device prevents excessive aerosols from contaminating the device, when sample and/or reagents are being provided through the matrix of the receptacle using positive pressure.
  • a process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples comprises the steps of: a) receiving a sample of the plurality of samples in a receptacle of a plurality of receptacles, b) binding a nucleic acid to a matrix, c) washing the matrix to remove residual reagent, and d) eluting the nucleic acid from the matrix; wherein a volume of sample used in the process and/or a volume of any reagent of a plurality of reagents used in the process are controlled using image processing; and wherein image processing is used to measure the volume of the sample and/or the volume of any reagent of a plurality of reagents received in the receptacle.
  • the use of the image processing for volume measurements directly in the receptacle enables for the process to be controlled and adjusted such that the accuracy and precision of the volumes used in the process of the invention does not change with time and exploitation of the devices used in the process.
  • the processes for automated, matrix-based extraction of nucleic acids known in the art predominantly relying on the use of either pipetting or syringe pumps for measuring the volume of the reagents suffer from the need for maintenance of the device to ensure the accuracy and precision of the measured volumes.
  • Measuring the volume of the sample and/or the reagent received in the receptacle enables for feedback to be used in the process such that the volume of the sample and/or the reagent can be readily adjusted to prevent decrease in the accuracy and precision of the volume of the reagents used in the process. Additionally, the use of volume measurements directly in the receptacle overcomes the issue wherein the air inside the syringe pump is expanded and compressed during the pumping process, resulting in lowered accuracy and precision of the volume delivered. The extent of air compression and expansion in the syringe pumps is also likely to change over time, resulting in further decreases in accuracy and precision of the volume delivered, which is again overcome with the use of volume measurements directly in the receptacle.
  • the control of the volume of the reagents is performed continuously. In preferred embodiments, the control of the volume of the reagents is performed using closed loop feedback control. In some embodiments, in addition to the use of the image processing for volume measurements, the process of the invention comprises using flow rate measurements of the reagent and/or samples throughout the process, in order to determine the volume of the sample and/or the volume of any reagent received in the receptacle.
  • the process comprises using a positive pressure to force the sample and/or the reagent through the matrix.
  • a positive pressure can be used in the process to force the sample and/or the reagent through the matrix.
  • the positive pressure is from +lkPa to +100 kPa, in some embodiments from +20kPa to +50 kPa, e.g +20kPa, +30kPa, +40kPa or +50kPa.
  • the positive air pressure provided on the analyte in the receptacle may be generated by any suitable means for generating positive air pressure.
  • the positive air pressure is generated by at least one syringe pump.
  • the positive air pressure is generated by the at least diaphragm pump.
  • the positive air pressure provided on the analyte is provided by means for storing compressed air, for example by a compressed air tank.
  • a presence of the leftover, undesired sample and/or reagent on the matrix is determined using an air pressure measurement in the receptacle.
  • the presence of the leftover, undesired sample and/or reagent on the matrix is determined using a backpressure measurement.
  • the backpressure measurement is used to adjust the number of wash cycles required to remove the leftover, undesired sample and/or reagent from the matrix.
  • the backpressure measurements are used to adjust a time for which the positive pressure is provided to force the sample and/or the reagents through the matrix, without the leftover, undesired sample and/or reagent staying on the matrix.
  • the matrix is a silica matrix (optionally a silica membrane), a glass fibre matrix or a zeolite.
  • the matrix is a silica membrane.
  • the silica membrane is comprised by a column.
  • the silica membrane is comprised by a spin column.
  • the silica membrane is comprised by a minispin column.
  • the device for automated extraction of nucleic acid samples from a plurality of samples comprises: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples, and d) a regeneration unit configured to regenerate the matrix in any receptacle of the plurality of receptacles; and ii) a multi-channel pump module, comprising: e) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples and a plurality of reservoirs for storing reagents for regeneration of the matrix, and f) at least one pump configured to deliver the plurality of samples, the reagents for
  • the at least one pump is a single pump. In some embodiments, the at least one pump is a plurality of pumps.
  • the extraction unit and the regeneration unit are part of the same unit. In other embodiments, the extraction unit and the regeneration unit are separate.
  • the regeneration unit increases the sample-processing capacity of the device without the need for increasing the number of the receptacles and/or matrices comprised by the device.
