Method for aqueous conjugation of a substrate

Abstract

A method for conjugating oligonucleotides to a bead carrier includes adding a carbodiimide to an aqueous suspension comprising a bead carrier having a carboxyl functional group; adding a hydroxysuccinimide to the aqueous suspension; and adding an amine-modified oligonucleotide to the aqueous suspension.

Description

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 616,502, filed December 29, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] Polymer particles are increasingly being used as components in separation techniques and to assist in the detection of analytes in both chemical and biological systems. For example, polymer particles have been used in chromatography to separate target molecules from solution. In another example, polymer particles with magnetic coatings are used in magnetic separation techniques. Recently, polymer particles have been used to enhance ELISA-type techniques and can be used to capture proteins or polynucleotides.

[0004] In particular, polymer particles conjugated with oligonucleotides can be used to capture complementary polynucleotides. Furthermore, such particles can be used to form multiple copies of the captured polynucleotides and used in sequencing reactions.

[0005] However, conventional methods for conjugating oligonucleotides to polymer particles are inefficient and require stringent process conditions. Therefore, an improved method for conjugating polymer particles is desired. Attached Figure Description

[0006] This disclosure will be better understood by referring to the accompanying drawings, and many features and advantages of this disclosure will become apparent to those skilled in the art.

[0007] Figure 1 Illustrations of exemplary sequencing systems are included.

[0008] Figure 2 The illustration includes an exemplary system comprising a sensor array.

[0009] Figure 3 Illustration including an exemplary sensor and associated aperture.

[0010] Figure 4 Illustrations of exemplary procedures for forming nucleic acid vectors and loading such vectors onto a substrate for sequencing.

[0011] Figure 5 Illustration of another exemplary procedure for forming nucleic acid vectors and loading such vectors onto a substrate for sequencing.

[0012] Figure 6 and Figure 7 An exemplary diagram for preparing bead assemblies is shown.

[0013] Figure 8 and Figure 9 An exemplary diagram for preparing bead carriers is shown.

[0014] Using the same reference numerals in different figures indicates similar or identical items. Detailed Implementation

[0015] In the example, a method for conjugating polymer particles (such as carboxyl-functionalized polymer particles) includes activating the carboxyl functional group and reacting the activated carboxyl functional group with a modified oligonucleotide. For example, the polymer particle may be a carboxyl-functionalized polyacrylamide particle. Activating the carboxyl functional group may include reacting the carboxyl functional group with a carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) followed by a reaction with a hydroxysuccinimide (such as N-hydroxysuccinimide or N-hydroxysulfosuccinimide). The modified oligonucleotide may include a modified 5' end comprising an amine, diene, or another reactive substance. In particular, the modified oligonucleotide includes an amine at its 5' end. The reaction may be carried out in an aqueous solution.

[0016] These oligonucleotide-conjugated polymer particles are particularly suitable for sequencing. For example, Figure 1 The system for performing pH-based nucleic acid sequencing is illustrated in the diagram. Each electronic sensor in the device generates an output signal whose value depends on a reference voltage. A fluid loop allows for the delivery of multiple reagents to the reaction chamber.

[0017] exist Figure 1 In this system 100, which includes a fluid loop 102, inlets are connected to at least two reagent reservoirs (104, 106, 108, 110, or 112), to a waste reservoir 120, and to a biosensor 134 via a fluid path 132 that connects a fluid node 130 to an inlet 138 of the biosensor 134 for fluid communication. Reagents from the reservoirs (104, 106, 108, 110, or 112) can be driven into the fluid loop 102 by various methods, including pressure, pumps such as syringe pumps, gravity feed, etc., and selected by a control valve 114. Reagents from the fluid loop 102 can be driven into the waste container 120 via valve 114, which receives a signal from the control system 118. Reagents from the fluid loop 102 can also be driven into the waste container 136 via the biosensor 134. The control system 118 includes controllers for the valves, which generate signals for opening and closing via electrical connections 116.

[0018] The control system 118 also includes controllers for other components of the system, such as a wash solution valve 124 and a reference electrode 128 connected thereto via an electrical connector 122. The control system 118 may also include control and data acquisition functions for the biosensor 134. In one operating mode, the fluid loop 102, under programmed control of the control system 118, delivers a series of selected reagents 1, 2, 3, 4, or 5 to the biosensor 134, such that the fluid loop 102 is prepared and washed between the selected reagent flows, and the biosensor 134 is washed. The fluid entering the biosensor 134 exits through an outlet 140 and is deposited in a waste container 136 by control of a clamp valve regulator. The valve is in fluid communication with the sensor fluid output 140 of the biosensor 134.

[0019] Devices including those defining a dielectric layer that defines an aperture formed by a first inlet and a second inlet and exposes a sensor pad are particularly suitable for detecting chemical reactions and byproducts, such as detecting the release of hydrogen ions in response to nucleotide incorporation, for genetic sequencing and other applications. In a particular embodiment, the sequencing system includes a flow cell in which a sensor array is disposed, communication circuitry in electronic communication with the sensor array, and a container and fluid controller in fluid communication with the flow cell. In an example, Figure 2 An enlarged cross-sectional view of the flow cell 200 is shown, and a portion of the flow chamber 206 is also shown. A reagent stream 208 flows through the surface of the aperture array 202, specifically through the open ends of the apertures in the aperture array 202. The aperture array 202 and the sensor array 205 can together form an integrated unit that forms the lower wall (or base plate) of the flow cell 200. A reference electrode 204 can be fluidly coupled to the flow chamber 206. Further, a flow cell cover 230 encloses the flow chamber 206 to contain the reagent stream 208 within a defined area.

[0020] Figure 3 As shown Figure 2An extended view of the aperture 301 and sensor 314 shown at position 210. The volume, shape, aspect ratio (such as the ratio of substrate width to aperture depth), and other dimensional characteristics of the aperture can be selected based on the nature of the reaction occurring and the reagents, byproducts, or labeling techniques used (if any). Sensor 314 can be a chemical field-effect transistor (chemFET) with a floating gate 318, more specifically, an ion-sensitive FET (ISFET) having a sensor plate 320 optionally separated from the aperture interior by a passivation layer 316. Sensor 314 can respond to the amount of charge 324 present on the passivation layer 316 opposite the sensor plate 320 (and generate an output signal associated with said amount). Changes in charge 324 can cause changes in the current between the source 321 and drain 322 in the chemFET. Furthermore, the chemFET can be used directly to provide a current-based output signal or indirectly used with additional circuitry to provide a voltage-based output signal. Reactants, washing solutions, and other reagents can enter and exit the aperture via a diffusion mechanism 340.

[0021] In this embodiment, the reaction performed in well 301 can be an analytical reaction used to identify or determine the characteristics or properties of a target analyte. Such reactions can directly or indirectly generate byproducts that affect the charge level of adjacent sensor plate 320. If such byproducts are generated in small amounts, decompose rapidly, or react with other components, multiple copies of the same analyte can be analyzed simultaneously in well 301 to increase the generated output signal. In this embodiment, multiple copies of the analyte can be attached to a solid support 312 before or after deposition into well 301. The solid support 312 can be microparticles, nanoparticles, beads, a solid containing a gel, or a porous material, etc. For simplicity and ease of interpretation, the solid support 312 is also referred to herein as particles or beads. For nucleic acid analytes, multiple linked copies can be prepared using rolling circle amplification (RCA), exponential RCA, or similar techniques to generate amplicones without the need for a solid support.

[0022] The pore wall structure 310 can be formed of one or more layers of material. In the example, the pore wall structure 310 can have a thickness (t) extending from the lower surface to the upper surface, ranging from 0.3 micrometers to 10 micrometers, such as from 0.5 micrometers to 6 micrometers. The pore 301 can have a feature diameter not exceeding 5 micrometers, such as not exceeding 3.5 micrometers, not exceeding 2.0 micrometers, not exceeding 1.6 micrometers, not exceeding 1.0 micrometers, not exceeding 0.8 micrometers, or even not exceeding 0.6 micrometers, the feature diameter being defined as 4 times the cross-sectional area (A) divided by the square root of Pi (e.g., sqrt(4*A / π)). In the example, the feature diameter is at least 0.01 micrometers.

[0023] Figure 4This illustration includes an exemplary method for forming nucleic acid vectors and loading such vectors onto a substrate for sequencing. Multiple target nucleic acids 402 and vectors 404 are provided in solution. Some of the target nucleic acids bind to vector 406, while others remain target-free. The target nucleic acids may be derived from one or more DNA or RNA samples, such as genomic DNA from different subjects. Target nucleic acids from each sample can be combined and processed as described below. Alternatively, target nucleic acids from each sample can be processed individually.

[0024] The target nucleic acid immobilized to vector 406 is amplified to form multiple copies of the target nucleic acid on vector 414. Vector 408 remains free of target nucleic acid. Amplification may include polymerase chain reaction (PCR) amplification, such as emulsion PCR, or isothermal amplification in an emulsion or bulk solution, such as recombinase-polymerase amplification (RPA).

[0025] A capture primer having a capture portion and complementary to the ends of the target nucleic acid immobilized to the vector can be used to bind the vector 414 to magnetic beads 410, the magnetic beads having a portion that binds to the capture portion. In an example, the capture portion is, for example, biotin that binds to streptavidin. As shown, the vector 414 comprising the target nucleic acid is combined with the magnetic beads 410 to form a complex 412. A magnetic field can be used to immobilize the magnetic beads 410 and the vector 414 comprising the target nucleic acid while washing away any remaining solution, including a vector 408 that does not contain the target nucleic acid.

