Oligonucleotide probe array with electronic detection system

Abstract

The present disclosure provides methods, devices and systems for hybridization detection of nucleic acid targets. The detection can use a probe array with addressable micropores. Each micropore can retain multiple copies of a probe that can be complementary to a portion of a specific nucleic acid target. Multiple copies of the probe can be generated from a pooled library of probe sequences by amplification techniques, including rolling circle amplification and emulsion PCR. The detection of hybridization may rely on methods using ion-sensitive field effect transistors (ISFETs) and optical detectors to detect signals indicating the presence of nucleic acid targets.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 512,855, filed May 31, 2017, which is incorporated herein by reference in its entirety. Background Art

[0003] The detection of different nucleic acid sequences in biological samples is crucial in many fields, including identification of microorganisms, diagnosis of infectious diseases, detection of genetic abnormalities, identification of biomarkers associated with various cancers, assessment of genetic susceptibility to selected diseases, and evaluation of patient response to medical treatment. Nucleic acid hybridization is a common technique for detecting different nucleic acid sequences.

[0004] Nucleic acid hybridization can be a molecular biology technique in which a single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule is annealed to a complementary DNA or RNA sequence. Specifically, the technique can measure the sequence similarity between DNA polymers (polynucleotides). The basic principle of hybridization may be to establish DNA polymer units (i.e., nucleotides) including specific nitrogenous nucleobases (guanine "G", adenine "A", thymine "T" and cytosine "C"), which are all able to pair with complementary nucleobases (A pairs with T, C pairs with G) to form hydrogen bonds (two (2) hydrogen bonds between AT and three (3) hydrogen bonds between CG). As a result, two single-stranded DNA portions with complementary sequences can bind to each other (hybridize) and form a DNA dimer (double-stranded DNA structure).

[0005] The sequence-dependent nature of the hybridization signal generated by a labeled probe upon successful hybridization of the probe to its target can be the basis for many research and diagnostic applications. This approach has enabled the detection and quantification of DNA / RNA on solid phase blots, cytogenetic localization of DNA / RNA on cells, detection and purification of specific DNA, and comparative gene expression analysis, among other applications. Recently, the principles of nucleic acid hybridization have been combined with next-generation sequencing technologies to create powerful new analytical platforms that are expected to expand the research tools for genomics and personalized medicine. Therefore, new hybridization methods may be of interest to the biomedical field. Summary of the invention

[0006] Using the methods, devices and systems provided in the present disclosure, hybridization between a target nucleic acid and multiple copies of a probe can be detected in a single sample chamber using a probe array on a solid surface and a matching sensor array. The sample chamber and the matching sensor array can be addressed separately. In addition, the probe array does not have to be permanently associated with the same addressing position on the solid surface.

[0007] One aspect of the present disclosure provides a method for analyzing the presence or absence of a target nucleic acid in a sample, the method comprising: (a) providing a chip, the chip comprising a sensor adjacent to a sample chamber, wherein the sample chamber is configured to retain multiple copies of a probe, wherein the probe is selectively coupled to the target nucleic acid, and wherein the sensor detects at least one signal indicating whether the target nucleic acid is present in the sample chamber; (b) providing the sample in the sample chamber under conditions that allow the probe to selectively couple to the target nucleic acid; (c) measuring the at least one signal; and (d) determining the presence or absence of the target nucleic acid in the sample under conditions where the multiple copies of the probe are not covalently attached to the surface of the sample chamber.

[0008] In some embodiments provided herein, the sensor is in an array of multiple sensors in the chip. In addition, the sample chamber is in an array of multiple sample chambers in the chip. In addition, each of the multiple sample chambers is adjacent to at least one sensor in the array of the multiple sensors. In some embodiments provided herein, the target nucleic acid is a fragment of a larger nucleic acid. In some embodiments provided herein, the probe is part of a library of multiple probes.

[0009] In some embodiments provided herein, the probe comprises: 1) a sequence of a nucleic acid fragment complementary to at least a portion of the target nucleic acid; 2) a barcode sequence attached to the first end of the nucleic acid fragment sequence in 1). In some embodiments provided herein, the method further comprises, before step (b): (a1) circularizing the probe template coupled with a linker; (a2) amplifying the probe template coupled with a linker to form a linearly amplified concatemer molecule comprising multiple copies of the probe. In some embodiments provided herein, the barcode sequence is 3 to 30 nucleotides in length. In some embodiments provided herein, the method further comprises, before step (b): (a1) delivering a single copy of the template of the probe, or a single copy of the double-stranded nucleic acid containing the probe, to an aqueous microreactor in an oil-in-water emulsion, wherein the microreactor comprises a plurality of primers capable of annealing to the probe, a single microbead, the microbead being capable of binding to the template of the probe and amplifying a first copy of the probe attached to the surface of the microbead, and an amplification reaction solution containing reagents sufficient to perform a nucleic acid amplification reaction; (a2) subjecting the microreactor to a nucleic acid amplification reaction under conditions that produce a first copy of the probe; (a3) ​​repeating step (a2) multiple times; (a4) destroying the oil-in-water emulsion and producing multiple copies of the probe attached to the microbead. The microreactor may be a bead, a partitioned area, a hole, etc.

[0010] In some examples of aspects provided herein, the sample includes a plurality of target nucleic acid molecules containing a target nucleic acid. In some examples of aspects provided herein, the sensor is part of an array of a plurality of sensors, wherein each of the plurality of sensors detects the presence or absence of at least one of the plurality of target nucleic acid molecules, and wherein each sensor in the array of the plurality of sensors is individually addressable. In some examples of aspects provided herein, each probe includes: 1) a sequence of a nucleic acid fragment complementary to a portion of one of the target nucleic acids; and 2) a barcode sequence attached to a first end of the nucleic acid fragment sequence; and wherein the method further includes in (d): (d1) decoding the barcode sequence on the probe retained in the corresponding sample chamber connected to the individually addressable sensor.

[0011] In some embodiments of aspects provided herein, wherein the chip includes additional sensors, wherein the sample comprises the target nucleic acid and additional target nucleic acid, and wherein the additional sensor detects the additional target nucleic acid. In some embodiments of aspects provided herein, the additional sensor is adjacent to an additional sample chamber, wherein the additional sample chamber is configured to retain multiple copies of the additional probe, wherein the additional probe is selectively coupled to the additional target nucleic acid.

[0012] In some embodiments of the aspects provided herein, the sensor comprises an ion sensitive field effect transistor (ISFET). In some embodiments of the aspects provided herein, the sensor comprises a chemical sensitive field effect transistor (chemFET). In some embodiments of the aspects provided herein, the sensor comprises an optical sensor. In some embodiments of the aspects provided herein, at least one signal comprises a change in pH.

[0013] Another aspect of the present disclosure provides a system for analyzing the presence of a target nucleic acid in a sample, the system comprising: a chip comprising a sensor adjacent to a sample chamber, wherein the sample chamber is configured to retain the sample having the target nucleic acid and multiple copies of a probe, wherein the probe is selectively coupled to the target nucleic acid, wherein the multiple copies of the probe are not covalently attached to a surface of the sample chamber, and wherein the sensor detects at least one signal from the sample, the at least one signal indicating the presence or absence of the target nucleic acid; a computer processor coupled to the chip, which is programmed to (i) measure at least one signal while contacting the chip with the sample; and (ii) determine the presence or absence of the target nucleic acid in the sample.

[0014] In some embodiments provided herein, the sensor is in an array of multiple sensors in the chip. In addition, the sample chamber is in an array of multiple sample chambers in the chip. In addition, each of the multiple sample chambers is adjacent to at least one sensor in the array of the multiple sensors, and each of the sensors is individually addressable. In some embodiments of the aspects provided herein, the computer processor is further programmed to (iii) map the array of the individually addressable sensors; and (iv) when the probe comprises a barcode sequence, associate the barcode sequence with the corresponding individually addressable sensor. In some embodiments of the aspects provided herein, the array of multiple sensors includes at least 96 sensors.

[0015] In some embodiments of aspects provided herein, the sensor is an ion-sensitive field effect transistor (ISFET), a chemical-sensitive field effect transistor (chemFET) or an optical sensor. In some embodiments of aspects provided herein, the chip includes an additional sensor, and wherein the sample includes an additional target nucleic acid. In some embodiments of aspects provided herein, the additional sensor detects at least one additional signal, and the additional signal indicates the presence or absence of the additional target nucleic acid.