  • the devices known in the art are characterized by sample processing capacity being dependent on the number of receptacles and/or matrices used, with one disposable matrix being used for extraction of nucleic acids from a single sample only in majority of said known devices.
  • the devices which do use matrix regeneration described for example in Preston C. et al.
  • Increasing the sample processing capacity by providing an increasingly larger number of disposable matrices in the device is undesirable and limited for several reasons, e.g., frequent necessity for waste disposal, high operating cost and increasing the footprint required for the process to operate with the desired processing capacity.
  • the sample processing capacity of the device is increased, which allows for the device to operate for longer without the need for providing replacement matrices and/or receptacles to the device. It further limits the need for providing waste disposal in the device as the matrices can be used for multiple extraction cycles prior to being eventually disposed of.
  • the reagents for regeneration of the matrix for nucleic acid binding are known in the art.
  • the reagents for regeneration of the matrix for nucleic acid binding can comprise any reagents for chemical regeneration of the matrix for nucleic acid binding known in the art.
  • the reagents for chemical regeneration of the matrix comprise a hypochlorite.
  • the reagents for chemical regeneration of the matrix comprise sodium hypochlorite, preferably 0.7% sodium hypochlorite.
  • the regeneration reagent is any suitable cleaning reagent such as bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IP A) and DNAZapTM PCR DNA.
  • the regeneration reagent is selected from bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IP A) and DNAZapTM PCR DNA Degradation reagent.
  • the reagents for regeneration of the matrix for nucleic acid binding comprise water, PBS or borate buffers. In such embodiments, the regeneration of the matrix is achieved by washing the membrane resulting in removal of the nucleic acid bound to the matrix.
  • the extraction unit and the regeneration unit each comprise a plurality of tubes configured to deliver the sample and/or the reagents to the matrix.
  • the extraction unit and the regeneration unit each comprise a nozzle configured to force the sample and/or the reagents through the matrix. To force the sample and/or the reagent through the matrix a positive pressure is provided on the analyte in the receptacle.
  • the positive pressure provided on the analyte is from +lkPa to +100 kPa, in some embodiments from +20kPa to +50 kPa, e.g. +20kPa, +30kPa, +40kPa or +50kPa.
  • the device comprises a valve for releasing and/or maintaining positive pressure, optionally a solenoid valve. The valve can be used to control the flow rate of the analyte through the matrix, by decreasing the air pressure in the receptacle and thus stopping the analyte from moving through the matrix.
  • the nozzle of the extraction unit and the nozzle of the regeneration unit can each form an airtight seal with any receptacle of the plurality of receptacles.
  • the airtight seal enables for the pressure in the receptacle to be increased such that the analyte can be forced through the matrix.
  • the extraction unit and/or the regeneration unit each comprise at least one linear actuator (e.g. an electric or pneumatic actuator) for docking each nozzle to the receptacle.
  • the device comprises an in-line sample lysis module for lysis of any sample of the plurality of samples.
  • the samples are lysed prior to delivery to the device.
  • the samples are lysed after delivery to the device.
  • Methods for lysis of samples for nucleic acid extraction are well known in the art.
  • the in-line sample lysis module comprises a mechanical vibration device for mixing of the sample with a lysis buffer.
  • the in-line sample lysis module comprises a load cell for measuring the volumes of the sample and the lysis buffer that are mixed together.
  • the lysis can be performed directly in the receptacle, by mixing of the sample and the lysis buffer in the receptacle.
  • the in-line sample lysis module comprises a magnetic stirrer.
  • the device comprises a waste reservoir for collection of waste liquid.
  • the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.
  • the reagents for the extraction can selected from the plurality of reagents well-known in the art. Examples of the reagents for nucleic acid extractions are described in for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012).
  • the matrix is a silica matrix (optionally a silica membrane), a glass fibre matrix or a zeolite. In preferred embodiments the matrix is a silica membrane.
  • the plurality of receptacles is a plurality of columns. In some embodiments, the plurality of receptacles is a plurality of spin columns. In preferred embodiments, the plurality of receptacles is a plurality of minispin columns. Minispin columns comprising silica membranes for nucleic acid extractions are well known in the art, available commercially and cost- effective.