[0026] The carrier 414 containing the target nucleic acid can be separated from the magnetic beads 410 by denaturing the capture portion or dehybridizing it with the target nucleic acid. For example, changes in ion concentration or increases in temperature can cause the capture primers to be released from the target nucleic acid. A magnetic field can be used to separate the magnetic beads 410 from the carrier 414 containing the target nucleic acid.

[0027] A vector 414 comprising the target nucleic acid can be applied to a substrate such as substrate 416. In the example, the substrate includes a set of wells 418 in which the vector 414 is deposited. Sequencing can be performed using the vector comprising the target nucleic acid, for example, by sequencing-by-synthesis.

[0028] In this example, sequencing primers can be added to well 418, or the bead carrier 414 can be pre-exposed to primers before being placed in well 418. Specifically, the bead carrier 414 may include bound sequencing primers. The sequencing primers and polynucleotides form a nucleic acid duplex containing a polynucleotide (e.g., a template nucleic acid) that has hybridized with the sequencing primers. The nucleic acid duplex is a polynucleotide that is at least partially double-stranded. Enzymes and nucleotides can be provided to well 418 to facilitate detectable reactions, such as nucleotide incorporation.

[0029] Sequencing can be performed by detecting nucleotide addition. Nucleotide addition can be detected using methods such as fluorescence emission or ion detection. For example, a set of fluorescently labeled nucleotides can be provided to system 416 and migrate to well 418. Excitation energy can also be provided to well 418. When the nucleotides are captured by polymerase and added to the ends of the extension primers, the nucleotide labeling can fluoresce, indicating which type of nucleotide has been added.

[0030] In an alternative example, a solution containing a single type of nucleotide can be added sequentially. In response to the addition of the nucleotide, the pH within the local environment of pore 418 can change. This pH change can be detected by an ion-sensitive field-effect transistor (ISFET). Therefore, the pH change can be used to generate a signal indicating the sequence of nucleotides complementary to the polynucleotides of particle 410.

[0031] Specifically, the sequencing system may include one or more holes disposed on a sensor pad of an ion sensor, such as a field-effect transistor (FET). In various embodiments, the system includes one or more polymer particles loaded into holes disposed on a sensor pad of an ion sensor (e.g., a FET), or into multiple holes disposed on a sensor pad of an ion sensor (e.g., a FET). In various embodiments, the FET may be a chemFET or an ISFET. A “chemFET” or chemical field-effect transistor includes a field-effect transistor used as a chemical sensor. The structure of a chemFET is similar to that of a MOSFET transistor, in which a charge is applied to the gate electrode via a chemical process. An “ISFET” or ion-sensitive field-effect transistor can be used to measure the concentration of ions in a solution; when the ion concentration (such as H+) changes, the current through the transistor changes accordingly.

[0032] In implementations, the FET can be a FET array. As used herein, an "array" is a planar arrangement of elements such as sensors or holes. The array can be one-dimensional or two-dimensional. A one-dimensional array can be an array having one column (or row) of elements in a first dimension and multiple columns (or rows) in a second dimension. The number of columns (or rows) in the first and second dimensions can be the same or different. A FET or array can contain 10... 2 10 3 10 4 10 5 10 6 10 7 Or more FETs.

[0033] In various embodiments, one or more microfluidic structures may be fabricated above the FET sensor array to provide containment or confinement for biological or chemical reactions. For example, in one embodiment, the microfluidic structure may be configured to provide one or more orifices (or orifices, or reaction chambers, or reaction orifices, as terms used interchangeably herein) above one or more sensors of the array, such that one or more sensors having predetermined orifices thereon detect and measure the presence, level, or concentration of an analyte in the predetermined orifice. In various embodiments, a 1:1 correspondence may exist between the FET sensors and the reaction orifices.

[0034] Return to Figure 4 In another example, the aperture 418 in the aperture array can be operatively connected to a measuring device. For example, in fluorescence emission methods, the aperture 418 can be operatively connected to a photodetector. In the case of ion detection, the lower surface of the aperture 418 can be disposed on the sensor pad of an ion sensor, such as a field-effect transistor.

[0035] One example system involving sequencing by detecting ionic byproducts of nucleotide incorporation is the Ion TorrentPGM™, Proton™, or S5™ sequencer (Thermo Fisher Scientific), an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions generated as byproducts of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporation that occur during template-dependent nucleic acid synthesis using polymerases. The Ion TorrentPGM™, Proton™, or S5™ sequencer detects nucleotide incorporation by detecting hydrogen ion byproducts of nucleotide incorporation. The Ion Torrent PGM™, Proton™, or S5™ sequencer may contain multiple template polynucleotides to be sequenced, each template disposed in a separate sequencing reaction well in an array. Each well in the array is coupled to at least one ion sensor that can detect the release of H+ ions or changes in solution pH as byproducts of nucleotide incorporation. The ion sensor includes a field-effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. Ion sensors provide an output signal indicating nucleotide incorporation, which can be represented as a voltage change whose magnitude is correlated with the H+ ion concentration in the respective well or reaction chamber. Different nucleotide types can flow into the reaction chamber sequentially and can be incorporated into the extension primers (or polymerization sites) in a sequence determined by the template sequence via polymerase. Each nucleotide incorporation may be accompanied by the release of H+ ions in the reaction well, and a corresponding change in local pH. The release of H+ ions can be recorded by the sensor's FET, which generates a signal indicating that nucleotide incorporation has occurred. Nucleotides that are not incorporated during a particular nucleotide flow may not generate a signal. The amplitude of the signal from the FET can also be correlated with the number of a particular type of nucleotide incorporated into the extended nucleic acid molecule, thus allowing homopolymer regions to be resolved. Therefore, during sequencer operation, the simultaneous inflow of multiple nucleotides into the reaction chamber and the monitoring of incorporation across multiple wells or reaction chambers allows the instrument to simultaneously resolve sequences from many nucleic acid templates.

[0036] In particular, solid-phase supports such as bead carriers can contain copies of polynucleotides. Figure 5 In the specific example shown, the polymer particles can be used as carriers of polynucleotides during sequencing. For example, such hydrophilic particles can immobilize polynucleotides for sequencing using fluorescence sequencing technology. In another example, the hydrophilic particles can immobilize multiple copies of polynucleotides for sequencing using ion sensing technology. Alternatively, the treatments described above can improve the adhesion of the polymer matrix to the sensor array surface. The polymer matrix can capture analytes, such as polynucleotides for sequencing.

[0037] Bead carriers can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyvinyl chloride, and polyacrylamide, as well as copolymers and grafts thereof. The carrier can also be inorganic, such as glass, silica, controlled-pore glass (CPG), or inverted silica. The carrier configuration can be in the form of beads, spheres, particles, fine particles, gels, or surfaces. The carrier can be porous or non-porous and can have swelling or non-swelling characteristics. In some embodiments, the carrier is an ionic sphere particle. Examples of bead carriers are disclosed in US 9,243,085, entitled "Hydrophilic polymer particles and methods for their preparation and use," and US 9,868,826, entitled "Polymer substrates formed from carboxyl-functionalized acrylamides," each of which is incorporated herein by reference.

[0038] In some embodiments, the solid carrier is a “microparticle,” “bead,” “microbead,” etc. (optionally, but not necessarily spherical), having a minimum cross-sectional length (e.g., diameter) of 50 micrometers or less, preferably 10 micrometers or less, 3 micrometers or less, about 1 micrometer or less, about 0.5 micrometers or less, such as about 0.1 micrometers, 0.2 micrometers, 0.3 micrometers, or 0.4 micrometers or less (e.g., less than 1 nanometer, about 1 nanometer to 10 nanometers, about 10 nanometers to 100 nanometers, or about 100 nanometers to 500 nanometers). In the example, the carrier is at least 0.1 micrometers. The microparticle or bead carrier can be made of a variety of inorganic or organic materials, including but not limited to glass (e.g., controlled-pore glass), silica, zirconium oxide, cross-linked polystyrene, polyacrylate, polymethyl methacrylate, titanium dioxide, latex, polystyrene, etc. Magnetization can facilitate the collection and concentration of reagents (e.g., polynucleotides or ligases) for post-amplification microparticle ligation, and can also facilitate additional steps (e.g., washing, reagent removal, etc.). In some implementations, groups of particles with different shapes, sizes, or colors are used. The particles may optionally be encoded, for example, with quantum dots, so that each particle or group of particles can be identified individually or uniquely.

[0039] Magnetic beads (e.g., Dynabeads from Dynal in Oslo, Norway) can have sizes ranging from 1 micrometer to 100 micrometers, such as 2 micrometers to 100 micrometers. Magnetic beads can be formed from inorganic or organic materials, including, but not limited to, glass (e.g., controlled-pore glass), silica, zirconium oxide, cross-linked polystyrene, polystyrene, or combinations thereof.

[0040] In some embodiments, the bead carrier is functionalized to connect to a first primer group. In some embodiments, the beads are of any size that can be placed in the reaction chamber. For example, one bead may be placed in the reaction chamber. In some embodiments, more than one bead may be placed in the reaction chamber. In some embodiments, the minimum cross-sectional length (e.g., diameter) of the bead is about 50 micrometers or less, or about 10 micrometers or less, or about 3 micrometers or less, approximately 1 micrometer or less, approximately 0.5 micrometers or less, such as approximately 0.1 micrometers, 0.2 micrometers, 0.3 micrometers, or 0.4 micrometers or less (e.g., less than 1 nanometer, about 1 nanometer to 10 nanometers, about 10 nanometers to 100 nanometers, or about 100 nanometers to 500 nanometers).