[0016] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the detailed description below. Wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure can include other, different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be considered illustrative in nature, rather than restrictive.

[0017] Incorporation by reference

[0018] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that an incorporated by reference publication, patent, or patent application contradicts the disclosure contained herein, this document is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments of the present invention, the principles used in the scheme, and the accompanying drawings (also referred to as "figures" or "diagrams"). Among them:

[0020] Figure 1An example probe 100 according to the present disclosure is shown.

[0021] Figure 2 Non-exclusive examples of circular DNA templates 100 ′ for synthesizing probe libraries according to the present disclosure are shown.

[0022] Figure 3 An illustrative example of a chip 200 according to the present disclosure is shown.

[0023] Figure 4 Illustrative examples of sample chambers 202A and 202B in accordance with the present disclosure are shown.

[0024] Figure 5 Shown is a schematic diagram for detecting a signal indicative of hybridization of a probe to a target nucleic acid according to the present disclosure.

[0025] Figure 6 A schematic diagram of an emulsion polymerase chain reaction according to the present disclosure is shown.

[0026] Figure 7 An example schematic diagram of a computer system programmed or otherwise configured to implement the methods provided herein is shown. DETAILED DESCRIPTION OF THE INVENTION

[0028] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may propose many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0029] Where values ​​are described as a range, it should be understood that the disclosure includes all possible sub-ranges within that range, as well as specific values ​​falling within that range, whether or not a specific value or sub-range is explicitly stated.

[0030] Scientists and researchers can use nucleotide sequence information in different ways to improve human life, such as increasing crop yields, producing better fuels, producing better vaccines, producing more effective drugs, preventing diseases or preventing outbreaks of dangerous pathogens, through clinical methods or material methods. (See: Ansorge, W., "Next-generation DNA sequencing techniques," New Biotech., 25(4): 195-203, 2009). In this effort, the determination and / or detection of specific nucleotide sequences may be crucial. Hybridization determination of nucleotide sequences may be an important tool for such research.

[0031] In many hybridization detections of nucleic acid target molecules, a probe or multiple copies of a probe can be directly attached or bound to a surface adjacent to a detector. This can provide a one-to-one correlation between the sequence of the probe and the signal captured by the corresponding detector. In some hybridization detections, because the concentration of the target nucleic acid molecule in the original sample is very low, a nucleic acid amplification reaction can be used to generate a higher copy number and / or a higher concentration of the target nucleic acid molecule to facilitate hybridization detection.

[0032] One type of nucleic acid amplification can be the polymerase chain reaction (PCR), which includes repeated cycles of denaturing the template strand of DNA, annealing a matching primer pair to the DNA template, and extending the DNA from the primers by a DNA polymerase. A popular PCR method used in DNA library construction is called emulsion PCR with microbeads (e-PCR). E-PCR methods can be used on platforms such as Roche's 454 (Margulies et al., "Genome Sequencing in Microfabricated High-density Picolitre Reactors," Nature 437(7057):376–380, 2005), Life Technologies' SOLiD (Valouev A et al., "A High-resolution, Mucleosome Position Map of C.elegans Reveals a Lack of Universal Sequence-dictated Positioning," Genome Res. 18(7):1051–1063, 2008), and Ion Torrent (Rothberg et al., "An Integrated Semiconductor Device Enabling Non-optical Genome Sequencing," Nature 475(7356):348–352, 2011). E-PCR may require PCR on billions of microbeads, each of which is separated in its own emulsion droplet, followed by emulsion fragmentation, template enrichment, and bead deposition before sequencing.

[0033] Another type of nucleic acid amplification can be rolling circle amplification (RCA) under isothermal conditions. RCA technology can use Phi29 DNA polymerase to amplify circular template nucleic acid to provide a longer (sometimes longer than 10kb) single-stranded linear DNA molecule that contains a tandem copy of the template nucleic acid sequence. (Blanco et al., "Highly Efficient DNA Synthesis by the Phageφ29 DNA Polymerase," J. Biol. Chem. 264, 8935, 1989; Drmanac et al., "Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays," Science 327 (5961): 78-81, 2010). The product thus formed can include hundreds of tandem copies of the desired nucleic acid sequence to form a concatemer for enhancing the signal intensity from subsequent hybridization reactions. In addition, the concatemer can include internal sequences that promote the formation of secondary structures, thereby causing densification through internal hybridization. (Drmanac et al., 2010). The resulting dense structure of nucleic acid molecule concatemers may sometimes be referred to as a "nanoball" (NB). A nucleic acid nanoball may generally refer to a nucleic acid particle having nanometer dimensions in at least one dimension.

[0034] Large-scale multiplex analysis of multiple target nucleic acids in biological samples may be required in forensics, diagnostics, and medical procedures. Therefore, an assay device and method with better performance, such as better signal-to-noise ratio, may be desired.

[0035] When faced with the problem of low concentration of target nucleic acid molecules in samples, current possible methods include using PCR or other amplification techniques to prepare multiple copies of target nucleic acid molecules and subjecting the amplicons to probes for detection. One benefit of this target amplification (amplification-of-target) method is that it can give the amplicon of the target nucleic acid molecule a better chance of hybridizing with the probe. However, the amplification reaction may require time and special equipment to complete. Further processing of the sample before the hybridization assay can extend the response time of the assay. It is best to perform a hybridization test and use the sample directly without the step of amplifying the target nucleic acid molecule.

[0036] After extensive experimentation, the applicant has discovered a new device and method for target nucleic acid hybridization assays. For example, instead of amplifying target nucleic acid molecules, probes can be amplified to produce multiple copies, and these probes can be positioned at specific locations on a detection device, so that even if the concentration of target nucleic acid molecules in the sample may be low, due to the absolute high number of probes at that location, the target nucleic acid molecules may find copies of the probes and hybridize with them.

[0037] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes a plurality of such molecules, and so forth.

[0038] As used herein, the term "fragment" generally refers to a portion of an original DNA sequence or RNA sequence of a specific region.

[0039] As used herein, the term "target nucleic acid" generally refers to a target nucleic acid fragment for detection using the hybridization method disclosed herein. The source of the target nucleic acid can be isolated from an organism including a mammal or a pathogen to be identified including a virus and a bacterium. In addition, the target nucleic acid can also be from a synthetic source. The target nucleic acid may or may not be amplified by a standard replication / amplification step to produce a nucleic acid sequence.

[0040] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" generally refers to a nucleic acid molecule having a given nucleotide sequence, and it may be desirable to determine the presence or absence or quantity of a given nucleotide sequence. The nucleotide sequence may comprise ribonucleic acid (RNA) or DNA, or a sequence derived from RNA or DNA. Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA, including genomic DNA and messenger RNA. The length of the sequence may be any length that can be amplified into a nucleic acid amplification product or amplicon, such as a length of up to about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 or more than 10,000 nucleotides.

[0041] As used herein, the term "template" generally refers to a single polynucleotide molecule from which another nucleic acid, including a complementary nucleic acid strand, can be synthesized by a nucleic acid polymerase. In addition, a template can be one or two strands of a polynucleotide that can serve as a template for a template-dependent nucleic acid polymerization reaction catalyzed by a nucleic acid polymerase. The use of this term should not be construed as limiting the scope of the present disclosure to the polynucleotide that is actually used as a template in a subsequent enzyme-catalyzed polymerization reaction.

[0042] As used herein, the term "analyte" generally refers to a substance to be detected or determined by the methods of the present disclosure. It can be broadly interpreted as any compound, molecule or other substance of interest to be detected, identified or characterized. The analyte may include nucleic acid fragments, including DNA, RNA or synthetic analogs thereof. Other types of analytes may be chemical substances in the sample or analytical solution before, during or after the addition of the target nucleic acid, including ions. One or more reactive ligands may be incorporated into the nucleic acid analyte, which may be members of a binding pair. Such ligands are incorporated into the analyte in such a way that the ligand is able to react with the second member of the binding pair. The ligand may be coupled at the 3' end, the 5' end or any point between the 3' and 5' ends of the nucleic acid analyte. In addition, the reporter molecule portion may also be incorporated into the nucleic acid analyte in a manner similar to the ligand incorporation. The nucleic acid analyte may be ligand-free, but may also be incorporated with sequence fragments complementary to the nucleic acid fragments of other reagents contained in the assay system.