  • the number of receptacles in the plurality of receptacles can vary depending on the desired sample-processing capacity of the device.
  • the plurality of receptacles is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 receptacles
  • the means for housing the plurality of receptacles is a cassette.
  • the means for housing the plurality of receptacles is a carousel.
  • tubing used to deliver the nucleic acid sample is manufactured form an inert material, which nucleic acid molecules will not stick to.
  • the carousel is configured to move in a single plane (through rotation). In other embodiments, the carousel is configured to move in two planes, the planes being perpendicular to each other.
  • the at least one pump comprises at least one peristaltic pump.
  • the at least one pump comprises at least one syringe pump.
  • the at least one pump comprises at least one diaphragm pump.
  • the at least one pump comprises at least one metering pump.
  • the at least one pump is at least one pump in communication with a valve terminal.
  • the at least one pump comprises at least one pump in communication with a valve terminal.
  • the valve terminal comprises a plurality of solenoids.
  • the positive air pressure provided on the analyte in the receptacle may be generated by any suitable means for generating positive air pressure.
  • the positive pressure is generated by the at least one pump.
  • the positive air pressure provided on the analyte in the receptacle is generated by the at least one syringe pump.
  • the positive air pressure provided on the analyte in the receptacle is generated by the at least one diaphragm pump.
  • the positive air pressure provided on the analyte is provided by means for storing compressed air, for example by a compressed air tank.
  • the device comprises means for measuring a volume of the analyte in any receptacle of the plurality of receptacles.
  • the volume measurement provided by the means for measuring the volume of the analyte is used to control the at least one pump.
  • the volume measurement is used to control the at least one pump using a closed loop feedback system.
  • the control of the at least one pump is performed periodically.
  • the control of the at least one pump is performed continuously.
  • the control of the at least one pump using the volume measurement allows for the adjustment of pump activity to deliver the sample and/or reagent through the matrix.
  • a plurality of embodiments can be employed to provide means for measuring the volume of analyte in any receptacle of the plurality of receptacles.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a weight-measurement system.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a load cell.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a sound-based system, optionally an ultrasound-based system.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles comprises a camera system.
  • the camera system can be positioned in sufficiently close proximity to the plurality of tubes configured to deliver the sample and/or the reagents to the matrix, in order to enable the volume of sample and/or reagents in the receptacle to be measured, and to enable the volume of sample and/or reagents delivered to the matrix to be controlled.
  • the camera system comprises a refraction target, which provides increased contrast between the background of the image and the meniscus of the liquid (analyte) in the receptacle.
  • the refraction target facilitates the measurements of the volumes directly in the receptacle.
  • the refraction target is a vertical, dark colour slip placed horizontally behind the receptacle, in the camera-receptacle-refraction target axis.
  • An exemplary set up of the camera system is provided in Figure 3 A.
  • the camera system does not comprise a refraction target.
  • a basic level (or line) detection is used to determine the position of the meniscus of the liquid in the sample receptacle and thus determine the volume of the liquid in the sample receptacle.
  • the means for measuring the volume of analyte in any receptacle of the plurality of receptacles further comprises means for measuring the flow rate of the reagents and/or the sample through the device such that the volume of the analyte in the receptacle can be determined from the flow rate measurements.
  • the nozzle of the extraction unit and the nozzle of the regeneration unit are further configured to measure the air pressure in the receptacle.
  • the air pressure measurements provided by the nozzle of the extraction unit and/or the nozzle of the regeneration unit are used together with the volume measurements to control the activity of the pumps.
  • the air pressure measurements are used to determine the presence of a leftover, undesired sample and/or reagent on the matrix.
  • the presence of the leftover, undesired sample and/or reagent on the matrix is determined using a backpressure measurement.
  • the backpressure measurement is used to adjust the number of wash cycles required to remove the leftover, undesired sample and/or reagent from the matrix.
  • the plurality of tubes of the device comprises projections (e.g. pipette tips), narrowing towards the end of the projections such that fine droplets can be created when a liquid (e.g. the samples or reagents) is delivered through the plurality of tubes.
  • projections e.g. pipette tips
  • the device of the invention comprises at least one actuator.
  • the device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 actuators.
  • at least one actuator is at least one electric actuator.