[0041] Generally, bead carriers can be processed to contain biomolecules, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), peptides, sugars, polysaccharides, lipids, or their derivatives or analogues. For example, polymer particles can be bound to or linked to biomolecules. The ends or any internal portions of the biomolecule can be bound to or linked to the polymer particles. Using linker chemistry methods, polymer particles can be bound to or linked to biomolecules. Linker chemistry methods involve covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. Linkage chemistry involves the affinity between binding partners, such as the affinity between: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or its immunoreactive fragment; an antibody and a hapten; a digoxigenin moiety and an anti-digoxigenin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operon and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement.

[0042] like Figure 5 As shown, multiple bead carriers 504 and multiple polynucleotides 502 (target polynucleotides or template polynucleotides) can be placed together in a solution. The target polynucleotides or template polynucleotides can be derived from one or more DNA or RNA samples, such as genomic DNA from different subjects. The target polynucleotides or template polynucleotides from each sample can be combined and processed as described below. Alternatively, the target polynucleotides or template polynucleotides from each sample can be processed individually.

[0043] These bead carriers 504 can be activated or otherwise pretreated to enable them to bind to polynucleotide 502. For example, the bead carrier 504 may contain an oligonucleotide (capture primer) complementary to a portion of the polynucleotide in a plurality of polynucleotides 502. In another example, the bead carrier 504 is modified with a target polynucleotide 502 using a technique such as biotin-streptavidin binding.

[0044] In some embodiments, the template nucleic acid molecule (template polynucleotide or target polynucleotide) may be derived from a sample that may be from a natural or non-natural source. The nucleic acid molecules in the sample may be derived from a living organism or cell. Any nucleic acid molecule can be used; for example, the sample may contain genomic DNA covering part or all of the genome, mRNA, or miRNA from a living organism or cell. In other embodiments, the template nucleic acid molecule may be synthetic or recombinant. In some embodiments, the sample contains nucleic acid molecules having substantially identical sequences or a mixture of different sequences. Illustrative embodiments are typically implemented using nucleic acid molecules generated within and from living cells. Such nucleic acid molecules are typically isolated directly from natural sources, such as cells or body fluids, without any in vitro amplification. Therefore, the sample nucleic acid molecules are used directly in subsequent steps. In some embodiments, the nucleic acid molecules in the sample may comprise two or more nucleic acid molecules with different sequences.

[0045] The method may optionally include a target enrichment step before, during, or after library preparation and prior to the pre-inoculation reaction. Target nucleic acid molecules containing target loci or regions of interest can be enriched, for example, by multiplex nucleic acid amplification or hybridization. Various methods can be used to perform multiplex nucleic acid amplification to generate amplicones, such as multiplex PCR, and these various methods can be used in the implementation scheme. A universal amplification reaction can be performed after enrichment by any method before adding the template nucleic acid molecule to the pre-inoculation reaction mixture. Any embodiment of the teachings of this invention may include enrichment of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 target nucleic acid molecules, target loci, or regions of interest. In any of the disclosed embodiments, the target locus or region of interest may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides long and comprises a portion or all of the template nucleic acid molecule. In other embodiments, the length of the target locus or region of interest may be between about 1 and 10,000 nucleotides, for example, between about 2 and 5,000 nucleotides, between about 2 and 3,000 nucleotides, or between about 2 and 2,000 nucleotides. In any embodiment of the teachings of this invention, multiplex nucleic acid amplification may comprise generating at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 copies of each target nucleic acid molecule, target locus, or region of interest.

[0046] In some embodiments, after library preparation and optional enrichment steps, the template nucleic acid molecule library can be templated onto one or more vectors. One or more vectors can be templated in two reactions: an inoculation reaction to generate a pre-inoculated solid vector and a templated reaction to further amplify the ligated template nucleic acid molecule using one or more pre-inoculated vectors. The pre-inoculation reaction is typically an amplification reaction and can be performed using various methods. For example, the pre-inoculation reaction can be performed in an RPA reaction, a template walking reaction, or PCR. In an RPA reaction, the template nucleic acid molecule is amplified using a recombinase, a polymerase, and optionally a recombinase helper protein in the presence of primers and nucleotides. The recombinase and optionally the recombinase helper protein dissociate at least a portion of the double-stranded template nucleic acid molecule to allow primer hybridization, after which the polymerase can bind to initiate replication. In some embodiments, the recombinase helper protein can be a single-stranded binding protein (SSB) that prevents rehybridization of the dissociated template nucleic acid molecule. Typically, the RPA reaction is performed at an isothermal temperature. In template walking reactions, a polymerase is used to amplify template nucleic acid molecules in the presence of primers and nucleotides under reaction conditions that allow at least a portion of the double-stranded template nucleic acid molecule to dissociate, enabling the primers to hybridize and allowing the polymerase to subsequently bind to initiate replication. In PCR, the double-stranded template nucleic acid molecule is dissociated by thermal cycling. After cooling, the primers bind to complementary sequences and can be used for replication by the polymerase. In any aspect of the invention, a pre-inoculation reaction can be performed in a pre-inoculation reaction mixture formed from components necessary for amplifying the template nucleic acid molecule. In any of the disclosed aspects, the pre-inoculation reaction mixture may include some or all of the following: a population of template nucleic acid molecules, a polymerase, one or more solid-phase carriers having a first group of primers linked together, nucleotides, and cofactors (such as divalent cations). In some embodiments, the pre-inoculation reaction mixture may further contain a second primer and optionally a diffusion-restricting agent. In some embodiments, the population of template nucleic acid molecules comprises template nucleic acid molecules that bind to at least one adaptor sequence that hybridizes with the first or second primer. In some embodiments, such as in emulsion RPA or emulsion PCR, the reaction mixture may form an emulsion. In pre-inoculation reactions via RPA, the pre-inoculation reaction mixture may contain recombinase and optionally recombinase accessory proteins. The various components of the reaction mixture are discussed in further detail herein.

[0047] In specific implementations of the inoculation, hydrophilic particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). For example, particle 504 contains a capture primer complementary to a portion of template polynucleotide 502. The template polynucleotide can hybridize with the capture primer. The capture primer can be extended to form bead 506 comprising the target polynucleotide linked thereto. Other beads may remain unlinked to the target nucleic acid, and other template polynucleotides may float freely in the solution.

[0048] In one example, a bead carrier 506 including a target polynucleotide can be linked to magnetic beads 510 to form a bead assembly 512. Specifically, the magnetic beads 510 are linked to the bead carrier 506 via a double-stranded polynucleotide linker. In another example, a separate probe including a linker portion can hybridize to a portion of the target polynucleotide on the bead carrier 506. The linker portion can be linked to a complementary linker portion on the magnetic beads 510. In yet another example, the template polynucleotide used to form the target nucleic acid linked to the bead 506 can include a linker portion linked to the magnetic beads 510. In yet another example, a template polynucleotide complementary to the target polynucleotide linked to the bead carrier 506 can be generated from primers modified to have a linker linked to the magnetic beads 510.

[0049] The linker portion attached to the polynucleotide and the linker portion attached to the magnetic bead can be complementary and mutually attached. For example, these linker portions have affinity and may include: an avidin portion and a biotin portion; an antigenic epitope and an antibody or its immunoreactive fragment; an antibody and a hapten; a digoxigenin portion and an anti-digoxigenin antibody; a fluorescein portion and an anti-fluorescein antibody; an operon and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement. In a particular example, the linker portion attached to the polynucleotide contains biotin, and the linker portion attached to the magnetic bead contains streptavidin.

[0050] The bead assembly 512 can be applied onto a substrate 516 of the sequencing device, including wells 518. In an example, a magnetic field can be applied to the substrate 516 to pull the magnetic beads 510 of the bead assembly 512 toward the wells 518. Bead carrier 506 enters the wells 518. For example, a magnet can be moved parallel to the surface of the substrate 516, thereby depositing bead carrier 406 in the wells 518.

[0051] The bead assembly 512 can be denatured to remove the magnetic beads 510, thereby leaving the bead carrier 506 in the pore 518. For example, the hybridized double-stranded DNA of the bead assembly 512 can be denatured using thermal cycling or an ionic solution to release the magnetic beads 510 and the template polynucleotide having a linker portion attached to the magnetic beads 510. For example, the double-stranded DNA can be treated with a low-ion-content aqueous solution such as deionized water to denature and separate the strands. In one example, foam washing can be used to remove the magnetic beads.

[0052] Optionally, target polynucleotide 506 (referred to herein as template) can be amplified while bead vector 514 containing multiple copies of the target polynucleotide is provided in well 518. Specifically, bead 514 contains a monoclonal population of the target polynucleotide. This amplification reaction can be performed using polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), or a combination thereof. Alternatively, amplification can be performed before depositing bead vector 514 into the well.