[0043] As used herein, the term "ligand" generally refers to one member of a binding pair that has been incorporated into a nucleic acid analyte, and may include, but is not limited to, an antibody, a lectin, a receptor, a binding protein, or a chemical reagent.

[0044] As used herein, the term "binding pair" can generally refer to any type of immunological binding pair, such as antigen / antibody or hapten / antihapten systems; and any type of non-immunological binding pair, such as biotin / avidin; biotin / streptavidin; folic acid / folate binding protein; complementary nucleic acid fragments; protein A or G / immunoglobulin; and binding pairs that form covalent bonds, such as sulfhydryl reactive groups, including maleimide and haloacetyl derivatives, and amine reactive groups, such as isotricyanate, succinimidyl ester and sulfonyl halide. (See Bobrow et al., "Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays," J. Immunol. Methods, 125: 279-85, 1989).

[0045] Specifically, for immunological binding pairs, the antibody members of the binding pair can be produced by various methods, whether polyclonal, monoclonal or immunoreactive fragments thereof. As used herein, the term "immunoreactive antibody fragment" or "immunoreactive fragment" generally refers to a fragment containing an antibody binding region. Such fragments can be Fab-type fragments defined as lacking the Fc portion, for example, Fab, Fab' and F(ab')2 fragments, or so-called "half-molecule" fragments obtained by reducing and cleaving the disulfide bonds connecting the heavy chain components of the intact antibody. If the antigen member of the specific binding pair is not immunogenic, such as a hapten, it can be covalently coupled to a carrier protein to make it immunogenic. A non-immunological binding pair refers to a system comprising two components that share a natural affinity for each other but are not antibodies.

[0046] As used herein, the term "reporter conjugate" generally refers to a conjugate comprising an enzyme, fluorescent molecule or radioactive label coupled to one member of a binding pair. The member of the binding pair can be an antibody, a nucleic acid sequence or some immunoreactive or affinity-reactive substrate.

[0047] As used herein, the term "reporter molecule" or "reporter moiety" generally refers to any entity capable of detection by enzymatic means or energy emission; including but not limited to a fluorescent moiety, a chemiluminescent moiety, a particle, an enzyme, a radioactive tag or a luminescent moiety or molecule.

[0048] As used herein, the term "particles" generally refers to latex particles that are dyed, submicron sized and uniform, but also includes other particles that can be detected.

[0049] As used herein, the term "conjugate" generally refers to two or more molecules (and / or materials such as nanoparticles) covalently linked to a larger structure. In some embodiments, the conjugate comprises one or more biomolecules (e.g., peptides, antibodies, nucleic acids, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, biopolymers (e.g., PEG) and lipoproteins) covalently linked to one or more other molecules, such as one or more other biomolecules. In other embodiments, the conjugate includes one or more specific binding molecules (e.g., antibodies and / or nucleic acid sequences) covalently linked to one or more detectable labels (e.g., fluorescent molecules, fluorescent nanoparticles, haptens, enzymes, and combinations thereof).

[0050] As used herein, the term "signal" generally refers to a variable that changes over time and is related to one or more properties of a sample being measured. A signal can be continuous or discrete in the time domain.

[0051] As used herein, the term "PCR" or "polymerase chain reaction" generally refers to the enzymatic replication of nucleic acids using, for example, thermal cycling to denature, extend, and anneal nucleic acids.

[0052] As used herein, the terms "probe", "capture probe" or "capture molecule" generally refer to a molecular species or other marker that can bind to a specific target nucleic acid sequence. The probe can be any type of molecule or particle. The probe can contain molecules and can be bound to a substrate or other solid surface directly or through a linker molecule. In the present disclosure, as described below, the probe may not be directly or indirectly bound to the surface of the sample chamber by covalent bonds. However, the probe may be restricted to move within the scope of the sample chamber where the probe is located. This restriction can be caused by charge-charge interactions or magnetic interactions. When the probe is a nucleic acid sequence, it can be a single-stranded sequence or a double-stranded sequence containing a single-stranded sequence of interest.

[0053] The association of two polynucleotides to form a stable duplex, e.g., under relevant assay conditions, can be referred to as "hybridization." Nucleic acids can hybridize due to a variety of well-known physicochemical forces, such as hydrogen bonding, solvent exclusion, base stacking, and the like. A comprehensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes (1993), Part I, Chapter 2, "Overview of Hybridization Principles and Nucleic Acid Probe Assay Strategies" (Elsevier, NY).

[0054] As used herein, the term "sensor" or "detector" generally refers to a device including optical, magnetic or electronic components that can detect a signal. In general, a sensor refers to a device that can be used to sense the presence or absence of an analyte. In particular, a sensor can be an environmental sensing device, such as an ion-sensitive and chemical-sensitive device, which generates an electrical signal (e.g., current, potential or conductivity) based on the presence or concentration of an analyte in a sample being measured. An optical sensor can be another type of sensor.

[0055] As used herein, the terms "about" or "almost" generally mean within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a specified value.

[0056] As used herein, the term "label" generally refers to a specific molecular structure that can be attached to a target molecule and makes the target molecule distinguishable and traceable by providing a unique feature that is not inherent to the target molecule. A label can include a molecular structure that provides unique features that are not inherent to those nucleic acid molecules once attached to a nucleic acid sequence. For example, a label with unique optical properties can be created. Sometimes, an optical label can be used. An optical label can be used alone as a signal generating entity, or in a bimolecular reporting system, as an energy donor, energy acceptor, or otherwise. Acceptors and donors can both be fluorophore molecules. Whether a fluorophore is a donor or an acceptor depends on its excitation and emission spectra, as well as the fluorophore paired with it.

[0057] In some cases, incorporated non-radioactive labels can be used for the detection of nucleic acids. For example, nucleic acids modified with biotin (U.S. Pat. No. 4,687,732; European Patent No. 063879), digoxigenin (European Patent No. 173251), and other hapten-modified nucleic acids can be used. For example, U.S. Pat. No. 5,344,757 uses a nucleic acid probe containing at least one hapten as a label to hybridize with a complementary target nucleic acid bound to a solid membrane. The sensitivity and specificity of these assays can be based on the incorporation of a single marker through an amplification reaction and detection with an antibody specific for the marker. In some cases, antibodies coupled to an enzyme may be involved. In some cases, the addition of a substrate produces a calorimetric or fluorescent change that can be detected by an instrument.

[0058] The term "complementary" refers to a polynucleotide that forms a stable duplex with its "complement" under relevant assay conditions. Two polynucleotide sequences that are complementary to each other have less than about 20% base mismatches, less than about 10% base mismatches, less than about 5% base mismatches, or no mismatches.

[0059] A "polynucleotide sequence", "nucleotide sequence" or "nucleic acid sequence" is a polymer of nucleotides (oligonucleotide, DNA, nucleic acid, etc.) or a string of characters representing a polymer of nucleotides, depending on the context. From any given polynucleotide sequence, a given nucleic acid or a complementary polynucleotide sequence (e.g., a complementary nucleic acid) can be determined.

[0060] As used herein, the term "DNA polymerase" generally refers to a cellular or viral enzyme that synthesizes DNA molecules using nucleotide building blocks.

[0061] As used herein, the term "chemically sensitive field effect transistor" or "chemFET" refers generally to the type of detector, regardless of the specific chemical system for which the device is suitable, or with which it interacts. A chemFET can be a chemical sensor that can detect a target chemical process by detecting a threshold voltage affected by modulation of the channel conductance of the device.

[0062] As used herein, the term "ion-sensitive field effect transistor" or "ISFET" generally refers to an impedance-converting device configured to selectively measure ionic activity in solution (e.g., hydrogen ions in solution). One operating principle of an ISFET is given in "30 Years of ISFETology: What Happened in the Past 30 Years and What Might Happen in the Next 30 Years" (Bergveld, Sens. Actuators, 88: 1-20 (2003)). The present disclosure is not limited to any particular class of chemFETs or ISFETs, and the use of these terms should not be interpreted in a limiting sense.