  • the at least one actuator is at least one pneumatic actuator.
  • the device comprises at least one linear actuator and at least one pneumatic actuator.
  • the device comprises an actuator configured to lower the end of the tube for delivery of any of the reagents and/or sample into the receptacle during delivery. Such lowering allows to prevent excessive splashing during delivery into the receptacle.
  • the device comprises a suction system.
  • the suction system comprises a suction pump, a waste reservoir for collection of waste liquid and a linear actuator configured to dock the waste reservoir of the suction system to the receptacle.
  • the waste reservoir is docked to the receptacle using a funnel.
  • the docking of the waste reservoir of the suction system to the sample receptacle is not air-tight. The use of the suction system during operation of the device prevents excessive aerosols from contaminating the device, when sample and/or reagents are being provided through the matrix of the receptacle using positive pressure.
  • the process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples using a plurality of matrices for nucleic acid binding comprises a nucleic acid extraction step and a matrix regeneration step, wherein the nucleic acid extraction step and the matrix regeneration step are performed in-parallel.
  • the processes known in the art are characterized by sample processing capacity being dependent on the number of receptacles and/or matrices used, with one disposable matrix being used for extraction of nucleic acids from a single sample only in majority of said processes.
  • the processes which do use matrix regeneration described for example in Preston C. et al. 2011 “Underwater application of quantitative PCR on an ocean mooring”, not only use a single column, but also require the use of custom-designed receptacles with matrices for nucleic acid binding, which increases the cost involved with the process.
  • sample processing capacity by providing an increasingly larger number of disposable matrices in the process is undesirable for several reasons, e.g., frequent necessity for waste disposal, high operating cost and increasing the footprint required for the process to operate with the desired processing capacity.
  • a matrix regeneration step in parallel to the nucleic acid extraction step in the processes, the sample processing capacity of the process is increased, which allows for the process to be performed for longer without the need for providing replacement matrices and/or receptacles during the process. It further limits the need for providing waste disposal in the process as the matrices can be used for multiple extraction cycles prior to being eventually disposed of.
  • the reagents for regeneration of the matrix for nucleic acid binding are known in the art.
  • the reagents for regeneration of the matrix for nucleic acid binding can comprise any reagents for chemical regeneration of the matrix for nucleic acid binding known in the art.
  • the reagents for chemical regeneration of the matrix comprise a hypochlorite.
  • the reagents for chemical regeneration of the matrix comprise sodium hypochlorite, preferably 0.7% sodium hypochlorite.
  • the regeneration reagent is any suitable cleaning reagent such as bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IP A) and DNAZapTM PCR DNA.
  • the regeneration reagent is selected from bleach, water, PBS, borate buffers, ethanol, isopropyl alcohol (IP A) and DNAZapTM PCR DNA Degradation reagent.
  • a cleaning solution for regenerating the matrix for nucleic acid binding does not comprise bleach-based reagents.
  • the reagents for regeneration of the matrix for nucleic acid binding comprise water, PBS or borate buffers. In such embodiments, the regeneration of the matrix is achieved by washing the membrane resulting in removal of the nucleic acid bound to the matrix.
  • the regeneration step comprises contacting the matrix with a regeneration reagent, followed by washing the matrix with water.
  • the volume of 0.7% sodium hypochlorite used for regeneration is 0.5mL.
  • the volume of water used for the washing is 20mL.
  • a presence of the leftover, undesired sample and/or reagent on the matrix is determined using an air pressure measurement in the receptacle.
  • the presence of the leftover, undesired sample and/or reagent on the matrix is determined using a backpressure measurement.
  • the backpressure measurement is used to adjust the number of wash cycles required to remove the leftover, undesired sample and/or reagent from the matrix.
  • the matrix is a silica matrix (optionally a silica membrane), a glass fibre matrix or a zeolite.
  • the matrix is a silica membrane.
  • the silica membrane is comprised by a column.
  • the silica membrane is comprised by a spin column.
  • the silica membrane is comprised by a minispin column.
  • sample processing capacity of the process will be a function of the number of matrices in the plurality of matrices used in the process and the number of times each matrix of the plurality of matrices is regenerated in the process.