[0053] In one particular embodiment, an enzyme (such as a polymerase) is present, bound to, or closely adjacent to a particle or bead. In one example, the polymerase is present in solution or in a pore to facilitate the replication of a polynucleotide. A variety of nucleic acid polymerases can be used in the methods described herein. In an example embodiment, the polymerase may comprise an enzyme, a fragment thereof, or a subunit thereof capable of catalyzing the replication of a polynucleotide. In another embodiment, the polymerase may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fusion or otherwise engineered polymerase, a chemically modified polymerase, a synthetic molecule, or an analogue, derivative, or fragment thereof. Examples of enzymes, solutions, compositions, and amplification methods can be found in WO2019 / 094,524, entitled “Methods and Compositions for Manipulating Nucleic Acids,” which is incorporated herein by reference in its entirety.

[0054] Although the polynucleotides in the bead carrier 514 are shown on the surface, they can extend within the bead carrier 514. Hydrogels and hydrophilic particles with a low concentration of polymers relative to water can include polynucleotide segments both inside and throughout the bead carrier 514, or the polynucleotides can be present in pores and other openings. In particular, the bead carrier 514 can allow the diffusion of enzymes, nucleotides, primers, and reaction products used for monitoring reactions. A large number of polynucleotides per particle produces a better signal.

[0055] In an example implementation, the bead carrier 514 can be used in a sequencing device. For example, sequencing device 516 may include an array of wells 518.

[0056] In this example, sequencing primers can be added to well 518, or the bead carrier 514 can be pre-exposed to primers before being placed in well 518. Specifically, the bead carrier 514 may include bound sequencing primers. The sequencing primers and polynucleotides form a nucleic acid duplex containing a polynucleotide (e.g., a template nucleic acid) that has hybridized with the sequencing primers. The nucleic acid duplex is a polynucleotide that is at least partially double-stranded. Enzymes and nucleotides can be provided to well 518 to facilitate detectable reactions, such as nucleotide incorporation.

[0057] Sequencing can be performed by detecting nucleotide addition. Nucleotide addition can be detected using methods such as fluorescence emission or ion detection. For example, a set of fluorescently labeled nucleotides can be provided to system 516 and migrate to well 518. Excitation energy can also be provided to well 518. When the nucleotides are captured by polymerase and added to the ends of the extension primers, the nucleotide labeling can fluoresce, indicating which type of nucleotide has been added.

[0058] In an alternative example, a solution containing a single type of nucleotide can be added sequentially. In response to the addition of the nucleotide, the pH within the local environment of pore 518 can change. This pH change can be detected by an ion-sensitive field-effect transistor (ISFET). Therefore, the pH change can be used to generate a signal indicating the sequence of nucleotides complementary to the polynucleotides of particle 510.

[0059] Specifically, the sequencing system may include one or more holes disposed on a sensor pad of an ion sensor, such as a field-effect transistor (FET). In various embodiments, the system includes one or more polymer particles loaded into holes disposed on a sensor pad of an ion sensor (e.g., a FET), or into multiple holes disposed on a sensor pad of an ion sensor (e.g., a FET). In various embodiments, the FET may be a chemFET or an ISFET. A “chemFET” or chemical field-effect transistor includes a field-effect transistor used as a chemical sensor. The structure of a chemFET is similar to that of a MOSFET transistor, in which a charge is applied to the gate electrode via a chemical process. An “ISFET” or ion-sensitive field-effect transistor can be used to measure the concentration of ions in a solution; when the ion concentration (such as H+) changes, the current through the transistor changes accordingly.

[0060] In implementations, the FET can be a FET array. As used herein, an "array" is a planar arrangement of elements such as sensors or holes. The array can be one-dimensional or two-dimensional. A one-dimensional array can be an array having one column (or row) of elements in a first dimension and multiple columns (or rows) in a second dimension. The number of columns (or rows) in the first and second dimensions can be the same or different. A FET or array can contain 10... 2 10 3 10 4 10 5 10 6 10 7 Or more FETs.

[0061] In various embodiments, one or more microfluidic structures may be fabricated above the FET sensor array to provide containment or confinement for biological or chemical reactions. For example, in one embodiment, the microfluidic structure may be configured to provide one or more orifices (or orifices, or reaction chambers, or reaction orifices, as terms used interchangeably herein) above one or more sensors of the array, such that one or more sensors having predetermined orifices thereon detect and measure the presence, level, or concentration of an analyte in the predetermined orifice. In various embodiments, a 1:1 correspondence may exist between the FET sensors and the reaction orifices.

[0062] Return to Figure 5 In another example, the aperture 518 in the aperture array can be operatively connected to a measuring device. For example, in fluorescence emission methods, the aperture 518 can be operatively connected to a photodetector. In the case of ion detection, the lower surface of the aperture 418 can be disposed on the sensor pad of an ion sensor, such as a field-effect transistor.

[0063] One example system involving sequencing by detecting ionic byproducts of nucleotide incorporation is the Ion TorrentPGM™, Proton™, or S5™ sequencer (Thermo Fisher Scientific), an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions generated as byproducts of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporation that occur during template-dependent nucleic acid synthesis using polymerases. The Ion TorrentPGM™, Proton™, or S5™ sequencer detects nucleotide incorporation by detecting hydrogen ion byproducts of nucleotide incorporation. The Ion Torrent PGM™, Proton™, or S5™ sequencer may contain multiple template polynucleotides to be sequenced, each template disposed in a separate sequencing reaction well in an array. Each well in the array is coupled to at least one ion sensor that can detect the release of H+ ions or changes in solution pH as byproducts of nucleotide incorporation. The ion sensor includes a field-effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. Ion sensors provide an output signal indicating nucleotide incorporation, which can be represented as a voltage change whose magnitude is correlated with the H+ ion concentration in the respective well or reaction chamber. Different nucleotide types can flow into the reaction chamber sequentially and can be incorporated into the extension primers (or polymerization sites) in a sequence determined by the template sequence via polymerase. Each nucleotide incorporation may be accompanied by the release of H+ ions in the reaction well, and a corresponding change in local pH. The release of H+ ions can be recorded by the sensor's FET, which generates a signal indicating that nucleotide incorporation has occurred. Nucleotides that are not incorporated during a particular nucleotide flow may not generate a signal. The amplitude of the signal from the FET can also be correlated with the number of a particular type of nucleotide incorporated into the extended nucleic acid molecule, thus allowing homopolymer regions to be resolved. Therefore, during sequencer operation, the simultaneous inflow of multiple nucleotides into the reaction chamber and the monitoring of incorporation across multiple wells or reaction chambers allows the instrument to simultaneously resolve sequences from many nucleic acid templates.

[0064] Oligonucleotide-conjugated polymer particles can be used for the process of extending oligonucleotides complementary to template nucleotides. The template is complementary to the target nucleic acid. As a result, the target nucleic acid sequence binds to the beads. The target nucleic acid can be amplified or templated onto oligonucleotide-conjugated polymer particles to form polymer particles with a monoclonal population of the target nucleic acid bound to it.

[0065] exist Figure 6In the example shown, a target polynucleotide BA' and its complementary template polynucleotide (A-B') are amplified in the presence of a bead vector with capture primers. The target polynucleotide has a capture portion (B) that is sequenced the same as or substantially similar to the capture primers coupled to the bead vector. A substantially similar sequence is a sequence whose complementary sequence can hybridize with each substantially similar sequence in the substantially similar sequence. The bead vector may have capture primers that have sequences that are the same as or substantially similar to the sequence of the B portion of the target polynucleotide to allow the complementary sequence of the capture portion (B) of the target polynucleotide to hybridize with the capture primers coupled to the bead vector. Optionally, the target polynucleotide may include a second primer position (P1) adjacent to the capture portion (B) of the target polynucleotide, and may also include a target region adjacent to the primers and binding to the sequencing primer portion (A) of the target polynucleotide via the complementary sequence portion (A').

[0066] When amplified in the presence of a bead vector including the capture primer, the template polynucleotide complementary to the target polynucleotide hybridizes with the capture primer (B). The target polynucleotide may remain in solution. The system cannot extend the capture primer B, which is complementary to the template polynucleotide, to obtain an extension of the target sequence bound to the bead vector.

[0067] Further amplification can be performed in the presence of a free primer (B), a bead carrier, and a free modified sequencing primer (A) with a linker portion (L) attached thereto. Primer (B) and the modified primer (LA) can interfere with the free floating target polynucleotide and template polynucleotide, hindering their binding to the bead carrier and to each other. Specifically, the modified sequencing primer (A) with the linker portion attached thereto can hybridize with the complementary portion (A') of the target polynucleotide linked to the bead carrier. Optionally, the linker-modified sequencing primer LA that hybridizes with the target polynucleotide can be extended to form a linker-modified template polynucleotide. This linker-modified template polynucleotide hybridizes with the target nucleic acid linked to the bead carrier, which can then be captured by magnetic beads and used as a magnetic load for the sequencing device.

[0068] Polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), or other amplification techniques can be used for amplification or extension. In a specific example, PCR amplification is used. Figure 6 Each step of the scheme shown.

[0069] exist Figure 7In another example shown, the alternative comprises a target polynucleotide (P1-A') and its complementary template polynucleotide (A-P1'). The target polynucleotide and template polynucleotide are amplified in a solution containing a linker-modified sequencing primer (LA) and a truncated P1 primer (trP1) having a portion of the sequence containing a capture primer (B). In this example, the truncated P1 primer (trP1) contains a subset or all of the P1 sequence. During subsequent amplification in the presence of the linker-modified sequencing primer (LA) and the truncated P1 primer (trP1-B), a single substance comprises a linker-modified template polynucleotide (LA-B') operable to hybridize with a bead vector containing the capture primer (B). Thus, the linker-modified template polynucleotide (LA-B') hybridizes with the capture primer (B) on the bead and extends to form the target polynucleotide (B-A') linked to the bead vector.

