Preparation of devices using condensed nucleic acid particles

Abstract

A method of sequencing nucleic acid strands includes receiving particles having nucleic acid strands coupled to a polymer matrix, exposing the particles to a solution including a coagulant, and applying the particles to a surface where the particles deposit on the surface.

Description

[0001] Related Applications Cross Reference

[0002] This application claims the benefit of U.S. Provisional Application No. 61 / 877,745, filed September 13, 2013, which is incorporated herein by reference in its entirety.

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 020,292, filed on July 2, 2014, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates generally to systems and methods for preparing devices suitable for nucleic acid sequencing, and in particular to systems and methods for preparing such devices utilizing particles containing nucleic acids. Background Art

[0005] The sequencing of nucleic acid chains (especially DNA) has become increasingly important in advanced fields including medicine, agriculture and biological research. However, conventional gel techniques for sequencing nucleic acid chains have proven to be time-consuming and expensive. The latest developments rely on the deposition of samples containing nucleic acids on substrates. According to sequencing techniques, the sequence of nucleic acid samples can be determined by measuring the ionic response to nucleic acid addition or by measuring the fluorescent emission caused by nucleic acid addition.

[0006] However, such techniques that rely on the deposition of nucleic acid samples on substrates have drawbacks caused by the failure of some nucleic acid strands to bind to the substrate surface or by the strands binding in close proximity to each other. Such drawbacks can result in underutilization of the substrate, inaccurate data, lost or incomplete samples, or other inaccuracies.

[0007] Thus, improved systems and methods for preparing sequencing devices would be desirable. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The present invention may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.

[0009] Figure 1 Included is a diagram of an exemplary method for depositing a nucleic acid sample.

[0010] Figure 2 Included are diagrams of exemplary apparatus for preparing nucleic acid particles and preparing the apparatus for sequencing.

[0011] Figure 3 Included are images of beads or particles during exposure to hexaaminecobalt.

[0012] Figure 4 , Figure 5 , Figure 6, Figure 7 and Figure 8 Included is a graphical illustration of the size response of beads or particles to exposure to hexaaminecobalt.

[0013] Fig. 9 Included are graphs of the dissociation kinetics of nucleic acid-bound beads or particles both with and without exposure to hexaaminecobalt.

[0014] Fig.10 and Fig.11 Included is a graphical illustration of the effect of coagulant on bead size.

[0015] The use of the same reference symbols in different drawings indicates similar or identical items. DETAILED DESCRIPTION

[0016] In one embodiment, the method includes preparing nucleic acid beads or particles, condensing nucleic acid beads, and depositing the condensed nucleic acid beads on a sensor substrate. In another example, the method includes depositing nucleic acid beads on a sensor substrate and condensing the deposited nucleic acid beads. The nucleic acid beads or particles include beads bound to one or more nucleic acid chains. Condensing nucleic acid beads may include condensing nucleic acids bound to beads, polymers that shrink beads, or a combination thereof, effectively reducing the size of the nucleic acid beads. After deposition, the condensed nucleic acid beads can be washed, which can unfold the condensed nucleic acid beads. Subsequently, the sensor substrate with the deposited nucleic acid beads can be used for nucleic acid testing, such as sequencing, such as synthetic sequencing. Condensing nucleic acid beads or particles may include exposing the nucleic acid beads or particles to a condensing agent. The condensing agent can condense aspects of the nucleic acid beads or particles, such as condensing nucleic acids, a polymer matrix of particles, or a combination thereof. In one example, the condensing agent includes a 3 + In another example, the coagulation agent includes an alkali metal salt or an alkaline earth metal salt, such as a magnesium salt. In another example, the coagulation agent includes a non-ionic polymer agent, such as a polyethylene glycol agent.

[0017] In another embodiment, the system implements a method for preparing a device suitable for sequencing. The system may include a solution container, a sample receiving port or a sample chamber, and a mixer coupled to the solution container and the sample receiving port or chamber. The mixer may be coupled to a substrate preparation unit. In one example, the solution in the solution container includes a coagulant reagent, which includes a coagulant. Alternatively, the methods and processes described herein may be performed manually or with the use of a stirring plate, a vortexer, a pipette, a centrifuge or other laboratory equipment.

[0018] The sample receiving port or sample chamber is used to receive nucleic acid beads or particles, which have a polymer matrix and a nucleic acid chain coupled to the polymer matrix. As in a mixer, a coagulation reagent and nucleic acid particles are mixed. As a result, the nucleic acid chain or polymer matrix coagulates to produce particles of reduced diameter or greater density. The processed particles are transferred to a substrate preparation unit, where the particles are deposited on the substrate. In a specific example, the substrate includes a layer defining a concave hole, and the particles are deposited in the concave hole. The substrate can be used to sequence the nucleic acid chain of the beads or particles. In an example, the substrate is transferred to a sequencing device that performs a function of sequencing that causes the nucleic acid chain.

[0019] Alternatively, nucleic acid beads may be applied to the sensor substrate and after deposition of the nucleic acid beads, the condensation agent is applied on the sensor substrate. In another example, the nucleic acid beads may be treated with a condensation agent prior to deposition, deposited, washed and treated with a condensation agent after deposition and washing.