  • the plurality of matrices is selected from at least 2 matrices, at least 3 matrices, at least 4 matrices, at least 5 matrices, at least 6 matrices, at least 7 matrices, at least 8 matrices, at least 9 matrices, at least 10 matrices, at least 11 matrices, at least 12 matrices, at least 13 matrices, at least 14 matrices, at least 15 matrices, at least 16 matrices, at least 17 matrices, at least 18 matrices, at least 19 matrices, at least 20 matrices, at least 30 matrices, at least 40 matrices, at least 50 matrices, at least 60 matrices, at least 70 matrices, at least 80 matrices, at least 90 matrices or at least 100 matrices.
  • each matrix of the plurality of matrices is regenerated at least 1 time. In some embodiments, each matrix of the plurality of matrices is regenerated at least 2 times.
  • each matrix of the plurality of matrices is regenerated at least 3 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 4 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 5 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 6 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 7 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 8 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 9 times. In some embodiments, each matrix of the plurality of matrices is regenerated at least 10 times.
  • the exemplary embodiment of the device for automated extraction of nucleic acid samples from a plurality of samples comprises: i) a sample module (200) comprising: a) a plurality of receptacles (210) for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, and c) an extraction unit (220) configured to extract the nucleic acid samples from any sample of the plurality of samples; and ii) a multi-channel pump module (300), comprising: d) a plurality of reservoirs (310) for storing reagents for extraction of nucleic acid samples, and e) at least one pump (320) configured to deliver the plurality of samples and/or the reagents for extraction of nucleic acid samples to and/or through the matrix of any receptacle of the
  • the sample module further comprises a regeneration unit (240) configured to regenerate the matrix in any receptacle of the plurality of receptacles
  • the multi-channel module further comprises a plurality of reservoirs (350) for storing reagents for regeneration of the matrix
  • the at least one pump is further configured to deliver the reagents for regeneration of the matrix to and/or through the matrix of any receptacle of the plurality of samples
  • the multi-channel pump module and the sample module are further connected to enable regeneration of any matrix of the plurality of receptacles
  • the means for housing the plurality of receptacles is further configured such that any of the plurality of the receptacles can be positioned for delivery of the reagents from the regeneration unit.
  • the sample module further comprises an in-line sample lysis module (230) for mixing of the sample with a lysis buffer.
  • Filling level sensors (231-233) determine the filling levels of the in-line sample lysis module.
  • An actuator (234) is configured to drive a magnetic stirrer comprised by the in-line sample lysis module.
  • An actuator (211) is configured to select any receptacle of the plurality of receptacles for delivery.
  • the multi-channel pump module further comprises a (solenoid) valve terminal (321) and the at least one pump is a syringe pump (322) in communication with the valve terminal.
  • the valve terminal comprises a plurality of solenoids.
  • the at least one pump and the valve terminal are in communication with the plurality of reservoirs.
  • the at least one pump provides positive pressure to the plurality of reagent reservoirs, such that the reagents can be delivered to the plurality of receptacles.
  • An actuator (311) is configured to select any reagent of the samples and/or reagents for delivery.
  • An actuator (312) is configured to move a plurality of tubes of the device vertically, in order to prevent excessive splashing during delivery of the reagent and/or the sample.
  • the plurality of tubes comprises projections (e.g. pipette tips), narrowing towards the end of the projections such that fine droplets can be created when a liquid (e.g. the samples or reagents) is delivered through the plurality of tubes.
  • the projections can be integrated into the end of the tube or detachable.
  • the at least one pump and the valve terminal are also in communication with the extraction unit such that positive pressure can be provided to the extraction unit in order for the sample and/or the reagents to be delivered through the matrix.
  • the at least one pump and the valve terminal are also in communication with the in-line sample lysis module such that the sample can be delivered from the in-line sample lysis module to the sample receptacle.
  • the at least one pump and the valve terminal are also in communication with the plurality of reservoirs for storing reagents for regeneration of the matrix and the in-line sample lysis module such that the in-line sample lysis module can be cleaned using the reagents for regeneration of the matrix.
  • the at least one pump and the valve terminal are also in communication with the regeneration unit such that positive pressure can be provided to the regeneration unit in order for the regeneration reagents to be delivered through the matrix.