[0070] The adapter-modified template polynucleotide for bead hybridization with the target polynucleotide can be used to ligate magnetic beads, which can then be used to magnetically load the beads into a sequencing device. As mentioned above, the adapter portion of the adapter-modified template polynucleotide can take various forms, such as biotin, which can bind to the adapter portion of the magnetic bead, such as streptavidin. Each of the amplification reactions can be performed using PCR, RPA, or other amplification techniques. Figure 7 In the example shown, three polymerase chain reaction (PCR) cycles can be used to implement the procedure. This series of PCR reactions produces a high percentage of bead vectors with a single target polynucleotide linked to them. Therefore, a larger population of single clones can be generated in the wells of the sequencing device.

[0071] Exemplary polymer particles include polyacrylamide polymer particles having carboxyl functional groups. Example particles are described in WO 2017 / 004556 entitled "Polymer substrate formed from carboxyl-functionalized acrylamide".

[0072] In an example embodiment, the polymer substrate (such as polymer particles) is formed from a carboxyl-functionalized monomer. In this example, the carboxyl-functionalized monomer has a protecting group (OH) that replaces the carboxyl group. The protecting group can protect the OH group during polymerization or make the monomer more miscible with a hydrophobic phase. Once the monomer has polymerized, the protecting group can be removed, thereby providing a polymer network with carboxyl-functionalized sites. Such sites can be used to attach functional groups to the polymer substrate, such as oligomer primers.

[0073] In certain examples, the monomer solution may be distributed into a dispersed hydrophobic phase within a hydrophilic phase or an aqueous continuous phase. In some examples, the dispersed hydrophobic phase may be formed from hydrophobic polymer beads. The monomer solution may include protected carboxyl-functionalized monomers, such as protected carboxyl-functionalized acrylamides. Optionally, the monomer solution may further include other monomers, crosslinking agents, pore-forming agents, catalysts, or any combination thereof.

[0074] In the example, the monomer may include a protected carboxyl-functionalized acrylamide monomer. Specifically, the protected carboxyl-functionalized acrylamide includes a protecting group that protects the hydrophilic OH group of the carboxyl functional group. The protecting group can protect the OH group, thereby preventing reactions during polymerization or making the monomer more miscible with a hydrophobic phase. In particular, the protecting group may be cleavable from the monomer or from the polymer network formed by the monomer. For example, the protecting group may be acid-cleavable, specifically, cleavable at a pH that will not cause hydrolysis of the polymer network.

[0075] For example, the protecting group may include a silyl group. In another example, the protecting group may include a straight-chain or branched alkyl group having at least three carbons. For example, the alkyl group may include 3 to 8 carbons, such as 3 to 6 carbons or 3 to 5 carbons. In particular, the protecting group may be a branched alkyl group, such as a branched alkyl group having between 3 and 5 carbons, such as a branched alkyl group with 4 carbons.

[0076] For example, a monomer can have the following formula (I):

[0077] (I)

[0078] Wherein R1 is an alkyl group having between 3 and 10 carbons, a polyether group having between 1 and 10 ether units, or another nonionic polar group; where R2 is a straight-chain or branched alkyl group having between 3 and 8 carbons, or a silyl group; and where R3 is hydrogen or an alkyl group having between 1 and 6 carbons. In a specific example, R1 is an alkyl group having between 3 and 10 carbons, or a polyether group having between 1 and 10 ether units. For example, R1 may be an alkyl group having 3 to 6 carbons, such as 3 to 5 carbons. In another example, R1 may be a polyether group including units ranging from 2 to 6 units, such as 2 to 4 units, such as ethylene oxide or propylene oxide units. In a further example, R1 may be a nonionic polar group, for example, including an amide. In the example, R2 is a branched alkyl group, which, for example, has 3 to 5 carbons, such as 4 carbons. Specifically, R2 can be an isopropyl, isobutyl, sec-butyl, or tert-butyl group, or any combination thereof. The silyl group can be a trialkylsilyl group, an organodimethylsilyl group, or an organotrimethylsilyl group. For example, the trialkylsilyl group can be a trimethylsilyl or triethylsilyl group. In a further example, R3 is hydrogen. In another example, R3 is a methyl or ethyl group.

[0079] In the example, the monomer can have equation (II):

[0080] (II)

[0081] Wherein R1 is an alkyl group having between 3 and 10 carbons, or a polyether group having between 1 and 10 ether units, and wherein R2 is a straight-chain or branched alkyl group having between 3 and 8 carbons, or a silyl group. For example, R1 may be an alkyl group having 3 to 6 carbons, such as 3 to 5 carbons. In another example, R1 may be a polyether group comprising units ranging from 2 to 6 units, such as 2 to 4 units, such as ethylene oxide or propylene oxide units. In the example, R2 is a branched alkyl group, which, for example, has 3 to 5 carbons, such as 4 carbons. In particular, R2 may be an isopropyl, isobutyl, sec-butyl, or tert-butyl group or any combination thereof. The silyl group may be a trialkylsilyl group, an organodisilyl group, or an organotrisilyl group. For example, a trialkylsilyl group may be a trimethylsilyl or triethylsilyl group.

[0082] In a specific example, the protected carboxyl functional monomer may be an acryloylaminobutyrate protected with a tert-butyl protecting group and having formula (III):

[0083] (III)

[0084] In the example, the protected carboxyl functional monomer of formula (I), (II), or (III) can be formed by reacting a protected aminoalkyl acid salt, such as an aminoalkyl ester hydrochloride, with acryloyl chloride. For example, a stoichiometric amount of an aminoalkyl ester hydrochloride (such as tert-butyl aminobutyrate hydrochloride) in a dichloromethane solvent can be mixed with an aqueous potassium carbonate solution in a temperature range such as -10°C to 5°C. An acryloyl chloride solution can be added, and the mixture can be stirred under the same thermal conditions. The mixture can be extracted with a solvent such as dichloromethane. The solvent can be removed under reduced pressure or vacuum.

[0085] In the example, the monomers described above can be polymerized to form a polymer substrate. For example, the polymer substrate can be a polymer coating or a film. In another example, the polymer substrate can be polymer particles or beads. For example, polymer particles can be formed using emulsion polymerization, or they can be formed in a dispersed hydrophobic phase within a hydrophilic continuous phase.

[0086] In the example, polymer particles can be converted into hydrophilic polymer particles by removing at least a portion of the hydrophobic protecting groups. For example, the hydrophobic protecting groups can be acid-cleaved from the polymer particles. In particular, such removal can remove substantially all of the hydrophobic protecting groups from the polymer particles, such as removing at least 80% or even at least 90% of the hydrophobic protecting groups.

[0087] In the example, the hydrophobic protecting group is acid-cleaved by adding an acid, such as an organic acid. Specifically, the organic acid can have a pKa in the range of 3.0 to 5.5. For example, the organic acid can include acetic acid, lactic acid, citric acid, or any combination thereof. Alternatively, an inorganic acid can be used. For example, a sulfuric acid solution can be used.

[0088] After removing at least a portion of the hydrophobic protecting groups, hydrophilic particles are formed. The hydrophilic particles include carboxyl functional groups. In the example, hydrophilic particle 112 may be a hydrogel particle comprising a hydrogel polymer. The hydrogel is a polymer capable of absorbing at least 20% of its weight in water, such as at least 45%, at least 65%, at least 85%, at least 100%, at least 300%, at least 1000%, at least 1500%, or even at least 2000% of its weight in water, but not exceeding 106%.

[0089] Biomolecules (such as oligonucleotides or proteins) can be conjugated with carboxyl-functionalized polymers (such as those described above). In particular, carboxyl functional groups can be activated to facilitate conjugation with biomolecules.

[0090] In the example, carbodiimides (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) and hydroxysuccinimides (such as N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (S-NHS)) can be used to activate the carboxyl functional group to form a functional group that can react with the modified oligonucleotide.

[0091] For example, such as Figure 8 As shown, carbodiimide can be added to a suspension of carboxyl-functionalized beads. In the example, the carbodiimide can be at least partially soluble in aqueous solution. For example, the carbodiimide can be an aminoalkyl carbodiimide, an aminocycloalkyl carbodiimide, or a variant thereof. For example, the carbodiimide can be an alkyl-aminoalkyl carbodiimide, a diaminoalkyl carbodiimide, an alkylaminocyclohexyl carbodiimide, a diaminocyclohexyl carbodiimide, or a variant or combination thereof. In a particular example, the carbodiimide is an alkyl-aminoalkyl carbodiimide, such as EDC. The suspension can be an aqueous suspension and optionally includes other solvents and buffer systems.

[0092] The carbodiimide can be dissolved in a solvent before being added to the suspension. Alternatively, a solvent can be added to the suspension. Example solvents include amides, ureas, carbonates, ethers, sulfoxides, sulfones, hindered alcohols, or combinations thereof. Exemplary amides or ureas include formamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone, N,N,N',N'-tetramethylurea, N,N'-dimethyl-N,N'-trimethyleneurea, or combinations thereof. Exemplary carbonates include dimethyl carbonate, propylene carbonate, or combinations thereof. Exemplary ethers include tetrahydrofuran. Exemplary sulfoxides or sulfones include dimethyl sulfoxide, dimethyl sulfone, or combinations thereof. Exemplary hindered alcohols include tert-butanol. In a particular example, the solvent includes N-methylpyrrolidone. In another example, the solvent includes dimethylformamide. In a further example, the solvent includes dimethyl sulfoxide.