[0063] When describing a device, system, sensor, sample chamber, etc., the term "array" as used herein generally refers to a one-dimensional or two-dimensional set of microstructures. The array can be of any shape. For example, the array can be a series of microstructures arranged in a line, such as a square array. The array can be arranged in a square or rectangular grid. Some parts of the array may be spatially separated from other parts. The array can have other shapes. For example, the array can be a series of microstructures arranged in a series of concentric circles, a series of concentric squares, a series of concentric triangles, a series of curves, etc. The spacing between the parts or between the microstructures in any array can be regular, or it can be different between specific parts or between specific pairs of microstructures. The microstructure array of the present disclosure may include microstructures having zero-dimensional, one-dimensional or two-dimensional shapes. Microstructures having a two-dimensional shape may have shapes such as squares, rectangles, circles, parallelograms, pentagons, hexagons, irregular shapes, etc.

[0064] Device and method

[0065] The present disclosure provides methods, devices and systems capable of multiplex detection of nucleic acid hybridization reactions. The methods, devices and systems of the present disclosure may include components including, but not limited to:

[0066] 1. A sample chamber, which may include an aqueous environment, in which there are multiple freely moving target nucleic acids to be analyzed. There may be an array of sample chambers. Each sample chamber in the array may be at an individually addressable location on a solid surface. Each addressable location (or "pixel") may retain multiple copies of a nucleic acid sequence that can specifically hybridize with a particular target. As described below, each addressable location may not accommodate more than one nucleic acid probe. The array of sample chambers may be an array of microwells on a semiconductor chip.

[0067] 2. A probe array, which may contain multiple nucleic acid probes. The probe array may be docked with a sample chamber array so that each probe corresponds to one sample chamber and thus becomes individually addressable. Each probe may contain multiple copies of a nucleic acid sequence that can specifically hybridize to a particular target; and

[0068] 3. A sensor that can measure the signal generated at each pixel in parallel. As the hybridization event progresses, the signal can be related to the presence and activity of the label or reporter in its vicinity. The signal can be a discrete (e.g., individually distinguishable) signal.

[0069] The probe array may include independently addressable positions, each position having one or more probes. The probe at a given independently addressable position of the array may be the same or different from the probe at other independently addressable positions of the array. In some cases, the probes of a group of positions in the array may be the same.

[0070] Additionally, the apparatus may include a computer system.

[0071] The methods, devices and systems of the present disclosure may employ variations of the above-described components assembled together to create a system capable of measuring nucleic acid hybridization reactions in parallel.

[0072] Targets for analysis

[0073] Genetic materials that can be used as targets of the present disclosure may include, but are not limited to, DNA and RNA. There may be many different types of RNA and DNA, all of which have been and will continue to be the subject of a large number of studies and experiments. DNA targets may include, but are not limited to, genomic DNA (gDNA), chromosomal DNA, mitochondrial DNA (mtDNA), plasmid DNA, ancient DNA (aDNA), all forms of DNA, including A-DNA, B-DNA and Z-DNA, branched DNA and non-coding DNA. The RNA forms that can be sequenced using the methods and compositions of the present invention include, but are not limited to, messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA, small RNA, snRNA and non-coding RNA. (See Limbach et al., "Summary: The modified nucleosides of RNA," Nuc. Acids Res., 22 (12): 2183-2196, 1994).

[0074] Nucleotides may include, but are not limited to, naturally occurring nucleotides G, C, A, T, and U, as well as rare forms such as inosine, xanthine, 7-methylguanosine, dihydrouridine, 5-methylcytosine, and pseudouridine, including methylated forms of G, A, T, and C, etc. (See, e.g., Korlach et al., "Going beyond five bases in DNA sequencing," Curr. Op. Struct. Biol., 22(3): 251-261, 2012; and U.S. Pat. No. 5,646,269). Nucleosides may also be non-naturally occurring molecules, such as molecules containing 7-deazapurine, pyrazolo[3,4-d]pyrimidine, propynyl-dN, or other analogs or derivatives. Exemplary nucleosides include ribonucleosides, deoxyribonucleosides, dideoxyribonucleosides, carbocyclic nucleosides, etc.

[0075] sample

[0076] Generally speaking, any sample containing genetic material having a nucleotide sequence of interest may be suitable for use in the present disclosure. Samples may be obtained from eukaryotes, prokaryotes, and archaea. For example, samples containing genetic material whose sequence may be determined using the present disclosure may be obtained from, for example, bacteria, bacteriophages, viruses, transposons, mammals, plants, fish, insects, and the like.

[0077] The sample may be of human origin and may be obtained from any human tissue containing genetic material. Typically, the sample may be a fluid sample, such as, but not limited to, normal and pathological body fluids and aspirates of these fluids.

[0078] Purification / isolation of DNA samples for analysis

[0079] In order to prepare a sample for determining or detecting the sequence of genetic information contained therein, the genetic material may be separated and / or purified from other components of the original sample. There may be methods for purifying nucleic acid material from a sample. (See, for example, Kennedy, S., "Isolation of DNA and RNA from soil using two different methods optimized with Inhibitor Removal (IRT), "BioTechniques, p.19, Nov.2009; "Molecular Cloning - Laboratory Manual" (4th Edition) Green, M. and J. Sambrook, Cold Spring Harbor Laboratory Press, USA, 2012; Methods and Tools in Biological Sciences and Medicine, Molecular Systems and Evolutionary Technologies, DeSalle et al., 2002, Verlag Basel / Switzerland; Keb-Llanes et al., Plant Molecular Biology Reports, 20:299a-299e, 2002).

[0080] Fragmentation of DNA samples to generate targets for analysis

[0081] Fragmentation of polynucleotide targets in a DNA sample may be performed prior to utilizing the various methods and devices disclosed in the present disclosure. These methods may include ultrasonic treatment, nebulization, hydraulic shearing and shearing by other mechanical methods, such as using microbeads, needle shearing, French press and sonic shearing, restriction endonuclease digestion and other enzymatic methods, such as using various combinations of nucleases (DNA enzymes, exonucleases, endonucleases, etc.), and transposon-based methods. (See Knierim et al., "Systematic comparison of three methods for fragmentation of long-range PCR products for next-generation sequencing," PLoS One, 6(11): e28240, 2011; Quail, MA, "DNA: Mechanical fragmentation," Nov. 15, 2010, eLS; Sambrook, J., "Nebulization fragmentation of DNA," Cold Spring Harb. Protoc., doi: 10.1101 / pdb.prot4539, 2006). Generally, the goal may be to obtain polynucleotides of a base pair (bp) size range suitable for the assay method selected. For example, the fragment can be about 50 bp, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp or more.

[0082] In one embodiment, the DNA sample can be fragmented by chemical, enzymatic or physical methods. Fragmentation can be performed by enzymatic or mechanical methods. Mechanical methods can be ultrasonic treatment or physical shearing. Enzymatic methods can be performed by digestion with nucleases (e.g., deoxyribonuclease I (DNase I)) or one or more restriction endonucleases. In some embodiments, fragmentation may result in unknown terminal sequences.

[0083] In another embodiment, the enzymatic method may use DNase I. DNase I may be an enzyme that non-specifically cleaves double-stranded DNA (dsDNA) to release 5'-phosphorylated dimers, trimers, and oligonucleotide products. 2+ ,Mg 2+ and Ca 2+The purpose of the DNase I digestion step may be to fragment a large DNA genome into a library of smaller fragments. When a manganese-based buffer is used, the cleavage properties of DNase I may result in random digestion of the substrate DNA (i.e., the digestion has no preference for the sequence of the DNA molecule), and when a magnesium-based buffer is used, it may result in a dominance of double-stranded DNA fragments with blunt ends (Melgar and Goldthwaite, "Deoxyribonuclease II. Effects of Metals on the Mechanism of Action of DNase I", J. Biol. Chem. 243 (17): 4409-16, 1968). The size range of the product produced after DNase I treatment of the genomic template may depend on three factors: i) the amount of enzyme used (enzyme activity units); ii) the digestion temperature (°C); iii) the incubation time (minutes). DNase I digestion can be optimized to produce a genomic library with a size range of about 50 to about 700 bp.

[0084] In one embodiment, DNase I can digest large substrate DNA or whole genomic DNA for about 1 or about 2 minutes to produce a fragmented polynucleotide population. In another embodiment, DNase I digestion can be performed at a temperature between about 10° C. and about 37° C. In another embodiment, the length of the digested DNA fragments can be between 50 bp and 700 bp.

