WO2019081259A1 - Support de réaction destiné à un dispositif microfluidique et procédé permettant de déterminer une séquence nucléotidique - Google Patents

Support de réaction destiné à un dispositif microfluidique et procédé permettant de déterminer une séquence nucléotidique

Info

Publication number
WO2019081259A1
WO2019081259A1 PCT/EP2018/078140 EP2018078140W WO2019081259A1 WO 2019081259 A1 WO2019081259 A1 WO 2019081259A1 EP 2018078140 W EP2018078140 W EP 2018078140W WO 2019081259 A1 WO2019081259 A1 WO 2019081259A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
solid phase
primers
capture
recess
Prior art date
Application number
PCT/EP2018/078140
Other languages
German (de)
English (en)
Inventor
Jochen Hoffmann
Original Assignee
Robert Bosch Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Bosch Gmbh filed Critical Robert Bosch Gmbh
Publication of WO2019081259A1 publication Critical patent/WO2019081259A1/fr

Links

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
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • 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
    • B01L2300/0896—Nanoscaled

Definitions

  • Reaction carrier for a microfluidic device and method for
  • microfluidic devices also called lab-on-a-chips
  • the invention relates to a reaction carrier for a microfluidic device for determining a nucleotide sequence.
  • the reaction carrier comprises at least a first recess and a second recess, wherein in the first recess first capture primer and in the second recess second capture primer are immobilized on a solid phase.
  • the first capture primers are different from the second capture primers.
  • the reaction carrier may in particular be a substrate, for example a
  • Plastic substrate include. Under a recess can be understood in particular a depression in a surface of the reaction carrier.
  • the nucleotide sequence is in particular a sequence of
  • nucleotides of a nucleotide strand or nucleic acid fragment of a nucleotide strand are nucleotides of a nucleotide strand or nucleic acid fragment of a nucleotide strand.
  • a virus in particular a virus, a bacterium or a unicellular.
  • Sequencing methods are understood as described above, in particular a sequencing-by-synthesis method. Both
  • Capture primers are, in particular, nucleotide strands which are designed to bind the nucleotide strands to be sequenced.
  • the capture primers are further designed for amplification of the nucleotide strands to be sequenced, in particular for one
  • the reaction support advantageously allows the amplification of the nucleotide strands to be sequenced, in particular by carrying out a PCR, on the reaction support, ie directly on a solid phase, to what
  • a sequencing on the solid phase can be carried out immediately afterwards.
  • the resulting PCR product is attached to the immobilized capture primer.
  • the too sequencing nucleotide strands are immobilized during PCR by binding to the capture primers and thus advantageously provided at a given location, namely at the site of the solid phase, for subsequent sequencing.
  • Nucleotide strands also allow easy removal of no longer required reaction components, especially by rinsing the solid phase. Furthermore, it is of particular advantage that no specific probes for detecting the nucleotide strands must be provided, but identification via the sequencing of the nucleotide strands bound to the capture primers is possible. In particular, it is advantageously possible to recognize known DNA signatures and also point mutations in such signatures.
  • the immobilized and different capture primers also advantageously allow parallel and spatially resolved sequencing and identification of different nucleotide strands.
  • the reaction carrier may preferably comprise further recesses with further immobilized capture primers, wherein the further capture primers may be partially identical and in part different from each other. This advantageously increases an effectiveness and efficiency or a parallelism of the determination of the nucleotide sequences.
  • a solid phase can be understood as meaning a material, a substance or a composition of materials and / or substances in a solid state of matter, including gels, in particular agarose gels or hydrogels.
  • the solid phase may be connected to the reaction carrier, in particular, the
  • Solid phase to be part of the reaction carrier.
  • the solid phase preferably comprises at least part of a wall or a bottom of the first and / or the second recess.
  • the solid phase can comprise particles, in particular magnetic particles, which can advantageously be immobilized with respect to the solid phase with the aid of a magnet.
  • magnetic particles can be understood as meaning particles having a magnetic or magnetizable material, for example paramagnetic polymer particles.
  • the use of particles as a solid phase has the advantage that at