  • the use of positive pressure provided by the at least one pump and the valve terminal for delivering positive pressure through the extraction and/or regeneration unit and through the matrices of the sample receptacles circumvents the need for conventional use of vacuum or centrifugal force to move the reagents and/or samples through the matrices.
  • pressurizing the matrix e.g. a silica membrane
  • the matrix leads to generation of aerosols when the liquids (i.e. the reagents and/or the sample) leave the matrix.
  • lysis reagents used for nucleic acid extraction can be harmful to the device handlers and/or result in damage of the device components over time.
  • the device further comprises a suction system (600) comprising a suction pump (610), a waste reservoir (620) for collection of waste liquid and a linear actuator (630).
  • the linear actuator is configured to dock the waste reservoir of the suction system to the sample receptacle (for example using a funnel (640)), such that aerosols are sucked into the waste reservoir.
  • the docking of the waste reservoir to the sample receptacle is not air-tight, in order to prevent the negative pressure created by the suction pump from causing the movement of the reagent and/or sample through the matrix of the receptacle.
  • the nucleic acid following extraction is collected in a nucleic acid collection container (700).
  • the nucleic acid container is an RNA collection container.
  • the exemplary embodiment of the device for automated extraction of nucleic acid samples from a plurality of samples comprises: i) a sample module (200) comprising: a) a plurality of receptacles (210) for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, and c) an extraction unit (220) configured to extract the nucleic acid samples from any sample of the plurality of samples; and ii) a multi-channel pump module (300), comprising: d) a plurality of reservoirs (310) for storing reagents for extraction of nucleic acid samples, and e) at least one pump (320) configured to deliver the plurality of samples and/or the reagents for extraction of nucleic acid samples to and/or through the matrix of any receptacle of the
  • the sample module further comprises a regeneration unit (240) configured to regenerate the matrix in any receptacle of the plurality of receptacles
  • the multi-channel module further comprises a plurality of reservoirs (350) for storing reagents for regeneration of the matrix
  • the at least one pump is further configured to deliver the reagents for regeneration of the matrix to and/or through the matrix of any receptacle of the plurality of samples
  • the multi-channel pump module and the sample module are further connected to enable regeneration of any matrix of the plurality of receptacles
  • the means for housing the plurality of receptacles is further configured such that any of the plurality of the receptacles can be positioned for delivery of the reagents from the regeneration unit.
  • the sample module further comprises an in-line sample lysis module (230) for mixing of the sample with a lysis buffer.
  • Filling level sensors (231-233) determine the filling levels of the in-line sample lysis module.
  • An actuator (234) is configured to drive a magnetic stirrer comprised by the in-line sample lysis module.
  • An actuator (211) is configured to select any receptacle of the plurality of receptacles for delivery.
  • the multi-channel pump module further comprises a (solenoid) valve terminal (321) and the at least one pump comprises a syringe pump (322) in communication with the valve terminal.
  • the valve terminal comprises a plurality of solenoids.
  • the at least one pump further comprises metering pumps (e.g. at least one metering pump (323), at least one metering pump (324) and a metering pump (325)).
  • the at least one metering pump (323) is in communication with the reagent reservoirs (310) and the sample receptacle and is configured to deliver the reagents for extraction of nucleic acids to the receptacle.
  • the at least one metering pump (323) is also in communication with the reagent reservoirs (310) and the in-line sample lysis module.
  • the at least one metering pump (323) is also in communication with the reagent reservoirs (350) and the in-line sample lysis module such that the in-line sample lysis module can be cleaned using the reagents for regeneration of the matrix.
  • the at least one metering pump (324) is in communication with the reservoirs (310) and is configured to deliver the reagents for extraction of nucleic acids to the receptacle.
  • the metering pump (325) is configured to deliver the sample for nucleic acid extraction from the in-line sample lysis module to the sample receptacle.
  • the metering pumps provide the means for measuring the volume of analyte.
  • the at least one pump and the valve terminal are in communication with the plurality of reservoirs.
  • An actuator (312) is configured to move a plurality of tubes of the device vertically, in order to prevent excessive splashing during delivery of the reagent and/or the sample.
  • the plurality of tubes comprises projections (e.g. pipette tips), narrowing towards the end of the projections such that fine droplets can be created when a liquid (e.g. the samples or reagents) is delivered through the plurality of tubes.