[0085] Furthermore, in some embodiments, in the case of Mn 2+ Digestion of genomic DNA (gDNA) substrate with DNase I in the presence of can produce DNA fragments with blunt ends or terminal overhangs of one or two nucleotides in length. In one embodiment, Pfu DNA polymerase can be used to increase the number of blunt ends produced. Fragment polishing using Pfu DNA polymerase may result in filling of 5' overhangs. In addition, Pfu DNA polymerase may remove mononucleotide and dinucleotide extensions, thereby further increasing the number of blunt-ended DNA fragments available for ligation to adapters (Costa and Weiner, "Protocols for cloning and analysis of PCR-generated blunt-ended DNA fragments," PCR Methods Appl 3(5):S95-106, 1994; Costa et al., "Cloning and analysis of PCR-generated DNA fragments," PCR Methods Appl 3(6):338-45, 1994; Costa and Weiner, "Polishing with T4 or Pfu polymerases improves cloning efficiency of PCR products," Nucleic Acids Res. 22(12):2423, 1994).

[0086] Amplification of nucleic acid sequences

[0087] Methods for amplifying genetic material can include whole genome amplification (WGA) (see, e.g., Lovmar et al., "Multiple displacement amplification to create a long-lasting DNA source for genetic research", Hum. Mutat., 27: 603-614, 2006). Amplification of nucleic acid sequences can be performed using any of a variety of PCR techniques and non-PCR techniques, including but not limited to e-PCR, RCA, simultaneous targeting of RNA and DNA for amplification in a transcription-mediated manner, nucleic acid sequence-based amplification (NASBA) for isothermal amplification, helicase-dependent isothermal amplification, strand displacement amplification (SDA), Q-beta replicase-based methods, ligase chain reaction, loop-mediated isothermal amplification (LAMP) and reactive displacement chimerism (RDC).

[0088] Sample room

[0089] The sample chamber may have a volume of about 10 nanoliters (nL) to about 10 milliliters (mL). In some cases, the sample chamber volume may be about 10nL to about 100nL, about 100nL to about 200nL, about 200nL to about 500nL, about 500nL to about 1 microliter (μL), and about 1μL to 100μL. The sample chamber volume may be at least about 10nL, 100nL, 1μL, 10μL, 100μL, 1mL or 10mL. The depth of the sample chamber may be controlled when considering the following factors: 1) the size of the nanospheres or microbeads retained in each sample chamber; 2) the amount of solution allowed to enter the sample chamber to facilitate hybridization and washing steps, and 3) the balance between retaining the nanospheres or microbeads and washing away unbound target nucleic acids after the hybridization step, etc. The depth of the sample chamber may be about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 1000 nm, or greater than 1000 nm.

[0090] The sample chamber may contain an aqueous solution. The aqueous solution in the sample chamber may include a buffer salt-based solution, such as an aqueous solution containing a mixture of a weak acid and its conjugate base, or vice versa. The solution may contain multiple target nucleic acid sequences, generally referred to herein as "targets". The sample chamber may accommodate multiple copies of a probe. The sample chamber may retain multiple copies of a probe, and may not accommodate two different probes, each of which has multiple copies. The sample chamber may take any 3-D shape that is configured to hold multiple copies of a probe. For example, the volume of the chamber may be adjusted when considering the average size of multiple probes to be retained. In addition, the design of the sample chamber may include a accommodating portion for an associated sensor, so that a signal generated by hybridization between an indication probe and a target nucleic acid within the sample chamber range may be detected by an associated sensor. Each sample chamber and its associated sensor may be individually addressable as a pixel.

[0091] The sample chamber can be in fluidic contact with other components, including but not limited to microfluidic channels. It can also allow the addition of other non-fluidic objects, such as microbeads or nanospheres (NB).

[0092] Probe array

[0093] A feature of the present disclosure may be that the device has a dynamic probe array rather than a static / fixed probe array. Many hybridization assays can construct their assay platforms by attaching probes or copies of the same probe to the pixel surface via covalent chemical bonds. The probe arrays thus obtained may be static or fixed, and the probes may be permanently associated with the same physical location and specific pixels, and will not change between different detection methods. Therefore, when a different probe array is needed, a new chip may have to be rebuilt and the old chip may be discarded.

[0094] In contrast, the present invention can use dynamic probe arrays, i.e., each probe or copy of each probe can change its physical position, or pixel, on the chip between different methods, so there is no need to abandon the array when using different detection methods. Therefore, the pixels associated with one or more probes may not be permanently fixed and may be determined separately during each detection process. One way to determine the pixels associated with one or more probes of a specific assay can be to rely on the use of a barcode sequence attached to each probe after the one or more probes are retained in a specific sample chamber. Other ways to determine the pixels associated with one or more probes may exist.

[0095] In some cases, oligonucleotides can be used as probes. "Oligonucleotides" as used herein can comprise a single-stranded nucleic acid. The length of the oligonucleotide can be 2 to about 1000 nucleotides, 2 to about 500 nucleotides, 10 to about 100 nucleotides, or 20 to about 50 nucleotides. In the methods, devices and systems disclosed herein, the probe can be attached to a solid substrate, or may not be attached to a solid substrate. The probe can be bound to the substrate directly or through a joint. The joint can comprise, for example, amino acids, polypeptides, nucleotides or oligonucleotides.

[0096] The solid matrix to which the probe is bound can be biological, non-biological, organic, inorganic or any combination thereof. The matrix can be, for example, in the form of one or more particles, segments, precipitates, gels, sheets, tubes, balls, containers, capillaries, pads, slices, films, plates or slides. However, the matrix may not be one side of the sample chamber or the surface of the sensor / detector. The solid matrix can be flat, or there can be other surface structures. For example, the solid matrix can include raised or depressed areas formed after synthesis or deposition thereon. In some examples, the solid matrix can be selected to provide appropriate light absorption characteristics. For example, the matrix can be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon or any of a variety of gels or polymers, such as polycarbonate, (poly) tetrafluoroethylene, polystyrene, (poly) vinylidene fluoride or a combination thereof.

[0097] Each probe or each of a plurality of probes can be held in an addressable sample chamber, which is identified or referred to as a "pixel" on the chip. In some cases, the chip can contain at least about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000, or more pixels holding probes. In some cases, a chip can contain up to about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000, or more pixels retaining probes. In some cases, a chip may contain about 2, 3, 4, 5, 6, or more than 7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000, or 1,000,000 pixels retaining probes.

[0098] In some cases, it may be useful to have some pixels without retaining probes. Such pixels can serve as control points to improve the quality of the measurement, for example, by using binding to the site to estimate and correct for nonspecific binding of the analyte. In some cases, such control points can be used to represent background signal.

[0099] In some cases, it may be useful to use a redundant pixel that has the same probe sequence as another pixel, but may not be physically adjacent or proximal to the other pixel. Data acquired by such a probe array may be less susceptible to non-ideal manufacturing and measurement errors.

[0100] Detectors and detection systems

[0101] The present disclosure provides a detector that can be used to detect a signal. Such a signal can be used to monitor the progress of nucleic acid hybridization. Such a detector can be an optical detector for measuring a light signal, an electrochemical detector for measuring an electrochemical signal, or an electrostatic detector for measuring an electric charge.

[0102] The signal detected by the detector may include information conveying the presence and / or amount of the marker, including the activity level of the marker at all pixels. The signal may be an optical signal, such as fluorescence or chemiluminescence. The signal may be an electrical signal, such as an electrochemical signal, an electrostatic signal, a resistance, a capacitance, or an inductance. The signal may be processed, including normalization based on a background signal.

[0103] Examples of optical detectors include, but are not limited to, a charge coupled device (CCD) array (including a cooled CCD), a complementary metal oxide semiconductor (CMOS) imager, an n-type metal oxide semiconductor (NMOS), an active pixel sensor (APS), or a photomultiplier tube (PMT). The detector may also include a wavelength selective component, such as an optical filter, to allow selection of a wavelength for measurement. Other examples of detectors include electrodes.

[0104] 53, 54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, 300, 400, 500, 1000, 10,000 times per minute.