  • Nucleotide strands a contact surface between the solid phase and the sample is greater than when using a planar phase, which provides for increased and thus improved reaction kinetics due to shortened diffusion paths.
  • the use of magnetic particles as a solid phase also has the advantage that, on the one hand, the particles can be retained by a magnetic force at well-defined and thus easily distinguishable locations in the microfluidic device.
  • the immobilized nucleotide strands can be transported to any location via magnetic actuation, for example for subsequent processing or analysis. Furthermore, it is advantageous that rinsing of the particulate solid phase can be carried out more effectively in comparison to a planar solid phase
  • the solid phase comprises porous material, wherein the first and / or the second capture primer are immobilized in pores of the porous material.
  • At least one of the recesses contains further primers for the determination of the nucleotide sequence in unbound form, in particular in dried form.
  • the further primers are preferably designed to support the PCR for the amplification of the nucleotide strands which bind to the capture primer.
  • the further primers are at least partially identical to primers for the PCR of these nucleotide strands.
  • the further primers in the first recess or in the second recess are at least partially identical to the first or the second catcher primer. For example, a sequence of the further primer in the first recess or in the second recess with a
  • Partial sequence starting at the 3 'end of the first or second capture primer identical.
  • Partial sequence starting at the 3 'end of the first or second capture primer identical.
  • Partial sequence starting at the 3 'end of the first or second capture primer identical.
  • are in at least one of the recesses further primer for the implementation of a
  • Amplification reaction before, especially in dried form is a method of amplification of
  • the further primers are designed for a multiplication of the nucleotide strands to be sequenced, that is to say, in particular, for the nucleotide strands to be sequenced and for the first or second capture primers.
  • the further primers may be reverse primers or forward primers if the primers are forward primers or reverse primers.
  • further reagents for carrying out the amplification reaction in particular a PCR, can be present in at least one of the recesses, in particular in dried form, for example additives for the amplification reaction.
  • the subject of the invention is also a microfluidic device with a reaction carrier according to the invention.
  • the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus.
  • the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus.
  • the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus.
  • the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus.
  • the reaction carrier is detachably arranged in the microfluidic device, for example via a latching mechanism or Einklippmechanismus.
  • Reaction carrier also be connected via a bond with the microfluidic device, which advantageously allows a particularly fluid-tight connection.
  • the reaction carrier can thus advantageously according to the
  • Production and immobilization of the first and second capture primer can be introduced into the device.
  • the microfluidic device comprises an area for pre-amplification of the nucleotide strands to be sequenced. This area may be
  • a fluidic connection between the chamber and the reaction carrier can be interrupted, for example by a valve.
  • Pre-duplication is a duplication of the sequences to be sequenced
  • nucleotide strands to understand before the nucleotide strands to be sequenced are brought into contact with the reaction support, for example a Duplication via PCR, especially as part of a nested PCR. This has the advantage that a large number of the to be sequenced
  • Nucleotide strands can be generated in the microfluidic device.
  • a dilution in particular with a buffer solution, take place, in particular after the pre-duplication.
  • the microfluidic device comprises a to the
  • Reaction support adjacent region wherein the region for receiving a magnet is formed such that a magnetic field of the magnet for fixing a magnetic or magnetizable solid phase described above in the recesses may extend into one or more of the recesses.
  • the magnet can be a permanent magnet or an electromagnet, which can generate a magnetic field suitable for the manipulation of the particles.
  • the reaction carrier may have a recess for receiving a magnet.
  • the device can in addition to the preferred latching or
  • Einklippmechanismus a region, in particular a recess, for receiving the magnet.
  • the invention further provides a method for determining a nucleotide sequence with a reaction carrier according to the invention.