  • the projections can be integrated into the end of the tube or detachable.
  • the single pump and the valve terminal are also in communication with the extraction unit such that positive pressure can be provided to the extraction unit in order for the sample and/or the reagents to be delivered through the matrix.
  • the single pump and the valve terminal are also in communication with the regeneration unit such that positive pressure can be provided to the regeneration unit in order for the regeneration reagents to be delivered through the matrix.
  • the single pump and the valve terminal are also in communication with the in-line sample lysis module such that the in-line sample lysis module can be emptied with pressurized air if needed.
  • the device further comprises a suction system (600) comprising a suction pump (610), a waste reservoir (620) for collection of waste liquid and a linear actuator (630).
  • the linear actuator is configured to dock the waste reservoir of the suction system to the sample receptacle (for example using a funnel (640)), such that aerosols are sucked into the waste reservoir.
  • the docking mode is as depicted in Figure 6.
  • the docking of the waste reservoir to the sample receptacle is not air-tight, in order to prevent the negative pressure created by the suction pump from causing the movement of the reagent and/or sample through the matrix of the receptacle.
  • the nucleic acid following extraction is collected in a nucleic acid collection container (700).
  • the nucleic acid container is an RNA collection container.
  • the device comprises a peristaltic pump (800) to provide the sample from a source to the in-line sample lysis module.
  • a device for automated extraction of nucleic acid samples from a plurality of samples comprising: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, and c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples; and ii) a multi-channel pump module, comprising: d) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples, and e) a plurality of pumps configured to deliver the plurality of samples and/or the reagents for extraction of nucleic acid samples to and/or through the matrix of any receptacle of the plurality of receptacles; and wherein: the device comprises means for measuring the volume of analyte in any receptacle
  • volume measurements are used to control the plurality of pumps, preferably using closed loop feedback control.
  • the extraction unit comprises a nozzle configured to force the sample and/or the reagents through the matrix.
  • the device comprises an in-line lysis module for lysis of any sample of the plurality of samples.
  • the in-line lysis module comprises a mechanical vibration device for mixing of the sample with a lysis buffer.
  • the matrix is a silica membrane, a glass fibre matrix or a zeolite.
  • the plurality of receptacles is a plurality of columns, preferably a plurality of mini spin columns.
  • the plurality of receptacles is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 receptacles.
  • the plurality of pumps comprises at least one diaphragm pump.
  • the device comprises a comprises a valve for releasing and/or maintaining positive pressure, optionally a solenoid valve.
  • the sample module further comprises a regeneration unit configured to regenerate the matrix in any receptacle of the plurality of receptacles
  • the multi-channel module further comprises a plurality of reservoirs for storing reagents for regeneration of the matrix
  • the plurality of pumps is further configured to deliver the reagents for regeneration of the matrix to and/or through the matrix of any receptacle of the plurality of samples
  • the multi-channel pump module and the sample module are further connected to enable regeneration of any matrix of the plurality of receptacles
  • the means for housing the plurality of receptacles is further configured such that any of the plurality of the receptacles can be positioned for delivery of the reagents from the regeneration unit.
  • the regeneration unit comprises a plurality of tubes configured to deliver the reagents to the matrix.
  • the regeneration unit comprises a nozzle configured to force reagents through the matrix, preferably wherein the nozzle can form an airtight seal with any receptacle of the plurality of receptacles.
  • the reagents for regeneration of the matrix comprise sodium hypochlorite, preferably 0.7% sodium hypochlorite.
  • a process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples comprising the steps of: i) receiving a sample of the plurality of samples in a receptacle of a plurality of receptacles, ii) binding a nucleic acid to a matrix, iii) washing the matrix to remove residual reagent, and iv) eluting the nucleic acid from the matrix; wherein a volume of sample used in the process and/or a volume of any reagent of a plurality of reagents used in the process are controlled using image processing; and wherein image processing is used to measure the volume of the sample and/or the volume of any reagent of a plurality of reagents received in the receptacle.