[0020] like Figure 1 As shown in , nucleic acid beads or particles 102 include a polymer matrix 104 and nucleic acid strands 106. Although nucleic acid strands 102 are shown extending from a surface of polymer matrix 104, polymer matrix 104 can be porous, such as a hydrogel, and nucleic acid strands 102 can be coupled to polymer matrix 104 and extend through polymer matrix 104. Nucleic acid beads or particles 102 can be treated to form treated nucleic acid beads or particles 108. Treated nucleic acid particles 108 can have a smaller diameter than nucleic acid particles 102.

[0021] The surface component 116 includes a substrate 110, which includes a layer 112 defining a well 114. The treated nucleic acid particles 108 can be deposited in the well 114. Subsequently, the treated nucleic acid particles 108 can be washed while being retained in the well 114. Washing can produce nucleic acid particles with an increased diameter. The nucleic acid beads or particles can be used for sequencing or other experiments while being retained in the well 114.

[0022] In one example, the nucleic acid beads or particles 102 include a polymer matrix 104 and one or more nucleic acid chains 106 coupled to the polymer matrix 104. The polymer matrix 104 can be formed of a hydrophobic polymer or can be formed of a hydrophilic polymer (such as a hydrogel matrix). Specifically, the polymer matrix 104 can include a polymer, such as a polysaccharide such as agarose, hyaluronic acid or methylcellulose; a polyoxyalkylene such as polyoxybutylene, polyoxyethylene or polyethylene glycol, or polyoxypropylene; an acrylamide such as dimethylacrylamide, polyacrylamide, N,N-polydimethylacrylamide, poly(N-isopropylacrylamide), poly-N-hydroxyacrylamide, poly-N-hydroxyalkylacrylamide or an amine-functional variant thereof; polyvinylpyrrolidone; polystyrene; silicone; poly(2-acrylamido-2-methyl-1-propanesulfonic acid); other acrylate polymers; polyvinyl alcohol; a copolymer or derivative thereof; or any combination thereof. In a specific example, the polymer matrix 104 can be formed of polystyrene. In another example, the polymer matrix 104 is formed of polyethylene oxide. In another example, the polymer matrix 104 is formed of acrylamide (eg, hydroxyalkyl acrylamide, amine-terminated acrylamide, or derivatives thereof).

[0023] For example, the polymer matrix 104 can be formed by monomers, which include free radical polymerizable monomers such as vinyl monomers. In one example, the monomers can include acrylamide, vinyl acetate, hydroxyalkyl methacrylate or any combination thereof. In a specific example, the hydrophilic monomer is acrylamide, such as acrylamide including a hydroxyl terminal group, an amino terminal group, a carboxyl terminal group or a combination thereof. In one example, the hydrophilic monomer is aminoalkyl acrylamide, acrylamide functionalized with amine-terminated polypropylene glycol (D, shown below), acrylopiperazine (C, shown below) or a combination thereof. In another example, acrylamide can be hydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. Specifically, hydroxyalkyl acrylamide can include N-tris (hydroxymethyl) methyl) acrylamide (A, shown below), N- (hydroxymethyl) acrylamide (B, shown below) or a combination thereof. In another example, a mixture of monomers can be used, such as a mixture of hydroxyalkyl acrylamide and amine functionalized acrylamide or a mixture of acrylamide and amine functionalized acrylamide. In one example, the amine functionalized acrylamide can include a hydroxyalkyl acrylamide:amine functionalized acrylamide ratio or acrylamide:amine functionalized acrylamide ratio in the range of 100:1 to 1:1, such as in the range of 100:1 to 2:1, in the range of 50:1 to 3:1, in the range of 50:1 to 5:1, or even in the range of 50:1 to 10:1.

[0024]

[0025] In one particular example, the polymer matrix 104 is a hydrogel bead substrate.

[0026] The polymer matrix 104 may include a coupling site that can hybridize the template polynucleotide. The coupling site may include a terminal group, such as a hydroxyl or amino terminal group. For example, the coupling site may each include a coupled oligonucleotide that is partially complementary to the template polynucleotide. The template polynucleotide may include a target polynucleotide or a fragment that is complementary to the target polynucleotide, in addition to a fragment that is complementary to the coupled oligonucleotide.

[0027] The coupled oligonucleotide can be bound to the polymer matrix 104. The polymer of the polymer matrix 104 can be activated to promote the binding with the target analyte (such as oligonucleotide or polynucleotide). For example, the functional group on the polymer matrix 104 can be enhanced to allow binding with the target analyte or analyte receptor. In a specific example, the functional group of the hydrophilic polymer can be modified by a reagent that can convert the hydrophilic polymer functional group into a reactive part that can undergo nucleophilic or electrophilic substitution. For example, the hydroxyl groups on the substrate can be activated by replacing at least a portion of the hydroxyl groups with a sulfonate group or chlorine. Exemplary sulfonate groups can be derived from trifluoroethane sulfonyl chloride (tresyl chloride), methanesulfonyl chloride, toluenesulfonyl chloride or p-toluenesulfonyl chloride (fosyl chloride) or any combination thereof. Sulfonate can be used to replace sulfonate with a nucleophilic reagent. Sulfonate can further react with the released chlorine to provide a chlorinated functional group, which can be used in the process of combining particles. In another example, the amine groups on the substrate can be activated.