  • the sequence of bound nucleotide strands bound to the first and / or second capture primers may have been amplified in an optional step, in particular by performing a PCR.
  • the sequence of bound nucleotide strands to be sequenced may have been amplified in an optional step, in particular by performing a PCR.
  • Nucleotide strands determined, in particular via a sequencing-by-synthesis method.
  • the immobilizing step comprises binding the
  • the solid phase can be a mobile solid phase
  • the binding of the capture primer to the solid phase takes place prior to the immobilization of the solid phase on the reaction support. This has the advantage that the binding of the capture primer to the solid phase can also take place separately from the reaction support and in particular outside the microfluidic device.
  • the method takes place during the binding of the nucleotide strands to the first and / or second capture primer, a closure of the recesses, in particular by a cover with a liquid.
  • a closure of the recesses in particular by a cover with a liquid.
  • the liquid may in particular be a non-polar liquid, for example an oil.
  • FIG. 1 and 2 an embodiment of the device according to the invention and
  • FIG. 3 shows a flowchart for an exemplary embodiment of the method according to the invention.
  • FIG. 1 schematically shows an exemplary embodiment of the microfluidic device 200 according to the invention with an exemplary embodiment of the invention
  • Reaction carrier 100 for determining a
  • the reaction carrier 100 comprises in this example a first recess 101, a second recess 102 and a third
  • Recess 103 which are each designed as depressions in the reaction carrier 100.
  • the depressions 101, 102, 103 each have a volume between 1 picoliter and 100 microliter, preferably between 1 nanoliter and 10 microliter.
  • All the recesses 101, 102, 103 comprise a solid phase 110, in this example magnetic particles 110.
  • the particles 110 may be
  • first capture primers 121 second capture primers 122 are bound to the particles 110 in the second recess 102 and third capture primers 123 are bound to the particles 110 in the third recess 103.
  • Different capture primers 121, 122, 123 thus enable parallel and spatially resolved detection of different nucleotide strands.
  • a part of the reaction support 100 in particular a part of a wall 131 or a bottom 132 of one or more
  • Reaction carrier 100 comprise porous silicon.
  • the recesses 101, 102, 103 may, as described above, further comprise substances for a PCR, for example in freeze-dried form, in particular polymerases, nucleotides, salts and also PCR enhancers.
  • substances for a PCR for example in freeze-dried form, in particular polymerases, nucleotides, salts and also PCR enhancers.
  • you can additional primers required for the PCR for example for a concentration between 0.1 and 1.5 micromolar, and further capture primers, for example for a concentration between 0.001 and 1 micromolar, in the recesses 101, 102, 103 be upstream, the latter to the PCR to support and in particular to allow a PCR in the liquid phase.
  • These substances and primers can be wholly or partially in dehydrated, especially in freeze-dried form in the recesses 101, 102, 103, to be re-hydrated and thus activated when filling a liquid sample to be sequenced with nucleotide strands.
  • Recesses stored substances should be superimposed with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • FIG. 1 also shows schematically that the reaction carrier 100 can preferably be releasably connected to the device 200, for example via latching hooks 141, 142. Furthermore, it is shown schematically that the reaction carrier 100 can preferably be releasably connected to the device 200, for example via latching hooks 141, 142. Furthermore, it is shown schematically that the reaction carrier 100 can preferably be releasably connected to the device 200, for example via latching hooks 141, 142. Furthermore, it is shown schematically that the
  • Device 200 has a region 210 for a duplication and preferably dilution of the nucleotide strands to be sequenced.
  • this region 210 can be embodied as a chamber 210 in the device 200, which is fluidically connected to the reaction carrier via a valve 211.
  • the magnetic particles 110 can be fixed in the depressions 102 by a magnet 300, wherein the magnet 300 can be arranged, for example, in a second region 105 adjoining the reaction region 101.
  • FIG. 2b by way of example, two particles 110 with immobilized capture molecules 121 are shown, to which sequenceable nucleotide strands 400 are bound. Sequencing primers 401 are already bound to the immobilized nucleotide strands 400 for sequencing by means of a sequencing-by-synthesis method on one of the two particles 110.
  • FIG. 3 shows a flowchart for an embodiment of the invention
  • the method 500 according to the invention for determining a nucleotide sequence which can be used, for example, with the exemplary embodiments described above Inventive Reaction carrier 100 and the inventive