[0105] The detector may include a light source. The light source may, for example, be used to excite fluorescence and / or colorimetric markers. The light source may include at least one lamp, such as an incandescent lamp, a halogen lamp, a fluorescent lamp, a gas discharge lamp, an arc lamp, or a light emitting diode (LED). The light source may include a laser. The light source may produce a specific wavelength or wavelength range, such as UV. The light source may include a filter for controlling the output spectrum, a single wavelength or multiple wavelengths. The light source may include multiple light sources of the same or different types, which may be used separately or in combination.

[0106] The detector may include various optical elements, including but not limited to filters, lenses, collimators, mirrors, reflectors, beam splitters, and diffusers. The detector may include one or more filters, including but not limited to wavelength filters (e.g., color filters, UV filters, IR filters), dichroic filter light sheets, and polarization filters. The filter may include multiple filters of the same or different types, which may be used separately or in combination. The detector may include elements (e.g., signal processing units) for eliminating image distortion or aberrations, such as barrel or fisheye distortion, pincushion distortion, whisker distortion, monochromatic aberrations (e.g., Piston, tilt, defocus, spherical aberration, coma, astigmatism, field curvature, image distortion) or chromatic aberrations (e.g., axial, longitudinal, transverse, lateral). Such elements may include a computer system programmed to implement instructions for partially or completely correcting image distortions. For example, Brown's deformation model or Brown-Conrady model may be used to correct radial distortion and tangential distortion.

[0107] In some examples, the detector can measure photons emitted from each pixel. These photons can be correlated to the presence and / or activity of the optical marker in that area.

[0108] In some cases, the detector may include an integrated biosensor array, which may be constructed using CMOS integrated circuit (IC) fabrication processes (Plummer et al., "Silicon Technology: Fundamentals, Practice, and Modeling," Prentice Hall Electronics and VLSI Series, 2000). In such a system, referred to herein as a "CMOS biochip," the sample chamber array may be placed on top of a CMOS biochip.

[0109] A detection system is also provided herein, having at least one detector configured to capture, detect and / or monitor signals from a probe array. Various signals can be generated, such as optical, electrical, electrochemical, magnetic, mechanical, acoustic or electromagnetic signals. The signal can be related to the presence, amount, concentration and / or binding characteristics of one or more analytes and reagents. The signal can reflect or indicate the progress of one or more reactions (e.g., hybridization). The signal can be detected at a single time point, multiple time points or in real time.

[0110] The detection system can include a single or multiple detectors (e.g., a detector array). The detector can be fixed or movable. The detector can scan the probe array so that a given detector detects signals from different addressable locations of the chip during hybridization. In the case where multiple detectors are included in the detection system, each detector can record a specific addressable location of a signal associated with multiple copies of the retained probe when detecting or measuring the signal.

[0111] Depending on the type of signal to be detected, various types of detectors can be used in the detection system. It can include, for example, an optical detector, an electrical detector, an electrochemical detector, or an electrostatic detector. Examples of optical detectors can include, but are not limited to, chemFETs, ISFETs, charge coupled device (CCD) arrays (including cooled CCDs), complementary metal oxide semiconductor (CMOS) imagers, n-type metal oxide semiconductors (NMOS), active pixel sensors (APS), or photomultiplier tubes (PMTs). The detector can also include a wavelength selection component, such as a filter, to allow measurement of selected wavelengths. Examples of other detectors can include electrodes.

[0112] The detection system may include a light source. The light source may include at least one lamp, such as an incandescent lamp, a halogen lamp, a fluorescent lamp, a gas discharge lamp, an arc lamp, or a light emitting diode (LED). The light source may include a laser. The light source may generate a specific wavelength or one or more specific wavelengths, such as ultraviolet light UV. The light source may include a filter for controlling the output spectrum, a single wavelength, or multiple wavelengths. The light source may include multiple light sources of the same or different types, which may be used separately or in combination.

[0113] sensor

[0114] Various sensors can be used in the present disclosure. They can include, but are not limited to, chemFETs, ISFETs, electronic sensors, magnetic sensors, and optical sensors.

[0115] 1. ChemFET

[0116] Electrochemical detection can be attractive because it can offer high sensitivity, small size, low cost, fast response, and compatibility with microfabrication techniques. These properties have led to the development of a variety of sensors based on amperometric, potentiometric, and impedance signals, and the assembly of sensors into array formats for chemical, biochemical, and cellular applications.

[0117] Various types of chemical sensors may be used in the detection of chemical processes. One type may be a chemically sensitive field effect transistor (chemFET). A chemFET may include a source and a drain separated by a channel region, and a chemically sensitive region coupled to the channel region. The operation of a chemFET may be based on the modulation of the channel conductance caused by a change in charge in the sensitive region due to a chemical reaction occurring nearby. The modulation of the channel conductance may change the threshold voltage of the chemFET, which may be measured to detect and / or determine characteristics of a target chemical reaction.

[0118] For example, chemFETs arranged in an array format can be used to monitor biological or chemical processes. Such chemFET arrays can involve the detection of analytes in solution and / or the detection of charges bound to a surface coupled to an active region of a chemFET. In some systems, the analyte can be distributed in a series of confined areas, such as microwells, where each confined area can be coupled to at least one chemFET.

[0119] 2.ISFET

[0120] An ion sensitive field effect transistor (ISFET) can be a type of chemFET. An ISFET can integrate an ion sensitive layer in the chemically sensitive region of a chemFET. The presence of ions in the analyte solution can change the surface potential at the interface between the ion sensitive layer and the analyte solution due to the protonation or deprotonation of surface charge groups caused by the ions present in the analyte solution. Changes in the surface potential of the chemically sensitive region of the ISFET can affect the threshold voltage of the device. The threshold voltage can be measured to indicate the presence and / or concentration of ions in the analyte solution. A change or no change in the threshold voltage can indicate the presence or absence of ions.

[0121] ISFET arrays can be used to monitor chemical reactions, such as DNA sequencing reactions, based on the detection of the presence, generation, or use of ions during the reaction. See, for example, U.S. Pat. No. 7,948,015 to Rothberg et al., which is incorporated herein by reference. More generally, large chemFETs or other types of chemical sensors can be used to detect and measure the static and / or dynamic amounts or concentrations of various forms of analytes (e.g., hydrogen ions, other ions, compounds, etc.) in various processes. These processes can be, for example, biological or chemical reactions, cell or tissue culture, or monitoring neural activity, nucleic acid sequencing, etc.

[0122] 3. Optical Sensor

[0123] In certain embodiments, fluorescent and light scattering labels can be used to indicate successful hybridization of the probe and target nucleic acid. Examples of suitable fluorescent labels can include, but are not limited to, fluorescent dyes such as 5-fluorescein (FITC), tetrachlorofluorescein (TET), and hexachlorofluorescein (HEX); green fluorescent protein, particularly enhanced GFP (eGFP); rhodamine dyes such as tetramethylrhodamine (TMR) and carboxytetramethylrhodamine (TAMRA); Cy3 and Cy5; and Nanocrystals (Quantum DotCorp., Hayward, CA). Fluorescently labeled nucleotides and custom probes are commercially available from many suppliers (e.g., Synthegen, Houston Tex.; Qiagen, Valencia, CA). Suitable light scattering labels can include luminescent labels, metal nanoparticles, such as gold nanoparticles (Storhoff et al., "Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scattering of gold nanoparticle probes", Nat Biotechnol. 22: 883-87, 2004)) and colloidal silver plasmon resonance particles (PRP) (Schultz et al., "Single target molecule detection using non-bleaching multicolor optical immunolabeling", Proc Natl Acad Sci USA 97: 996-1001, 2000).

[0124] Fluorescent labels can be incorporated into the target nucleic acid before or after target / probe hybridization. The label can be directly bound or incorporated into the target nucleic acid, or can be attached to the target nucleic acid through a joint such as biotin-streptavidin. In one embodiment, the label can be fluorescent, which is attached to the target nucleic acid through different joints. In the detection step, the label can be distinguished based on the different optical effects of different joints on the label. In another embodiment, the label can be fluorescent and attached to the target nucleic acid through different joints. In the detection step, the label can be distinguished based on the different optical effects of different joints on the label, wherein the joint can be a carbon chain with different lengths, and the fluorescence intensity of the polarization label is inversely correlated with the length of the label.