[0028] For example, the target analyte or analyte receptor can be bound to the hydrophilic polymer by nucleophilic substitution with sulfonate groups. In one specific example, the target analyte receptor capped with a nucleophile such as an amine or thiol can undergo nucleophilic substitution to displace the sulfonate groups on the surface of the polymer matrix 104.

[0029] In another example, the sulfonated polymer matrix can be further reacted with a monofunctional or polyfunctional mono- or poly-nucleophilic reagent (such as maleimide) which can form an attachment to the particle while maintaining nucleophilic activity toward oligonucleotides containing electrophilic groups, such as maleimide). Additionally, residual nucleophilic activity can be converted to electrophilic activity by attaching to a reagent containing poly-electrophilic groups, which are then attached to oligonucleotides containing nucleophilic groups.

[0030] In another example, monomers containing functional groups can be added during polymerization. The monomers can include, for example, acrylamides containing carboxylic acid, ester, halogen or other amine reactive groups. The ester groups can be hydrolyzed prior to reaction with the amine-terminated oligonucleotides.

[0031] Other conjugation techniques include the use of monomers containing amines. Amine groups are nucleophilic groups that can be further modified with amine-reactive bifunctional di-electrophiles, which generate monofunctional electrophilic groups after attachment to beads or particles. Such electrophilic groups can react with oligonucleotides having nucleophilic groups (e.g., amines or thiols), resulting in attachment of the oligonucleotide by reaction with a vacant electrophile.

[0032] If the polymer matrix is ​​prepared from a combination of amino-acrylamide and hydroxy-acrylamide, then the substrate may include a combination of nucleophilic amino groups and neutral hydroxyl groups. The amino groups may be modified with difunctional di-electrophilic moieties such as di-isocyanates or bis-NHS esters to produce hydrophilic particles reactive toward nucleophiles. Exemplary bis-NHS esters include bis-succinimidyl C2-C12 alkyl esters such as bis-succinimidyl suberate or bis-succinimidyl glutarate.

[0033] Other activation chemical reactions include incorporating multiple steps of converting designated functional groups to supply specific required bonds. For example, sulfonate-modified hydroxyl groups can be converted into nucleophilic groups by several methods. In one example, sulfonate reacts with azide anions to produce azide-substituted hydrophilic polymers. Azide can be directly used to combine with acetylene-substituted biomolecules by "CLICK" chemical methods, which can be carried out with or without copper catalysts. Optionally, azide can be converted into amines by, for example, hydrogen catalytic reduction or reduction with organic phosphine. Then, the resulting amine can be converted into an electrophilic group with a variety of reagents (such as diisocyanates, bis-NHS esters, cyanuric chloride or a combination thereof). In one example, a urea bond between a polymer and a linking group is produced using diisocyanates, which produces a residual isocyanate group, which can react with amino-substituted biomolecules to produce a urea bond between a linking group and a biomolecule. In another example, bis-NHS esters are used to generate an amide bond between the polymer and the linking group and a residual NHS ester group, which can react with an amino-substituted biomolecule to generate an amide bond between the linking group and the biomolecule. In another example, cyanuric chloride is used to generate an amino-triazine bond between the polymer and the linking group and two residual chloro-triazine groups, one of which can react with an amino-substituted biomolecule to generate an amino-triazine bond between the linking group and the biomolecule. Other nucleophilic groups can be incorporated into the particles through sulfonate activation. For example, the reaction of sulfonated particles with thiobenzoate anions and subsequent hydrolysis of thiobenzoate esters incorporates thiols into the particles, which then react with maleimide-substituted biomolecules to generate sulfanyl-succinimide bonds with biomolecules. Thiols can also react with bromo-acetyl groups.

[0034] Alternatively, acryloyl oligonucleotides may be used to incorporate the oligonucleotide during polymerization.Exemplary acryloyl oligonucleotides may include ion exchange oligonucleotides.

[0035] The diameter of the polymer matrix 104 may be in the range of 0.1 μm to 15 μm. For example, the diameter of the polymer matrix 104 may be in the range of 0.1 μm to 10 μm, such as in the range of 0.1 μm to 5.0 μm, in the range of 0.1 μm to 3.0 μm, or in the range of 0.1 μm to 1 μm. In a specific example, the diameter of the core 104 may be in the range of 0.1 μm to 0.8 μm, such as in the range of 0.1 μm to 0.5 μm.

[0036] The length of nucleic acid chain 106 may be at least 50 bases. For example, the length of nucleic acid chain may be at least 100 bases. In a specific example, the length of nucleic acid chain may be between 100 bases and 10,000 bases, such as between 100 bases and 8,000 bases, between 100 bases and 5,000 bases, between 100 bases and 1,000 bases, or between 150 bases and 500 bases. Nucleic acid beads or particles 102 may include more than one nucleic acid chain bound to polymer matrix 104. In a specific example, the nucleic acid chains on nucleic acid particles are the same. For example, particles may include at least 100, such as at least 1000 or even at least 10,000 identical copies of nucleic acid chains. Specifically, nucleic acid particles 102 may include at least 100,000 copies of nucleic acid chain 106, such as at least 1,000,000 copies of nucleic acid chains. In one example, the nucleic acid particle 102 includes no more than 100,000,000 copies of a nucleic acid strand.