  • Device 200 can be performed.
  • the first capture primer 121 of the first recess 101 and second capture primer 122 are immobilized in the second recess 102 of the reaction support 100. If available, further capture primers can be immobilized in further recesses of the reaction support. As described above, this can preferably take place via a binding of the capture primers 121, 122, 123 to a mobile phase in the form of magnetic particles 110, which can be held in the recesses 101, 102, 103 of the reaction carrier 100 by means of a magnet 300.
  • the binding of the capture primers 121, 122, 123 to the solid phase can be carried out, for example, via silanes, for example APTES, to which linkers are attached, for example 1,4-phenylene diisothiocyanate (PDITC), glutaraldehyde, s-SIAB, s-MBS, s-silane. GMBS, s-MBP. Also a direct one
  • Immobilization using poly (C, T) -tagged nucleic acids in combination with UV-initiated immobilization is possible (Y. Sun, R. Dhumpa, D. Bang, J. Hogberg, K. Handberg, A. Wolff, "A lab -on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR ", Lab Chip, vol. 11, pp. 1457-1463, 2011).
  • nucleotide strands 400 to be sequenced are bound to the capture primers, preferably as part of a solid-phase PCR.
  • the recesses are during binding of the
  • Nucleotide strands 400 are closed to the capture primer, in particular by covering with a liquid, such as, for example, with an oil layer or a silicone layer, in order to ensure a contamination-free bond as possible.
  • a liquid such as, for example, with an oil layer or a silicone layer
  • the nucleotide strands to be sequenced may have been amplified in an optional step, in particular by performing a PCR. This has the advantage that depending on the number of
  • a rinse of the particles 110 with a wash buffer for example with a phosphate buffered saline solution (PBS for short), in a third Step 503, to optionally remove reaction components of the PCR.
  • PBS phosphate buffered saline solution
  • Nucleotide strands may have two hydrogen-bonded sequences of nucleotides that are complementary to each other, and one end of a sequence is each bound to a capture primer or is the extension thereof. In other words, each are two mutually complementary nucleotide strands, one of the two nucleotide strands being directly bound to the capture primer.
  • the nucleotide strand not directly bound to the primer can be removed by dissolving the hydrogen bonds, for example using a sodium hydroxide solution.
  • the caustic soda can be used both for the removal of these nucleotide strands and for the flushing described above.
  • the caustic soda can a
  • a fourth step 504 the sequence of the bound nucleotide strands is determined, in particular via a sequencing-by-synthesis method, which is described in US Pat
  • the fourth step 504 may preferably comprise the following steps five to ten.
  • a fifth step 505 the sequencing primer 401 is added to the bound first nucleotide strands, wherein the sequencing primer 401 is designed to bind to a free end of the first nucleotide strands 400, as shown in Figure 2b.
  • nucleotides of one of a plurality of predetermined types are added to the bound first nucleotide strands, the added nucleotides having a label, preferably a fluorophore, and a polymerase terminator.
  • the prescribed varieties are adenosine triphosphate, guanosine triphosphate, cytidine triphosphate and
  • Thymidine triphosphate examples of suitable fluorophores are:
  • Cyanine dyes such as Cy3, or Cy5 or Atto equivalents.
  • the polymerase terminator is an esterification of the 3 ' Hydroxyl group of a nucleotide.
  • the addition is carried out with the addition of polymerase, so that each binding of one of the added nucleotides to the bound nucleotide strands takes place when a complementarity of the added nucleotide with the bound in relation to the
  • Sequencing primer nearest nucleotide is present without complementary nucleotide of a respective bound first nucleotide strand.
  • the addition of the polymerase can be carried out together or before the addition of the nucleotides.
  • unbound nucleotides are removed, preferably via a rinse of the solid phase, for example with PBS, before, in an eighth step 508, a detection of the bound nucleotides takes place via detection of the label.
  • a conventional optical system consisting of excitation light, excitation filter, detection filter and detector can be used.
  • a ninth 509 step for example via rinsing of the bound first nucleotide strands with Na 2 PdCl 4 / P (PhSO 3Na) 3 as a buffer when using 3'-0-allyl groups as a polymerase terminator. This will be tied to the first
  • nucleotide strands generated free 3'-hydroxy ends, which allows incorporation of the next complementary nucleotide. Subsequently, in a tenth step 510, a new addition of polymerase and of nucleotides of one of the other prescribed varieties with label and