[0125] In order to detect the fluorescent signal, the label can be irradiated with light, for example, using a high-intensity mercury vapor lamp, a laser, etc. In some cases, the illumination method can be a laser with an illumination beam with a wavelength between 488 and 550 nm. In other cases, the dye polynucleotide can be irradiated with a laser generated by an argon ion laser, in particular the 488 and 514 nm emission lines of an argon ion laser, or the 532 emission lines of a neodymium solid-state YAG laser. Several argon ion lasers are commercially available and can emit the above several radiation lines simultaneously. For example, the 2001 model produced by Cyonics, Ltd (Sunnyvale, California), etc. Fluorescence can be detected by a photosensitive detector such as a photomultiplier tube, a charged coupling device, etc.

[0126] As described above, luminescence is the emission of light from excited electronic states of luminescent atoms or molecules. Luminescence generally refers to all types of luminescence other than incandescence, and can include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, which includes fluorescence and phosphorescence, excited electronic states are produced by the absorption of electromagnetic radiation. In chemiluminescence, which includes bioluminescence, excited electronic states are produced by the transfer of chemical energy. In electrochemiluminescence, excited electronic states are produced by an electrochemical process. In the present disclosure, photoluminescence may, but is not limiting, be used interchangeably with luminescence and fluorescence, and luminophore may be used interchangeably with fluorophore.

[0127] Here, cold luminescence is emitted by a luminophore associated with a reporter molecule incorporated into a target nucleic acid. The luminescence lifetime of the luminophore may be shorter (0.1-10 nanoseconds) or longer (10 nanoseconds-1+ seconds). Such luminophores may be external, such as cyanine dyes, phenanthridines (e.g., ethidium bromide), acridines (e.g., acridine orange), indoles (e.g., DAPI), imidazoles, psoralens and luminescent metal-ligands, lanthanide complexes and oxalates (clyptates), and others. Other luminophores are listed in Richard P. Haugland's Handbook of Fluorescent Probes and Research Chemistry (Sixth Edition, 1996), which is incorporated herein by reference for all purposes. Exogenous luminophores may be covalently or non-covalently bound to polynucleotides. Luminophores can be covalently bound using various reactive groups, especially if amines or thiols are incorporated into nucleotides during synthesis. The luminophore can be non-covalently bound via specific binding pairs, such as avidin and biotin, protein A and immunoglobulins, lectins and sugars (e.g., concanavalin A and glucose). The luminophore can also be non-covalently bound by intercalation into the polynucleotide or binding to a helical groove in the polynucleotide.

[0128] Luminophores can be used in a variety of luminescence assays, including fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), total internal reflection (TIR) ​​fluorescence, fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP), as well as their analogs in phosphorescence and higher transitions.

[0129] Preparation of probe arrays

[0130] Probe arrays can be designed and synthesized based on a collection of synthetic oligonucleotide capture probes. Figure 1 See Figure 1 , each copy of the capture probe 100 may include a 5'-terminal adapter A1, a coding sequence CS adjacent to A1 as a non-interactive probe address, a probe sequence PS complementary to a specific target nucleic acid, and a 3'-terminal adapter A2.

[0131] In order to prepare the probe array, a library of nucleic acid templates can be prepared. The nucleic acid templates can be amplified to form a synthetic oligonucleotide capture probe library, which may generally include open-loop or closed-loop nucleic acid template molecules. A "closed loop" can be a covalently closed circular nucleic acid molecule, such as a circular DNA or RNA molecule. An "open loop" can be a linear single-stranded nucleic acid molecule with a 5' phosphate group and a 3' hydroxyl group.

[0132] In one embodiment, the single-stranded nucleic acid template can include at least one copy of a specific nucleic acid template sequence. In some embodiments, an open loop can be formed in situ by a linear double-stranded nucleic acid molecule. The end of a given open-loop nucleic acid molecule can be connected by DNA ligase. The sequence at the 5' and 3' ends of the open-loop molecule can be complementary to two regions formed by adjacent nucleotides in the second nucleic acid molecule, such as the joint region (sometimes referred to as the joint region) of an anchor primer, or two regions almost adjacent in the second DNA molecule. Therefore, DNA ligase can be used to connect the ends of the open-loop molecule, or extended by DNA polymerase in a gap-filling reaction. Open-loop molecules are described in detail in Lizardi's U.S. Patent No. 5,854,033, which is incorporated herein for all purposes. In the presence of DNA ligase (for DNA) or RNA ligase, an open loop can be converted into a closed loop, for example, after the open loop is annealed and combined with a primer.

[0133] A starting nucleic acid template library can be constructed using single-stranded or double-stranded nucleic acid template molecules, provided that the nucleic acid sequence includes a region that, if present in the library, is available, or potentially available, for annealing to an anchor primer sequence. For example, when used as a template for rolling circle amplification, a region of a double-stranded template can be at least transiently single-stranded in order to serve as a template for extension of an anchor primer.

[0134] The library template may include multiple elements, including but not limited to one or more regions complementary to the anchor primer. For example, the template library may include a region complementary to the sequencing primer, a control nucleotide region, and an insert sequence consisting of a sequencing template to be subsequently characterized. For example, the control nucleotide region can be used to calibrate the relationship between the amount of byproducts and the number of nucleotides incorporated.

[0135] In one embodiment, an index oligonucleotide or barcode sequence can be attached to a member of a template library to allow the template nucleic acid and its amplified product, i.e., a probe, to be subsequently associated with a nucleic acid population from which the template nucleic acid is derived. For example, a sample of one or more starting DNA populations can be fragmented using methods such as restriction digestion or ultrasonic treatment. An index oligonucleotide sequence that specifically identifies each sample is attached to, for example, the end of a fragmented population member. The index oligonucleotide can be used for cyclization, amplification, and optionally sequencing steps, which allows it to be used to index or encode nucleic acids, thereby identifying the starting sample from which it originates. Due to the complementarity of nucleic acid chains during chain replication, a barcode attached to a template may cause another barcode to be attached to an amplified probe.

[0136] Different template libraries made with multiple distinguishable index primers can be mixed together for subsequent reactions. Determining the sequence of the members in the library can allow identification of the sequence corresponding to the index oligonucleotide. Based on this information, the origin of any given fragment can be inferred.

[0137] The design of the end adapters A1 and A2 can vary depending on various factors, including, for example, the amplification method selected for making multiple copies of the capture probe. The probe sequence can be customized for each probe array so that the combined library can selectively bind and capture the desired target nucleic acid and detect it.

[0138] Furthermore, by the detection method of the present disclosure, the copy number of the probe at each addressable position of the chip can be amplified to generate a sufficient number of copies of the probe to generate a detectable signal.Any suitable nucleic acid amplification method can be used.

[0139] Many in vitro nucleic acid amplification techniques have been described. These amplification methods can be divided into the following categories of methods: (i) those requiring temperature cycling - polymerase chain reaction (PCR) (see, e.g., Saiki et al., Science 230:1350-54, 1995), ligase chain reaction (see, e.g., Barany, Proc. Natl. Acad. Sci. USA 88:189-193, 1995; Barringer et al., Gene 89:117-122, 1990) and transcription-based amplification (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-77, 1989) and (ii) isothermal amplification systems - self-sustaining, sequence replication (see, e.g., Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-78, 1990); Qβ replicase system (see, e.g., Lizardi et al., BioTechnology 6:1197-1202, 1988); strand displacement amplification (see, e.g., Walker et al., Nucleic Acids Res. 20(7):1691-96, 1992).

[0140] 1. Rolling circle amplification

[0141] Rolling circle amplification (RCA) can generate long single-stranded DNA and RNA using unique DNA and RNA polymerases (Phi29, Bst and VentExo-DNA polymerases for DNA, and T7 RNA polymerases for RNA). In RCA, polymerases can continuously add nucleotides to primers that bind to circular templates. The final result may be a long single-stranded DNA (ssDNA) with many repetitions. In one embodiment, a probe sequence or a fragment thereof can be used as an RCA primer. In another embodiment, a probe sequence or a fragment thereof can be used as a ligation template to cyclize a padlock sequence of a circular template for RCA. The circular DNA template of RCA can be synthesized by intramolecular connection of terminal phosphate and ribose hydroxyl groups by enzymatic or chemical methods. For example, template-mediated enzymatic connection, such as T4 DNA ligase, or template-free connection using, for example, CircLigase can be used to construct the circular DNA template of RCA.