[0093] Example buffer systems include 2-(N-morpholino)ethanesulfonic acid (MES) buffer, phosphate-buffered saline (PBS) buffer, or TE buffer system. Optionally, the suspension may include a surfactant, such as Tween-20. For example, MES may be used at concentrations in the range of 0.1M to 2M, such as in the range of 0.2M to 0.7M or in the range of 0.2M to 0.5M. Optionally, a chloride salt, such as NaCl, may be added to the solution. For example, NaCl may be used at concentrations in the range of 0.01M to 1M, such as in the range of 0.05M to 0.6M or in the range of 0.1M to 0.5M. In particular, the suspension may have a pH in the range of 4 to 9, such as in the range of 4 to 7 or in the range of 5 to 6.

[0094] Carbodiimide can be added in equivalence ratios relative to the initial number of carboxyl functional groups on the beads or substrate, ranging from 1 eq. to 1000 eq., such as 10 eq. to 800 eq., 100 eq. to 700 eq., or 200 eq. to 550 eq.

[0095] Hydroxysuccinimide (such as NHS or S-NHS) is added to the suspension. For example, hydroxysuccinimide can be added in equivalent amounts to the initial number of carboxyl functional groups on the beads or substrate, in the range of 0.5 eq. to 75 eq., such as 0.5 eq. to 50 eq., 0.75 eq. to 20 eq., or 1 eq. to 10 eq.

[0096] The reaction mixture can be maintained at a temperature ranging from 0°C to 25°C, such as 0°C to 15°C or 0°C to 10°C. The reaction mixture can be incubated for periods ranging from 10 minutes to 10 hours, such as 10 minutes to 5 hours, 30 minutes to 3 hours, or 1 hour to 3 hours.

[0097] Therefore, the carboxyl functional group is coupled to the nitrogen of the attached NHS or S-NHS. In the example, such a functional group may be conjugated to an amine-modified oligonucleotide or further processed to couple with other modified oligonucleotides.

[0098] Optionally, the activated beads can be separated or washed. For example, the suspension can be centrifuged to granulate the beads, and the solution can be separated from the granulated beads. The granulated beads can be resuspended in an aqueous solution. In this example, the aqueous solution includes a buffer, such as PBS buffer or TE buffer. Alternatively, the activated beads can be kept in suspension.

[0099] After activation, modified oligonucleotides can be conjugated to activated beads. For example... Figure 9 As shown, one conjugation pathway involves using an amine-modified oligonucleotide. Alternatively, NHS can be replaced with an amino-maleimide, thereby forming an amide from the carboxyl group. Conjugation can also be performed using diene-modified oligonucleotides.

[0100] For example, activation can increase the pH of the suspension. An optional base can be added to the reaction mixture. Example bases include sodium hydroxide or amines, such as hindered amines, such as triethylamine or diisopropylethylamine (DIPEA). The pH can be increased to a range of 6 to 10, such as 7 to 10 or 8 to 9.5.

[0101] In the example, modified oligonucleotides can be added to the suspension. The modified oligonucleotides may include an amine group at the 5' end, for example, as shown below. Figure 9 As shown. The amine moiety can be linked to the phosphate ester group of the oligonucleotide via an intermediate structure (such as an alkyl group, aryl group, or glycol ether group). For example, the intermediate structure may include an alkyl group, such as an ethyl, propyl, butyl, or pentyl group. In the example, the modified oligonucleotide may include an aminopropyl-modified oligonucleotide.

[0102] Modified oligonucleotides can have between 10 and 50 nucleotides. For example, modified oligonucleotides can have between 15 and 35 nucleotides, such as between 20 and 35 nucleotides.

[0103] Amine-modified oligonucleotides can be added in equivalences relative to the initial number of carboxyl functional groups on the beads or carrier, in the range of 1 eq to 20 eq, such as 2 eq to 10 eq or 3 eq to 7 eq.

[0104] The reaction can be maintained at temperatures ranging from 20°C to 50°C, such as 20°C to 40°C or 20°C to 30°C, for example, at room temperature. The reaction can be maintained for periods ranging from 30 minutes to 50 hours, such as 1 hour to 35 hours, 3 hours to 20 hours, or 8 hours to 20 hours. As a result of the reaction, oligonucleotides are immobilized into beads.

[0105] In the example, each of the aminoalkylcarbodiimide, hydroxysuccinimide, and amine-modified oligonucleotides was added to the aqueous suspension simultaneously, with each added to the same container within a short period of time. The suspension was then incubated after addition.

[0106] The reaction mixture can be maintained at a temperature ranging from 0°C to 25°C, such as 0°C to 15°C or 0°C to 10°C. The reaction mixture can be incubated for periods ranging from 10 minutes to 10 hours, such as 10 minutes to 5 hours, 30 minutes to 3 hours, or 1 hour to 3 hours.

[0107] After low-temperature incubation, the pH of the reaction mixture can be adjusted to a range of 7 to 10, such as 8 to 9.5 or 8.5 to 9. For example, sodium hydroxide can be added to the reaction mixture. The reaction mixture is mixed at a temperature in the range of 20°C to 30°C, such as 20°C to 25°C or room temperature, for a period of 5 to 25 hours, such as 10 to 20 hours or 12 to 18 hours.

[0108] Alternatively, the activated carboxyl-functionalized beads can react with an amino-maleimide to replace NHS. The amino-maleimide can be an aminoalkylmaleimide, an aminophenylmaleimide, or a similar amino-functionalized maleimide. For example, the amino-maleimide can be an aminoalkylmaleimide, such as aminoethylmaleimide, aminopropylmaleimide, or aminobutylmaleimide. In a specific example, the amino-maleimide is an aminoethylmaleimide, such as N-(2-aminoethyl)maleimide.

[0109] For example, an amino-maleimide is added to the suspension. The amino-maleimide can be added in equivalent amounts to the initial number of carboxyl functional groups, in the range of 1 eq. to 150 eq., such as 2 eq. to 100 eq., 8 eq. to 75 eq., or 20 eq. to 60 eq.

[0110] The reaction mixture can be maintained at temperatures ranging from 3°C to 35°C, such as 5°C to 28°C or 20°C to 25°C. The reaction mixture can be incubated for periods ranging from 10 minutes to 100 hours, such as 10 minutes to 60 hours, 30 minutes to 16 hours, or 1 hour to 3 hours.

[0111] Diene-modified oligonucleotides can be added to the suspension. The modified oligonucleotide may include a diene group at its 5' end. In examples, the modified oligonucleotide may include a terminal portion, such as a furan moiety, a cyclopentadiene moiety, an anthracene moiety, a butadiene moiety, an isoprene moiety, a combination thereof, or equivalents thereof. For example, the modified oligonucleotide may include a butadiene-terminal portion or an isoprene-terminal portion. In an example, the modified oligonucleotide includes a butadiene-terminal portion.

[0112] The terminal portion can be linked to the phosphate ester group of the oligonucleotide via an intermediate structure (such as an alkyl group, aryl group, or glycol ether group). For example, the intermediate structure may include an alkyl group, such as an ethyl, propyl, butyl, or pentyl group. In the example, diene-modified oligonucleotides may include 1,3-hexadiene-modified oligonucleotides.

[0113] Modified oligonucleotides can have between 10 and 50 nucleotides. For example, modified oligonucleotides can have between 15 and 35 nucleotides, such as between 20 and 35 nucleotides.

[0114] Diene-modified oligonucleotides can be added at concentrations ranging from 0.1 mM to 5 mM, such as 0.5 mM to 3.5 mM or 1.5 mM to 3.0 mM. In another example, diene-modified oligonucleotides can be added at equivalent amounts relative to the initial number of carboxyl functional groups, ranging from 1 eq to 50 eq, such as 2 eq to 30 eq or 5 eq to 20 eq.

[0115] The reaction can be maintained at temperatures ranging from 23°C to 80°C, such as 35°C to 75°C or 40°C to 70°C. The reaction can be maintained for periods ranging from 10 minutes to 70 hours, such as 30 minutes to 60 hours, 1 hour to 50 hours, or 16 hours to 48 hours. As a result of the reaction, oligonucleotides are immobilized into beads.

[0116] Example

[0117] Example 1

[0118] Conjugated carboxyl-functionalized beads (manufactured by Dynal) have an average size in the range of 0.5 μm to 0.65 μm and an initial carboxyl functional group count in the range of 0.84 M COOH / bead to 1.43 M COOH / bead.

[0119] EDC (500 eq.) and NHS (1 eq.) were added to an aqueous suspension of beads (pH 5) comprising MES buffer (0.25 M) and NaCl (0.1 M). The reaction was carried out at 0 °C for 1 hour. Carboxyl functional groups above 300 K were activated.

[0120] The pH of the aqueous suspension was increased to 9, and amino-modified oligonucleotides were added at a concentration of 5 eq. The reaction was carried out at 23°C for 16 hours to obtain beads with conjugated groups exceeding 300K.

[0121] Example 2

[0122] Reagent and Sample Preparation

[0123] Set the hot mixer to 23°C and 750 rpm, and insert a 2 mL tube.