  • a device for automated extraction of nucleic acid samples from a plurality of samples comprising: i) a sample module comprising: a) a plurality of receptacles for receiving the plurality of samples, wherein each receptacle of the plurality of receptacles comprises a matrix for nucleic acid binding, b) means for housing the plurality of receptacles, c) an extraction unit configured to extract the nucleic acid samples from any sample of the plurality of samples, and d) a regeneration unit configured to regenerate the matrix in any receptacle of the plurality of receptacles; and ii) a multi-channel pump module, comprising: e) a plurality of reservoirs for storing reagents for extraction of nucleic acid samples and a plurality of reservoirs for storing reagents for regeneration of the matrix, and f) a plurality of pumps configured to deliver the plurality of samples, the reagents for extraction of nucleic acid samples
  • the device of embodiment any of embodiments 31 to 34, wherein the reagents for regeneration of the matrix comprise sodium hypochlorite, preferably 0.7% sodium hypochlorite.
  • the device of any of embodiments 31 to 35 wherein the device comprises an in-line lysis module for lysis of any sample of the plurality of samples.
  • the device of embodiment 36 wherein the in-line lysis module comprises a mechanical vibration device for mixing of the sample with a lysis buffer.
  • the device of any of embodiments 31 to 37 wherein the nucleic acid is DNA or RNA.
  • the device of any of embodiments 31 to 38 wherein the matrix is a silica membrane, a glass fibre matrix or a zeolite.
  • the device of any of embodiments 31 to 39, wherein the plurality of receptacles is a plurality of columns, preferably a plurality of mini spin columns.
  • the device of embodiment 40 wherein the plurality of receptacles is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 receptacles.
  • the device of any of embodiments 31 to 42, wherein the plurality of pumps comprises peristaltic pumps.
  • the device of any of embodiments 31 to 43, wherein the plurality of pumps comprises syringe pumps.
  • the plurality of pumps comprises at least one diaphragm pump.
  • the device comprises a valve for releasing and/or maintaining positive pressure, optionally a solenoid valve.
  • a process for automated, matrix-based extraction of nucleic acid samples from a plurality of samples using a plurality of matrices for nucleic acid binding comprising a nucleic acid extraction step and a matrix regeneration step, wherein the nucleic acid extraction step and the matrix regeneration step are performed in-parallel.

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Abstract

La présente invention concerne des dispositifs utiles pour l'extraction automatisée d'échantillons d'acides nucléiques à partir d'une pluralité d'échantillons. La présente invention concerne en outre des procédés d'extraction automatisée d'échantillons d'acide nucléique à partir d'une pluralité d'échantillons, qui peuvent être réalisés à l'aide des dispositifs de l'invention.
PCT/EP2024/062942 2023-05-10 2024-05-10 Extraction automatisée d'échantillons d'acides nucléiques à partir d'une pluralité d'échantillons Pending WO2024149910A2 (fr)

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EP24723920.5A EP4611937A2 (fr) 2023-05-10 2024-05-10 Extraction automatisée d'échantillons d'acides nucléiques à partir d'une pluralité d'échantillons
AU2024208619A AU2024208619A1 (en) 2023-05-10 2024-05-10 Automated extraction of nucleic acid samples from a plurality of samples

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GBGB2306923.0A GB202306923D0 (en) 2023-05-10 2023-05-10 Device
GB2306923.0 2023-05-10
GBGB2314626.9A GB202314626D0 (en) 2023-09-25 2023-09-25 Device
GB2314626.9 2023-09-25

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JP2001516731A (ja) * 1997-09-17 2001-10-02 ジエントラ・システムズ・インコーポレーテツド 核酸を単離するための装置および方法
WO2002078847A1 (fr) * 2001-03-28 2002-10-10 Hitachi, Ltd. Instrument et procede permettant de recuperer de l'acide nucleique
EP2177915A4 (fr) * 2007-06-19 2014-01-08 Universal Bio Research Co Ltd Appareil de traitement de puces en colonne et procédé de traitement de puces en colonne
CA3097861A1 (fr) * 2011-01-21 2012-07-26 Labrador Diagnostics Llc Systemes et procedes de maximisation d'utilisation d'echantillon

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"The Merck Manual of Diagnosis and Therapy", 2006, MERCK RESEARCH LABORATORIES
BENJAMIN LEWIN: "Current Protocols in Protein Sciences", 2009, JONES & BARTLETT PUBLISHING
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