[0037] When mixed with a solution including a coagulant, the nucleic acid strands or polymer matrix coagulate. In one example, the coagulant includes a metal complex. The metal complex may have 3 + Charge and can be provided to the solution as a salt. For example, the metal complex salt can be a halogen salt, such as a chloride salt or an iodide salt. Specifically, the metal complex includes cobalt, for example, forming a cobalt organic complex, such as a cobalt-ammine complex. In one example, the metal complex can include hexaammine cobalt. In another example, the metal complex includes tri(ethylenediamine)cobalt. In another example, the metal complex includes 1,3,6,8,10,13,16,19-octaazabicyclo-[6.6.6]eicosanecobalt.

[0038] Specifically, the solution may include a concentration of metal complex such that when mixed with the sample, the concentration of the metal complex in the resulting solution is at least 1 μM, such as at least 10 μM, at least 20 μM, or at least 50 μM. In one example, the concentration may be in the range of 10 μM to 1 μM, such as in the range of 20 μM to 500 μM or even in the range of 20 μM to 350 μM. In an alternative example, the concentration is at least 200 μM, such as at least 500 μM or at least 1 mM. For example, the concentration may be at least 4.7 mM, such as at least 9.8 mM, at least 14.9 mM, or even at least 19.7 mM. Specifically, the concentration is not greater than 100 mM, such as not greater than 50 mM. When expressed relative to the concentration of DNA, where the concentration of DNA is expressed in μg / ml, the ratio of the concentration of metal complex to the concentration of DNA is at least 0.5, such as at least 1.0, at least 1.5, at least 2.0 or even at least 3.0, but not greater than 100.

[0039] Alternatively or additionally, the solution may include a coagulant that affects the density of the polymer matrix 104 of the beads or particles 102. For example, the solution may include an alcohol, such as methanol, ethanol, or isopropyl alcohol (IPA), which may affect the density of the polymer matrix 104 of the particles 102. Specifically, the solution may include an alcohol (such as methanol) in a concentration ranging from 0.1 vol.% to 60 vol.%, such as 0.1 vol.% to 50 vol.%, 0.1 vol.% to 30 vol.%, 0.1 vol.% to 20 vol.%, or even 0.1 vol.% to 10 vol.%. For example, the concentration of the alcohol in the solution may be in the range of 0.1 vol.% to 5 vol.%, such as 1 vol.% to 5 vol.%. In another example, the solution can include an alcohol (such as ethanol or IPA) in a concentration range of 0.1 vol.% to 60 vol.%, such as 1.0 vol.% to 60 vol.%, 5.0 vol.% to 60 vol.%, 10 vol.% to 60 vol.%, or even 40 vol.% to 60 vol.%.

[0040] In another example, the coagulant includes a concentrated alkali metal salt or alkaline earth metal salt, such as a halide salt. In additional examples, the coagulant may include, for example, magnesium chloride at a concentration in the range of 1 mM to 1 M, such as in the range of 30 mM to 1 M, in the range of 50 mM to 1 M, in the range of 50 mM to 800 mM, or even in the range of 50 mM to 500 mM.

[0041] The coagulant may also be a nonionic polymer, such as a polyethylene glycol polymer. In a specific example, the molecular weight of the polyethylene glycol polymer is in the range of between 1000 and 100000, such as in the range of between 2000 and 20000, between 5000 and 15000, or between 8000 and 12000. The nonionic polymer may be included in a concentration in the range of 0.1 wt% to 20.0 wt%, such as in the range of 0.5 wt% to 15.0 wt%, in the range of 0.5 wt% to 10 wt%, or in the range of 2.5 wt% to 7.5 wt%.

[0042] The remainder of the solution may include a buffer solution, such as a buffered saline solution. For example, the remainder of the solution may include a phosphate buffered saline solution. Specifically, the solution may include a sodium halide or a potassium halide, a sodium phosphate or a potassium phosphate and a polysorbate. Such salts may be included, for example, in an amount in the range of 1mM to 500mM, such as 50mM to 350mM or even 150mM to 250mM. In one example, potassium chloride may be included in a concentration in the range of 0.5M to 2M, such as 0.8M to 1.5M or even 0.8M to 1.2M. In addition or alternatively, other buffers may be used, such as amine buffers, such as tris (hydroxymethyl) aminomethane. Such amine buffers may be used in a concentration in the range of 50mM to 1M, such as 100mM to 1M, 100mM to 800mM or even 150mM to 500mM. Optionally, the solution may include other ionic components such as calcium or magnesium from salts. In addition, the pH of the solution may be between 6 and 9, such as between 6.5 and 8.5 or between 7 and 8.5.

[0043] The solution may include a surfactant. For example, the surfactant may be a nonionic polymeric surfactant, such as an ether of polyethylene glycol, for example, octylphenyl ether of polyethylene glycol. The nonionic polymeric surfactant may be included in a range of 0.01% to 1.0%, such as a range of 0.05% to 0.8%, a range of 0.05% to 0.5%, or even a range of 0.08% to 0.15%. An exemplary surfactant is TritonX-100.

[0044] In one example, the coagulation solution includes a coagulation agent such as MgCl in a range of 30 mM to 500 mM. 2 ; Salt, such as KCl in the range of 0.8M to 1M; Buffer, such as tris(hydroxymethyl)aminomethane in the range of 150mM to 500mM; and Surfactant, such as TritonX-100 in the range of 0.05% to 0.5%. The pH is in the range of 7 to 9.