[0124] Remove the hydrogel bead stock (MA980, Dynal®) (using water as solvent) from the 4°C freezer and vortex for 2 minutes. Remove the Av4 oligonucleotide stock solution from the freezer and place it on ice.

[0125] Prepare a 1M EDC stock solution in NMP. Weigh 20 mg of EDC powder and add 130 μL of NMP. Heat to 60°C to ensure complete dissolution.

[0126] Prepare a 0.1 M NHS stock solution in NMP. Weigh 5 mg of NHS powder and add 434 μL of NMP.

[0127] activation

[0128] Use a P1000 electronic pipette to aspirate the hydrogel bead reserve, wiping away any excess material from the edge of the bead reserve tube. Dispense 200 µL onto the sidewall of a labeled 2 mL centrifuge tube. Place the tube back on ice.

[0129] Using a P200 electronic pipette, aspirate 162.5 µL of MES buffer (pH 5.0) into a 2 mL centrifuge tube. Dispense the buffer onto the side of the tube. Place the tube back on ice.

[0130] Use a P1000 electronic pipette to aspirate the Av4 oligonucleotide stock solution and allocate 257.1 µL to a 2 mL centrifuge tube. Vortex for 5 seconds and place the tube back on ice.

[0131] Using a P200 electronic pipette, aspirate the EDC stock solution and dispense 103.4 µL into a 2 mL centrifuge tube. Vortex for 5 seconds and place the tube back on ice.

[0132] Using a P10 electronic pipette, aspirate the NHS stock solution and dispense 2.1 µL into a 2 mL centrifuge tube. Vortex for 5 seconds and return the tube to ice. The molar ratio of EDC:NHS:-COOH is 500:1:1. The molar ratio of -NH2:-COOH is 5:1.

[0133] Incubate the mixture on ice for 1 hour.

[0134] Adhesion

[0135] Adjust the pH of the mixture to 8.5 to 9.0 using freshly prepared NaOH solution (0.5 M). Place the sample tubes in a hot mixer at 23°C and 750 rpm overnight (16 hours).

[0136] Quenching and post-processing

[0137] The reaction was quenched by adding 700 µL of 125 mM NaOH solution. The sample tube was centrifuged at 250,000 rpm for 1 hour to precipitate the hydrogel beads. After rotation, the supernatant was gently poured off, and 1 mL of water was added to the precipitate. The sample tube was sonicated to resuspend the beads. This process was repeated twice to wash away unreacted reagents. The hydrogel precipitate was resuspended in low TE buffer for storage.

[0138] Primer loading characterization

[0139] Primer load was determined by FAM assay. The conjugated hydrogel bead solution was diluted to 1 M / µL. In a PCR tube, 45 µL of annealing buffer, 5 µL of hydrogel bead solution, and 1 µL of FAM-Av4' probe (100 µM) were mixed. The PCR tube was loaded onto a thermal cycler for one probe hybridization cycle. The tube was heated to 97 °C for 2 minutes, then gradually cooled to 37 °C and held for 2 minutes, and then held at 4 °C. The hybridization sample was further washed with Guava buffer to remove excess FAM-Av4' probe, and the sample was measured by Guava flow cytometry to determine the primer load.

[0140] Example 3

[0141] activation

[0142] Using a P1000 electronic pipette, aspirate the hydrogel bead reserve and wipe away any excess material from the edge of the bead reserve tube. Dispense 200 µL onto the sidewall of a labeled 2 mL centrifuge tube. Place the tube back on ice. Using a P200 electronic pipette, aspirate the MES buffer (pH 5.0) and dispense 162.5 µL into a 2 mL centrifuge tube. Dispense the buffer onto the sidewall of the tube. Place the tube back on ice. Using a P200 electronic pipette, aspirate the EDC reserve solution and dispense 103.4 µL into a 2 mL centrifuge tube. Vortex for 5 seconds and place the tube back on ice. Using a P10 electronic pipette, aspirate the NHS reserve solution and dispense 2.1 µL into a 2 mL centrifuge tube. Vortex for 5 seconds and place the tube back on ice. The molar ratio of EDC:NHS:-COOH is 500:1:1. Incubate the mixture on ice for 1 hour.

[0143] Using a P1000 electronic pipette, aspirate 383.1 µL of PBS buffer (pH 6.5) into a 2 mL centrifuge tube. Apply the buffer solution to the side of the tube. Vortex for 5 seconds and return the tube to ice. Using a P10 electronic pipette, aspirate 4.5 µL of DIPEA stock solution into a 2 mL centrifuge tube. Apply the solution to the side of the tube. Vortex for 5 seconds and return the tube to ice. Using a P200 electronic pipette, aspirate 27.3 µL of maleimide stock solution into a 2 mL centrifuge tube. Apply the solution to the side of the tube. Vortex for 5 seconds and return the tube to ice.

[0144] Incubate the mixture on a hot mixer at room temperature for 16 hours.

[0145] Adhesion

[0146] Using a P1000 electronic pipette, aspirate 400 µL of PBS buffer (pH 6.5) into a 2 mL centrifuge tube. Apply the buffer solution to the side of the tube. Using the P1000 electronic pipette, aspirate 400 µL of the diene oligonucleotide stock solution (5 mM, in PBS buffer) into a 2 mL centrifuge tube. Apply the buffer solution to the side of the tube. Vortex for 5 seconds and place the tube back on the heat mixer. Set the heat mixer temperature to 40°C and 750 rpm. Allow the conjugation reaction to proceed for 16 hours.

[0147] Quenching and post-processing

[0148] The reaction was quenched by adding 700 µL of 125 mM NaOH solution. The sample tube was centrifuged at 250,000 rpm for 1 hour to precipitate the hydrogel beads. After rotation, the supernatant was gently poured off, and 1 mL of water was added to the precipitate. The sample tube was sonicated to resuspend the beads. This process was repeated twice to wash away unreacted reagents. The hydrogel precipitate was resuspended in low TE buffer for storage.

[0149] In a first aspect of this disclosure, a method for conjugating oligonucleotides to a bead carrier is provided. The method includes adding an aminoalkylcarbodiimide to an aqueous suspension comprising a bead carrier having a carboxyl functional group; adding a hydroxysuccinimide to the aqueous suspension; and adding an amine-modified oligonucleotide to the aqueous suspension.

[0150] In one example of the first aspect and in the examples above, the aminoalkyl carbodiimide includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0151] In another example of the first aspect and the example above, the method further includes dissolving an aminoalkylcarbodiimide in a solvent and then adding the aminoalkylcarbodiimide to the suspension.

[0152] In yet another example of the first aspect and in the examples above, the solvent includes N-methylpyrrolidone.

[0153] In an additional example of the first aspect and in the examples above, the addition of an aminoalkylcarbodiimide includes the addition of an aminoalkylcarbodiimide in the range of 1 equivalent to 1000 equivalents. In one example of the first aspect and in the examples above, the addition of an aminoalkylcarbodiimide includes the addition of an aminoalkylcarbodiimide in the range of 100 equivalents to 700 equivalents.

[0154] In another example of the first aspect and in the examples above, hydroxysuccinimide includes N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (S-NHS).

[0155] In yet another example of the first aspect and in the examples above, the addition of hydroxysuccinimide comprises the addition of hydroxysuccinimide in the range of 0.5 equivalents to 75 equivalents. In one example of the first aspect and in the examples above, this range is 0.75 equivalents to 20 equivalents.

[0156] In an additional example of the first aspect and in the examples above, the method further includes incubating the suspension at a temperature ranging from 0°C to 25°C after adding the hydroxysuccinimide. In one example of the first aspect and in the examples above, the incubation includes a period of time ranging from 10 minutes to 10 hours.

[0157] In another example of the first aspect and in the examples above, the method further includes adjusting the pH of the suspension to a range of 6 to 10 after incubation. In one example of the first aspect and in the examples above, the pH is in the range of 7 to 10.

[0158] In another example of the first aspect and in the examples above, the method further includes incubating the suspension at a temperature ranging from 20°C to 50°C after adjusting the pH. In one example of the first aspect and in the examples above, the temperature is in the range of 20°C to 40°C.

[0159] In the additional examples of the first aspect and the examples above, incubation at temperatures ranging from 20°C to 50°C includes a period of incubation lasting from 30 minutes to 50 hours. In one example of the first aspect and the examples above, this period of incubation ranges from 1 hour to 35 hours.

[0160] In another example of the first aspect and in the examples above, adding an amine-modified oligonucleotide includes adding an amine-modified oligonucleotide to a quantity ranging from 1 equivalent to 20 equivalents. In one example of the first aspect and in the examples above, this range is from 2 equivalents to 10 equivalents.

[0161] In another example of the first aspect and in the examples above, the addition of each of the aminoalkylcarbodiimide, hydroxysuccinimide and amine-modified oligonucleotides is carried out simultaneously.

[0162] In a second aspect of this disclosure, a method for conjugating oligonucleotides to a bead carrier is provided. The method includes adding an aminoalkylcarbodiimide to an aqueous suspension comprising a bead carrier having a carboxyl functional group; adding a hydroxysuccinimide to the aqueous suspension; adding an amino-maleimide to the suspension; and adding a diene-modified oligonucleotide to the aqueous suspension.

[0163] In one example of the second aspect and in the examples above, the aminoalkyl carbodiimide includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0164] In another example of the second aspect and the example above, the method further includes dissolving an aminoalkylcarbodiimide in a solvent and then adding the aminoalkylcarbodiimide to the suspension.