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Dispersion Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Physics & Mathematics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un support de réaction (100) destiné à un dispositif microfluidique (200) permettant de déterminer une séquence nucléotidique, comprenant au moins une première cavité (101) et une seconde cavité (102), dans la première cavité (101), des premières amorces de capture (121) étant immobilisées sur une phase solide (110), et dans la seconde cavité (102), des secondes amorces de capture (122) étant immobilisées sur une phase solide (110), les premières amorces de capture (121) étant différentes des secondes amorces de capture (122). L'invention concerne en outre un tel dispositif microfluidique (200) et un procédé associé (500) permettant de déterminer une séquence nucléotidique.
PCT/EP2018/078140 2017-10-23 2018-10-16 Support de réaction destiné à un dispositif microfluidique et procédé permettant de déterminer une séquence nucléotidique WO2019081259A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102017218849.9 2017-10-23
DE102017218849.9A DE102017218849A1 (de) 2017-10-23 2017-10-23 Reaktionsträger für eine mikrofluidische Vorrichtung und Verfahren zur Bestimmung einer Nukleotidsequenz

Publications (1)

Publication Number Publication Date
WO2019081259A1 true WO2019081259A1 (fr) 2019-05-02

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WO (1) WO2019081259A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE102019216438A1 (de) * 2019-10-25 2021-04-29 Robert Bosch Gmbh Verfahren zum Erzeugen von hydrophilen Oberflächen oder Oberflächenbereichen auf einem Träger
DE102022210371A1 (de) 2022-09-30 2024-04-04 Robert Bosch Gesellschaft mit beschränkter Haftung Mikrofluidische Kartusche, mikrofluidische Vorrichtung und Verfahren zu ihrem Betrieb

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US20040151631A1 (en) * 2001-03-09 2004-08-05 Rudolf Rigler Determination of analytes by means of fluorescence correlation spectroscopy
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US20040151631A1 (en) * 2001-03-09 2004-08-05 Rudolf Rigler Determination of analytes by means of fluorescence correlation spectroscopy
WO2003102231A1 (fr) * 2002-05-29 2003-12-11 Axaron Bioscience Ag Procede de sequençage parallele d'un melange d'acide nucleique a l'aide d'un systeme d'ecoulement
WO2005108604A2 (fr) * 2004-05-06 2005-11-17 Clondiag Chip Technologies Gmbh Dispositif et procede pour de detecter des interactions moleculaires

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BENTLEY ET AL.: "Accurate whole human genome sequencing using reversible terminator chemistry", NATURE, vol. 456, no. 7218, 6 November 2008 (2008-11-06), pages 53 - 59, XP002572505, DOI: doi:10.1038/nature07517
JOHN H. LEAMON ET AL: "A massively parallel PicoTiterPlate(TM) based platform for discrete picoliter-scale polymerase chain reactions", ELECTROPHORESIS, vol. 24, no. 21, 1 November 2003 (2003-11-01), pages 3769 - 3777, XP055091684, ISSN: 0173-0835, DOI: 10.1002/elps.200305646 *
JU ET AL.: "Fourcolor DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators", PNAS 2006, vol. 103, no. 52, 26 October 2006 (2006-10-26), pages 19635 - 19640
Y. SUN; R. DHUMPA; D. BANG; J. HOGBERG; K. HANDBERG; A. WOLFF: "A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR", LAB CHIP, vol. 11, 2011, pages 1457 - 1463

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