[0045] In another example, the coagulation solution includes a combination of coagulation agents, such as MgCl in the range of 30 mM to 500 mM. 2 and polyethylene glycol in the range of 0.5 wt% to 10.0 wt%. The molecular weight of the polyethylene glycol may be in the range of 5000 to 15000. The coagulation solution may also include a salt, such as KCl in the range of 0.8 M to 1 M; a buffer, such as tris (hydroxymethyl) aminomethane in the range of 150 mM to 500 mM; or a surfactant, such as Triton X-100 in the range of 0.05% to 0.5%. The pH is in the range of 7 to 9.

[0046] In response to exposure to the solution, the diameter of the nucleic acid bead or particle 102 including the nucleic acid strand 106 can decrease. For example, in response to exposure to the solution, the diameter of the bead or particle can decrease by at least 1%. In one example, the diameter decreases by at least 5%, such as at least 10%, at least 15%, or even at least 19%. In a specific example, the diameter decreases by no more than 75%. In addition, in response to exposure to the coagulant, the density of the bead or particle can increase. For example, the density can increase by at least 2%, such as at least 8%, at least 14%, at least 21%, or even at least 25%. Specifically, the density increases by no more than 75%.

[0047] Although nucleic acid strands 106 or polymer matrix 104 condense in response to exposure to a condensation solution or condensation agent, the nucleic acid strands after exposure to the solution may exhibit a similar enzyme dissociation constant when compared to the constant value of the enzyme dissociation constant of the nucleic acid strands prior to exposure. For example, the nucleic acid strands after exposure may exhibit a similar dissociation constant when in the presence of a polymerase when compared to the nucleic acid strands prior to exposure.

[0048] Return to Figure 1 , after exposure to the solution, the treated nucleic acid particles 108 can be applied to a surface component 116 of a sensor substrate, such as a sequencing device. For example, a suspension including the treated nucleic acid particles 108 can be applied to the surface of the surface component 116, such as by flowing the suspension over the surface, by foaming the suspension and applying the foam across the surface, or by applying the suspension and centrifuging the component 116. In a particular example, the surface includes areas where the particles are fixed, fixed, or bonded. For example, the surface can include treated areas that respond to a surface agent on a bead or particle. Specifically, the surface component 116 can include a pattern of treated areas, such as a pattern of metal deposition.

[0049] In another example, the surface component 116 includes a layer 112 defining a recess 114, into which the processed nucleic acid beads or particles 108 can be deposited. Alternatively, the surface component 116 can include a dispersed portion, such as a pit, groove, channel, depression or other recessed portion. In a specific example, the surface component 116 can define a recess 114 corresponding to an active circuit for measuring ion concentrations such as pH. Alternatively, the active circuit can measure heat, fluorescence or phosphate concentrations. In another example, the recess 114 can be deposited on a transparent layer of the surface component 116 for receiving or transmitting electromagnetic radiation or fluorescent emission.

[0050] The effective diameter of the recessed hole 114 may be no greater than 10 μm, such as no greater than 7 μm, no greater than 4 μm, no greater than 3 μm, no greater than 2 μm, no greater than 1.5 μm, no greater than 1.0 μm or even no greater than 0.8 μm. The effective diameter is the square root of the result of four times the cross-sectional area divided by π (pi) (i.e., d 有效 =sqrt(4A / π)). Specifically, the effective diameter may be no greater than 1.0 μm or no greater than 0.8 μm. The effective diameter may be at least 0.3 μm. In addition, the depth of the recessed hole 114 may be no greater than 10 μm, such as no greater than 5 μm, no greater than 3 μm, no greater than 1.5 μm, or even no greater than 1 μm. For example, the depth may be no greater than 500 nm, such as no greater than 200 nm or even no greater than 150 nm. The depth may be at least 100 nm.

[0051] The well 114 may define a volume of no greater than 1 microliter, such as no greater than 100 nanoliters, no greater than 10 nanoliters, or no greater than 1 nanoliter. For example, the well may define a volume of no greater than 100 picoliters, such as no greater than 10 picoliters, or even no greater than 1 picoliter. Specifically, the well may define a volume of no greater than 100 femtoliters, such as no greater than 50 femtoliters, such as no greater than 20 femtoliters, no greater than 10 femtoliters, no greater than 5 femtoliters, no greater than 1 femtoliter, or even no greater than 0.6 femtoliters, but may be at least 0.01 femtoliters.

[0052] After the particles are deposited on the surface, the surface component 116 can be placed in a sequencing system. Optionally, the surface component 116 can be washed to remove the coagulant before placing it in the sequencing system or can be washed once placed in the sequencing system. In response, the diameter of the nucleic acid beads or particles can increase or the nucleic acid chains can unfold from the coagulated state. For example, the beads or particles can be washed with a buffer solution (such as a buffered saline solution). Specifically, the solution can be a solution similar to the solution containing the coagulant, a solution without the coagulant.

[0053] Alternatively, the nucleic acid beads may be loaded prior to exposure to the coagulant. For example, the nucleic acid beads may be applied to the sensor substrate and a solution comprising the coagulant may be applied over the deposited nucleic acid beads. In another alternative example, the nucleic acid beads may be treated with a coagulant prior to deposition, deposited, washed, and treated with a coagulant after being deposited.