[0165] In another example of the second aspect and in the examples above, the solvent includes N-methylpyrrolidone.

[0166] In one example of the first or second aspect and in the examples above, the bead carrier comprises a polyacrylamide polymer matrix.

[0167] In one example of the second aspect and in the examples above, the addition of an aminoalkylcarbodiimide includes the addition of an aminoalkylcarbodiimide in the range of 1 equivalent to 1000 equivalents. In one example of the second aspect and in the examples above, the addition of an aminoalkylcarbodiimide includes the addition of an aminoalkylcarbodiimide in the range of 100 equivalents to 700 equivalents.

[0168] In one example of the second aspect and in the examples above, hydroxysuccinimide includes N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (S-NHS).

[0169] In one example of the second aspect and in the examples above, the addition of hydroxysuccinimide includes the addition of hydroxysuccinimide in the range of 0.5 equivalents to 75 equivalents. In one example of the second aspect and in the examples above, this range is 0.75 equivalents to 20 equivalents.

[0170] In one example of the second aspect and in the examples above, the method further includes incubating the suspension at a temperature ranging from 0°C to 25°C after adding the hydroxysuccinimide. In one example of the second aspect and in the examples above, the incubation includes a duration ranging from 10 minutes to 10 hours.

[0171] In one example of the second aspect and in the examples above, the method further includes incubating the suspension at a temperature ranging from 3°C to 35°C after adding the amino-maleimide. In one example of the second aspect and in the examples above, incubating the suspension after adding the amino-maleimide includes a period of incubation ranging from 30 minutes to 16 hours.

[0172] In one example of the second aspect and in the examples above, the addition of diene-modified oligonucleotides includes adding diene-modified oligonucleotides in the range of 1 equivalent to 50 equivalents. In one example of the second aspect and in the examples above, this range is 5 equivalents to 20 equivalents.

[0173] In one example of the second aspect and in the examples above, the method further includes incubating the suspension at a temperature ranging from 23°C to 80°C after adding the diene-modified oligonucleotide. In one example of the second aspect and in the examples above, the temperature is in the range of 35°C to 70°C.

[0174] In one example of the second aspect and in the examples above, incubation at a temperature ranging from 23°C to 80°C includes a period of incubation lasting from 30 minutes to 60 hours. In one example of the second aspect and in the examples above, this period of incubation ranges from 1 hour to 50 hours.

[0175] It should be noted that not all activities described above in the general description or examples are required. Some activities may not be required, and one or more additional activities may be performed besides those described. Furthermore, the order in which the activities are listed is not necessarily the order in which they are performed.

[0176] In the foregoing specification, the concepts have been described with reference to specific embodiments. However, those skilled in the art will understand that various modifications and changes can be made without departing from the scope of the invention as set forth in the appended claims. Therefore, the specification and drawings should be considered illustrative rather than restrictive, and all such modifications are intended to be included within the scope of the invention.

[0177] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of features is not necessarily limited to those features, but may include other features not expressly listed or inherent to such a process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” means inclusive “or” rather than exclusive “or.” For example, conditions A or B are 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).

[0178] Furthermore, the terms "a" or "an" are used to describe the elements and components described herein. This is done merely for convenience and to give a general meaning regarding the scope of the invention. The description should be understood to include one or at least one, and the singular includes the plural, unless it is obvious otherwise.

[0179] The benefits, other advantages, and solutions to problems have been described above with respect to specific embodiments. However, the benefits, advantages, solutions to problems, and any features that may cause any benefit, advantage, or solution to occur or become more significant should not be construed as key, essential, or necessary features of any or all claims.

[0180] Upon reading this specification, those skilled in the art will understand that, for clarity, certain features described herein in the context of separate 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. Furthermore, references to values ​​within a scope include every value within that scope.

Claims

1. A method for conjugating oligonucleotides to a bead carrier, the method comprising: An aminoalkyl carbodiimide is added to an aqueous suspension comprising a bead carrier having a carboxyl functional group. Hydroxysuccinimide was added to the aqueous suspension; as well as An amine-modified oligonucleotide was added to the aqueous suspension.

2. The method according to claim 1, wherein the bead carrier comprises a polyacrylamide polymer matrix.

3. The method according to claim 1 or claim 2, wherein the aminoalkyl carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

4. The method of claim 3, further comprising dissolving the aminoalkylcarbodiimide in a solvent and then adding the aminoalkylcarbodiimide to the suspension.

5. The method according to claim 4, wherein the solvent comprises N-methylpyrrolidone.

6. The method according to any one of claims 1 to 5, wherein the addition of the aminoalkylcarbodiimide comprises the addition of the aminoalkylcarbodiimide in the range of 1 eq. to 1000 eq.

7. The method of claim 6, wherein the addition of the aminoalkylcarbodiimide comprises the addition of the aminoalkylcarbodiimide in the range of 100 eq to 700 eq.

8. The method according to any one of claims 1 to 7, wherein the hydroxysuccinimide comprises N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (S-NHS).

9. The method according to any one of claims 1 to 8, wherein the addition of the hydroxysuccinimide comprises the addition of the hydroxysuccinimide in the range of 0.5 eq. to 75 eq.

10. The method of claim 9, wherein the range is from 0.75 eq. to 20 eq.

11. The method according to any one of claims 1 to 10, further comprising incubating the suspension at a temperature in the range of 0°C to 25°C after adding hydroxysuccinimide.

12. The method of claim 11, wherein the incubation comprises a period of time ranging from 10 minutes to 10 hours.

13. The method of claim 12, further comprising adjusting the pH of the suspension to a range of 6 to 10 after incubation.

14. The method of claim 13, wherein the pH is in the range of 7 to 10.

15. The method of claim 13, further comprising, after adjusting the pH, incubating the suspension at a temperature in the range of 20°C to 50°C.

16. The method of claim 15, wherein the temperature is in the range of 20°C to 40°C.

17. The method of claim 15, wherein incubation at a temperature in the range of 20°C to 50°C comprises incubation lasting for a period of time in the range of 30 minutes to 50 hours.

18. The method of claim 17, wherein the time period is in the range of 1 hour to 35 hours.

19. The method according to any one of claims 1 to 18, wherein adding the amine-modified oligonucleotide comprises adding the amine-modified oligonucleotide to a range of 1 eq to 20 eq.

20. The method according to any one of claims 1 to 19, wherein the range is 2 eq to 10 eq.

21. The method according to any one of claims 1 to 20, wherein the addition of each of the aminoalkylcarbodiimide, the hydroxysuccinimide, and the amine-modified oligonucleotide is performed simultaneously.

22. A method for conjugating oligonucleotides to a bead carrier, the method comprising: An aminoalkyl carbodiimide is added to an aqueous suspension comprising a bead carrier having a carboxyl functional group. Hydroxysuccinimide was added to the aqueous suspension; Add amino-maleimide to the suspension; as well as Diene-modified oligonucleotides are added to the aqueous suspension.

23. The method of claim 22, wherein the aminoalkyl carbodiimide comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

24. The method of claim 23, further comprising dissolving the aminoalkylcarbodiimide in a solvent and then adding the aminoalkylcarbodiimide to the suspension.

25. The method of claim 24, wherein the solvent comprises N-methylpyrrolidone.

26. The method according to any one of claims 22 to 25, wherein the addition of the aminoalkylcarbodiimide comprises the addition of the aminoalkylcarbodiimide in the range of 1 eq. to 1000 eq.

27. The method of claim 26, wherein the addition of the aminoalkylcarbodiimide comprises the addition of the aminoalkylcarbodiimide in the range of 100 eq to 700 eq.

28. The method according to any one of claims 22 to 27, wherein the hydroxysuccinimide comprises N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (S-NHS).

29. The method according to any one of claims 22 to 28, wherein the addition of the hydroxysuccinimide comprises the addition of the hydroxysuccinimide in the range of 0.5 eq. to 75 eq.

30. The method of claim 29, wherein the range is from 0.75 eq. to 20 eq.

31. The method according to any one of claims 22 to 30, further comprising incubating the suspension at a temperature in the range of 0°C to 25°C after adding hydroxysuccinimide.

32. The method of claim 31, wherein incubation comprises a period of time ranging from 10 minutes to 10 hours.

33. The method of claim 32, further comprising, after adding amino-maleimide, incubating the suspension at a temperature in the range of 3°C to 35°C.

34. The method of claim 33, wherein incubating the suspension after adding the amino-maleimide comprises a period of incubation lasting from 30 minutes to 16 hours.

35. The method according to any one of claims 22 to 34, wherein adding the diene-modified oligonucleotide comprises adding the diene-modified oligonucleotide to a range of 1 eq to 50 eq.

36. The method according to any one of claims 22 to 35, wherein the range is 5 eq to 20 eq.

37. The method of claim 36, further comprising, after adding the diene-modified oligonucleotide, incubating the suspension at a temperature in the range of 23°C to 80°C.

38. The method of claim 37, wherein the temperature is in the range of 35°C to 70°C.

39. The method of claim 37, wherein incubation at a temperature in the range of 23°C to 80°C comprises incubation lasting for a period of time in the range of 30 minutes to 60 hours.

40. The method of claim 39, wherein the time period is in the range of 1 hour to 50 hours.

41. The method according to any one of claims 22 to 40, wherein the bead carrier comprises a polyacrylamide polymer matrix.

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