[0054] Once placed in a sequencing system, the nucleic acid chains attached to the particles deposited on the surface components can be sequenced. In one example, sequencing can include measuring the ion concentration in response to the addition of nucleotides. In this example, a solution comprising a single type of nucleotide is contacted with the nucleic acid chain and the sequencing system monitors the change in ion concentration, such as the change in pH. Subsequently, a solution with different types of nucleotides can be applied to the surface and the ion concentration is monitored again. In this way, the addition of a single nucleotide can be monitored and detected, so as to determine which nucleotide to attach and in what order to attach. In an alternative example, sequencing can include measuring the fluorescent emission in response to the addition of nucleotides. For each nucleotide or for nucleotides with different fluorescent substances, the different fluorescent substances fluoresce at different wavelengths, which is related to each type of nucleotide, and nucleotides can be continuously supplied in a separate solution, and nucleotides can be supplied to the sequencing system in a combined solution.

[0055] This approach can be implemented in systems such as Figure 2 . For example, the system 200 may include a solution container 202. In addition, the system 200 includes a sample port or sample receiving chamber 204. Both the solution container 202 and the sample port or receiving chamber 204 are fed into a mixer 206. The mixer 206 may be a stirring mixer, an ultrasonic mixer, or an in-line mixer in a tube extending to a surface preparation unit 208. Specifically, the mixing device 206 feeds the processed particles received at the sample port or chamber 204 to the surface part to be prepared for use by the sequencing device. Alternatively, the solution container 202 and the sample port or receiving chamber 204 may be fed into the surface preparation unit 208 without entering the mixer or being mixed.

[0056] The surface preparation unit 208 may include a chamber in which the sensor substrate forms a major surface and a solution including a coagulant and nucleic acid beads or particles including nucleic acid chains flow through the chamber over the surface to promote the deposition of beads or particles on the sensor substrate. In a specific example, the sensor substrate may include a flow channel defined on the sensor surface including a concave hole. The surface preparation unit may also include an agitator, an aspirator, a centrifuge, a pipette or a vortexer to further promote the deposition of beads or particles into the concave holes of the sensor substrate. Alternatively, the process may be performed manually using the above-mentioned equipment.

[0057] As a result of the method, particles may be deposited on the surface feature to a desired surface density. For example, such a method may provide a surface feature having an occupancy of at least 60%, such as at least 80%, at least 85%, at least 87% or even at least 89%, the occupancy being defined as the percentage of recesses that include DNA-containing particles. In particular, the occupancy may be at least 90%, such as at least 93% or even at least 95%.

[0058] It is generally believed that the increase in density and decrease in diameter facilitates the deposition of particles on the surface. After deposition, the particles can be washed in the surface preparation device 208 or once placed in the sequencing system.

[0059] Examples

[0060] Example 1

[0061] Particles formed from polymer particles containing DNA (ION Spheres after PCR amplification) TM ) were exposed to hexaaminecobalt in a concentration range of 300 μM to 20 mM. Figure 3 The change in particle diameter in response to the change in the concentration of hexaaminecobalt is shown. Initially, the particles are in phosphate buffered saline with Tween-20 (PBST) solution (e.g., including 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24g KH 2 PO 4 The particles were then washed with PBST solution and the diameter of the particles increased.

[0062] The DNA-containing particles were tested as prepared or in contact with a polymerase. Figure 4 As shown in , the diameter of the DNA-containing particles decreases as the concentration of hexaaminecobalt increases. Figure 5 As shown in , when in contact with the enzyme, the diameter increases. When the particles in contact with the enzyme are exposed to hexaaminecobalt (5 mM Co-complex), the diameter decreases and then increases when washed with a buffer solution (PBST).

[0063] Figure 6 , Figure 7 , Figure 8 The response of DNA-containing particles formed using a nucleic acid library to contact with different enzymes is shown. In each case, the diameter of the DNA-containing particles increases when contacted with the enzyme, and then the diameter of the DNA-containing particles decreases in response to increasing concentrations of hexaaminecobalt. In addition, the diameter of the particles increases when washed with a buffer solution for the first and second time and when contacted with nucleic acid phosphate.

[0064] The test involves FAM-labeling of the particles with a hairpin structure to measure the dissociation kinetics from the enzyme Bst1. Fig. 9 As shown in , the dissociation kinetics as a function of ionic strength are similar for samples exposed to hexaaminecobalt and samples not exposed to hexaaminecobalt.

[0065] DNA-containing particles having an average core diameter of 0.7 microns were provided in both a suspension without a metal complex and a suspension comprising hexaamine cobalt. A substrate defining recessed pores having an effective diameter of less than 1 micron was exposed to one of the two suspensions. The substrate was washed with a PBST solution and observed to determine how many recessed pores were occupied by the DNA-containing particles. Those substrates exposed to the suspension comprising DNA-containing particles without a metal complex exhibited an occupancy of less than 60%, which was defined as the percentage of recessed pores comprising DNA-containing particles. Those substrates exposed to the suspension comprising DNA-containing particles and hexaamine cobalt exhibited an occupancy of greater than 85%, with some substrates exhibiting an occupancy of greater than 90%.

[0066] Example 2

[0067] The aqueous coagulation reagent may include 1M KCl, 230mM MgCl 2 , 200 mM tris-HCl and 0.1% Triton X-100. The pH of the reagent may be about 8.0. When the polyacrylamide beads (obtained from Ion Torrent TM ) loaded into Priton I TM Chip (obtained from ION Torrent TM ) When a coagulant is used during the above step, magnesium salts have been found to promote the loading of beads into the recesses. While not limiting the solution to a particular theory, it is generally believed that the solution causes coagulation of the hydrogel polymer matrix.

[0068] Example 3

[0069] Aqueous coagulation reagent including coagulation agent 100 mM MgCl 2 and 5 wt% polyethylene glycol. The average molecular weight of polyethylene glycol is 10000. The coagulation solution also includes 0.7 M KCl and 200 mM tris(hydroxymethyl)aminomethane. The pH is 8.0.

[0070] Example 4

[0071] Hydrogel beads (ION Spheres from ION Torrent) were treated with a dye (SBYR). TM), and microscopy was used to observe the hydrogel beads that responded to coagulants including magnesium and polyethylene glycol (PEG). The bead size is expressed as a ratio relative to the bead size in low ionic strength saline solution.

[0072] Fig.10 The response of bead size to different concentrations of magnesium in the presence of 5% PEG is shown. As shown, the relative size initially decreases rapidly with increasing magnesium concentration. As magnesium concentration increases, the rate of decrease slows.

[0073] Fig.11 Shown in 100 mM MgCl 2 or 50 mM MgCl 2 Response of bead size to different concentrations of PEG in the presence of PEG. As shown, the relative size decreases with increasing PEG concentration. Polyethylene glycol can create an osmotic pressure difference between the hydrogel beads and the external solution, causing water to leave the beads. PEG may not enter the beads, but may remain outside the beads, resulting in an osmotic pressure difference.

[0074] In a first aspect, a method of sequencing a nucleic acid strand comprises receiving a particle having a polymer matrix bound to the nucleic acid strand, exposing the particle to a solution comprising an agglomerating agent, and applying the particle to a surface, the particle being deposited on the surface.

[0075] In an example of the first aspect, the coagulant includes a metal complex having a 3+ charge. For example, the metal complex may include cobalt. In another example of the first aspect and the above examples, the metal complex is a metal-amine complex. For example, the metal complex may include hexaamine cobalt. In another example, the metal complex includes tri(ethylenediamine)cobalt. In another example, the metal complex includes 1,3,6,8,10,13,16,19-octaazabicyclo-[6.6.6]cobalt eicosane (cobaltsepulchrate). In an additional example, the metal complex is derived from a chloride salt. In additional examples of the first aspect and the above examples, the solution includes a metal complex at a concentration of at least 1 μM. For example, the concentration may be at least 10 μM, such as at least 1 mM, at least 4.7 mM or even at least 14.9 mM. Specifically, the concentration is not greater than 100 mM, such as not greater than 50 mM.

[0076] In another example of the first aspect and the above examples, the surface includes a layer defining wells and wherein applying the particles to the surface includes applying the particles into the wells.

[0077] In another example of the first aspect and the above examples, the coagulant includes a nonionic polymer. For example, the nonionic polymer includes a polyethylene glycol polymer. The molecular weight of the polyethylene glycol polymer may be in the range of 2000 to 20000. In one example, the nonionic polymer surfactant is included in a concentration of 0.1 wt% to 10.0 wt%.

[0078] In another example, the coagulant includes an alkali metal salt or an alkaline earth metal salt. For example, the coagulant includes a potassium salt at a concentration of 0.5 M to 2.0 M. In another example, the coagulant includes a magnesium salt at a concentration of 30 mM to 1.0 M.

[0079] In additional examples of the first aspect and the above examples, the coagulant includes an alkali metal salt or an alkaline earth metal salt and a nonionic polymer surfactant.

[0080] In another example of the first aspect and the above examples, in response to exposure, the diameter of the particle decreases by at least 1%. For example, the diameter can decrease by at least 5%, such as at least 10%, at least 15%, or at least 19%. Specifically, the diameter can decrease by no more than 75%.

[0081] In another example of the first aspect and the above examples, in response to exposure, the density of the particles increases by at least 2%. For example, the density may increase by at least 8%, such as at least 14% or at least 21%. Specifically, the density may increase by no more than 75%.

[0082] In additional examples of the first aspect and the above examples, after exposure the nucleic acid strands exhibit dissociation similar to that of the nucleic acid strands before exposure.

[0083] In another example of the first aspect and the above examples, the polymer matrix includes a polymer including agarose, polyoxybutylene, dimethylacrylamide, polyoxyethylene or polyethylene glycol, polyacrylamide, polyoxypropylene, N,N-polydimethylacrylamide, poly(N-isopropylacrylamide), polyvinylpyrrolidone, poly-N-hydroxyacrylamide, poly-N-hydroxyalkylacrylamide, polystyrene, copolymers or derivatives thereof, or any combination thereof.

[0084] In additional examples of the first aspect and the above examples, the method further includes washing the surface to remove the coagulant.

[0085] In another example of the first aspect and the above examples, the method further comprises sequencing the nucleic acid strand. For example, sequencing may comprise determining ion concentration in response to nucleotide addition. In another example, sequencing may comprise determining radiation emission in response to nucleotide addition.

[0086] In a second aspect, a method of preparing a surface comprises applying a solution to the surface, the solution comprising particles and a coagulant, the particles being deposited on the surface. The method further comprises washing the surface with a solution free of metal complexes.

[0087] In a third aspect, a device includes a solution container including a solution containing an agglutinating agent, a sample receiving port for receiving particles including nucleic acid strands, and a mixer in fluid communication with the solution container and the sample receiving port.

[0088] In an example of the third aspect, the device further comprises a surface preparation unit for preparing a surface component of the sequencing device and for receiving the processed particles from the mixer.

[0089] In a fourth aspect, the aqueous reagent solution comprises a magnesium salt in the range of 30 mM to 500 mM; a potassium salt in the range of 0.8 M to 1.0 M; a buffer in the range of 150 mM to 500 mM; and a surfactant in the range of 0.05% to 0.5%.

[0090] In one example of the fourth aspect, the pH is in the range of 7 to 9. In another example of the fourth aspect and the above examples, the buffer comprises tris(hydroxymethyl)aminomethane. In another example of the fourth aspect and the above examples, the surfactant comprises a polyethylene glycol-based surfactant.

[0091] In a fifth aspect, the aqueous reagent solution comprises a magnesium salt in the range of 30 mM to 500 mM; polyethylene glycol in the range of 0.5 wt% to 10.0 wt%; a potassium salt in the range of 0.8 M to 1.0 M; and a buffer in the range of 150 mM to 500 mM.

[0092] In one example of the fifth aspect, the pH is in the range of 7 to 9. In another example of the fifth aspect and the above examples, the buffer comprises tris(hydroxymethyl)aminomethane.

[0093] It should be noted that not all activities described in the general description or examples above are required, a portion of a specific activity may not be required, and one or more other activities in addition to those described may be performed. Furthermore, the order in which the activities are listed is not necessarily the order in which they are performed.

[0094] In the foregoing description, the concepts have been described with reference to specific embodiments. However, it will be appreciated by those skilled in the art that various modifications and variations may be made without departing from the scope of the invention as set forth in the claims. Accordingly, the description and drawings are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.

[0095] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited to only those features, but may include other features not expressly listed or other features inherent to the process, method, article, or apparatus. In addition, unless expressly specified to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, condition A or condition B is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0096] In addition, the use of "a" or "an" is used to describe the elements and components described herein. This is done only for convenience and to give a general sense of the scope of the present invention. Unless otherwise clearly intended, this description should be understood to include one or at least one, and the singular also includes the plural.

[0097] Benefits, other advantages, and solutions to problems have been described above with respect to specific embodiments. However, these benefits, advantages, solutions to problems, and any (one or more) features that may make any benefit, advantage, or solution occur or become more significant should not be construed as important, necessary, or required features of any or all claims.

[0098] After reading this specification, those skilled in the art will appreciate that, for clarity, certain features described herein in the context of independent embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features described in the context of a single embodiment may also be provided individually or in any sub-combination. In addition, numerical values ​​expressed as ranges include each or every value within the range.

Claims

1. A method for sequencing a nucleic acid chain, the method comprising: receiving a particle having a polymer matrix bound to a nucleic acid strand, the polymer matrix comprising polyacrylamide, N,N-polydimethylacrylamide, poly(N-isopropylacrylamide), poly-N-hydroxyacrylamide, poly-N-hydroxyalkylacrylamide, copolymers or derivatives thereof, or any combination thereof; exposing the particles to a solution comprising a coagulant to reduce the size of the particles, the solution comprising a magnesium salt in a range of 50 mM to 1.0 M and a nonionic polymer in a range of 0.5 wt % to 10.0 wt %; applying the particles to a surface including a layer defining pores in which the particles are deposited; and The surface was washed with PBST solution to remove the aggregating agent and to spread the particles in the wells.

2. The method of claim 1, wherein the nonionic polymer comprises a polyethylene glycol-based polymer.

3. The method according to claim 2, wherein the molecular weight of the polyethylene glycol-based polymer is in the range of 2,000 to 20,000.

4. The method of any one of claims 1 to 3, wherein in response to the exposing, the diameter of the particles decreases by at least 1%.

5. The method of claim 4, wherein the diameter is reduced by at least 5%.

6. The method of claim 5, wherein the diameter is reduced by at least 10%.

7. The method of claim 6, wherein the diameter is reduced by at least 15%.

8. The method of claim 7, wherein the diameter is reduced by at least 19%.

9. The method of claim 8, wherein the diameter is reduced by no more than 75%.

10. The method of any one of claims 1 to 3, wherein in response to the exposing, the density of the particles increases by at least 2%.

11. The method of claim 10, wherein the density is increased by at least 8%.

12. The method of claim 11, wherein the density is increased by at least 14%.

13. The method of claim 12, wherein the density is increased by at least 21%.

14. The method of claim 13, wherein the density increase does not exceed 75%.

15. The method of any one of claims 1 to 3, wherein after exposure the nucleic acid strands exhibit dissociation similar to that of the nucleic acid strands before exposure.

16. The method according to any one of claims 1 to 3, further comprising: The nucleic acid strands are sequenced.

17. The method of claim 16, wherein sequencing comprises measuring ion concentration in response to nucleotide addition.

18. The method of claim 17, wherein sequencing comprises measuring radiation emission in response to nucleotide addition.

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