[0142] If RCA is used to amplify the pooled library, the templates of adapters A1 and / or A2 may be incorporated into the sequences required to achieve tandem replication of relatively long synthetic probe sequences. In addition, one or more auxiliary sequences may contain sequences complementary to the templates of adapters A1 and A2 so that the linear template of the desired capture probe can be circularized by base filling and ligation to form a single-stranded circularized template nucleic acid ("circular DNA template") for the RCA reaction. Figure 2 Non-exclusive examples of circular DNA templates 100' are shown. Sequence PS' can be a template for probe sequence PS; sequence CS' can be a template for coding sequence CS; sequence A1' can be a template for adapter A1; sequence A2' can be a template for adapter A2.

[0143] The resulting circular DNA template library can be amplified with Phi29 DNA polymerase via RCA reaction in the presence of appropriate primers and amplification solution to form concatemers of capture probes.

[0144] As used herein, the term DNA "nanoball" or "rolony" generally refers to a monoclonal, dense concatemer molecule produced by isothermal amplification of a DNA template molecule. As described above, in the present disclosure, RCA technology can be one of the methods for amplifying a circular template nucleic acid to generate a long single-stranded linear DNA concatemer of a capture probe.

[0145] In one embodiment, adapter sequences can be attached at the 3' and 5' ends to modify a linear single-stranded DNA ("ssDNA") that can serve as a capture probe template. The 5' end of the template ssDNA so modified can be further modified to include a phosphate group. In some embodiments, the adapter sequence can be specifically designed to include desired features, including but not limited to facilitating directional ligation to the ends of the template ssDNA.

[0146] After linking adapters to both ends of the template ssDNA, the 3' and 5' ends of the linear ssDNA can be ligated to form a circular template using CircLigase ligase commercially available from Epicentre Biotechnologies (Illumina, Madison, WI).

[0147] In some cases, linear ssDNA can be circularized to generate circular template ssDNA. TM IIssDNA ligase can be used to circularize linear template ssDNA. This ligase is a thermostable ligase that catalyzes the intramolecular ligation (i.e., circularization) of single-stranded DNA (ssDNA) substrates with 5'-monophosphate and 3'-hydroxyl groups. The circularization conditions can be formulated according to the method provided by the manufacturer. For example, in a total volume of 10 μL of a reaction buffer containing 33 mM Tris-acetate (pH 7.5), 66 mM potassium acetate, and 0.5 mM DTT, the concentrations of the components can be 0.5 pmol / μL single-stranded DNA, 2.5 mM MnCl2, 1 M trimethylglycine (Betaine), 5 U / μL CIRCLIGASE TMII single-stranded DNA. The reaction can be incubated at 60°C for 1 hour or longer. After circularization is complete, the reaction can be incubated at 60°C for 10 minutes to inactivate the ligase.

[0148] Then, a forward amplification primer complementary to the linker region of the circular template single-stranded DNA can be annealed to the template to generate a circular template single-stranded DNA. The annealed primer can be extended in the presence of a polymerase with high strand displacement activity (e.g., Phi29 DNA polymerase) to generate multiple capture probes in tandem form. A tandem form of the capture probe can have about 100, about 200, about 500, and about 1000 copies or more of the probe sequence.

[0149] In some cases, the circular template obtained above can be amplified. The amplification can be rolling circle amplification (RCA). For example, Phi29 DNA polymerase (0.5 U / μl, Fermentas) can be used in 1× Phi29 reaction buffer (33 mM Tris-acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v / v) Tween 20, 1 mM dithiothreitol (DTT); Fermentas) containing a deoxynucleotide triphosphate (dNTP) mixture (1 mM each dNTP; Fermentas) and increased single-stranded binding protein T4 gene 32 concentration (0–100 ng / μl, NEB), incubated at 30°C for 24 h to amplify circular template single-stranded DNA (0.25 ng / μl).

[0150] Intramolecular hydrogen bond or hybridization can be formed between the joint region, and the region has a suitable hydrogen bond forming sequence or complementary sequence for hybridization. The probe sequence can also include a region suitable for forming intramolecular hydrogen bonds. Therefore, the linear concatemer can be folded into a secondary structure, which compresses the linear single-stranded DNA into a very compact three-dimensional nanosphere structure. Appropriate buffer conditions may also contribute to compressing the linear single-stranded DNA concatemer into a dense nanosphere. Such nanospheres can have a radius of about 50nm, about 100nm, about 150nm or about 200nm. The size of the nanosphere formed can be adjusted by changing the amount of deoxynucleotide triphosphates (dNTPs) added to the RCA reaction.

[0151] In one embodiment, the linker in the linear concatemer may contain a short palindromic sequence, which can help form a compact nanoball through reversible intramolecular hybridization, thereby promoting the coiling of the single-stranded DNA concatemer.

[0152] After the nanoball is retained in the addressable position on the chip and contacts with the solution containing the copy of the desired target nucleic acid, the nanoball of the capture probe concatemer can hybridize with more than one copy of the target nucleic acid. Therefore, the nanoball of the coenzyme of the capture probe using the target nucleic acid can provide signal amplification, indicating the successful hybridization with the target nucleic acid. Therefore, using the concatemer nanoball to target the desired target nucleic acid, signal amplification can be provided and the success rate of the probe hybridization with the target nucleic acid can be promoted.

[0153] 2. Microbead emulsion PCR amplification

[0154] In one embodiment of the present disclosure, the amplification of microbead emulsion can be carried out by attaching the nucleic acid template to be amplified (e.g., DNA template) to a solid support in the form of spherical microbeads. Microbeads can be connected to a large number of single primers complementary to a region of template DNA. As a result of amplification, beads can be connected to many copies of the desired capture probe, which is an amplification product obtained by extending the template DNA by the attached primer on the microbead. Alternatively, beads can be connected to chemical groups (e.g., biotin) that can be combined with chemical groups (e.g., streptavidin) included on the template DNA. Again, as a result of amplification, beads can be connected to many copies of the desired capture probe, which are amplified copies of the template DNA. Beads can be suspended in an aqueous reaction mixture and then encapsulated in an oil-in-water emulsion. Template DNA can be combined with beads before emulsification, or template DNA can be included in the solution of the amplification reaction mixture. In one embodiment, an amplification step can be performed before the nucleic acid template is distributed to the bead emulsion PCR amplification process.

[0155] An example of a representative emulsion PCR (e-PCR) technique is Figure 6 As shown in FIG. 6 . The nucleic acid template 600 for e-PCR may include two adapters A2' and A1', a template sequence PS' for the probe sequence and a template sequence CS' for the coding sequence. After the library of nucleic acid templates 600 is synthesized, multiple copies of the probe sequence may be attached to a single bead using the e-PCR method. Figure 6As shown, this can be achieved by dividing a nucleic acid template 600, a microbead 602 with a plurality of connected first primers P1 (e.g., n primers, where n is an integer greater than 2), a second primer P2 and other reagents for PCR (e.g., dNTPs) in the same microreactor for e-PCR (e.g., a microcapsule of a droplet or emulsion). At the end of the amplification of the primer P1 extending according to the nucleic acid template 600, a microbead 604 can be obtained, on which m copies of a nucleic acid sequence 606 (m is an integer greater than 2, but not greater than n) can be attached. The nucleic acid sequence 606 can include a primer P1, a coding sequence CS, a probe sequence PS and a primer sequence A2. The probe sequence PS can be designed to be complementary to a selected target nucleic acid sequence.

[0156] In certain embodiments, the emulsion can be composed of discrete aqueous phase droplets, such as droplets having an average diameter of about 60 μm to about 200 μm and surrounded by a thermally stable oil phase. Each droplet can contain an amplification reaction solution (i.e., reagents sufficient for nucleic acid amplification). An example of an amplification reaction solution can include a PCR reaction mixture (polymerase, salts and dNTPs) and a pair of PCR primers. In some cases, template DNA may be included in the reaction mixture. A subset of the droplets can include DNA beads and templates. This subset of droplets may be the basis for emulsion PCR amplification. The remaining microcapsules may not contain template DNA and may not participate in amplification.

[0157] In one embodiment, the amplification technique used can be PCR, and the PCR primers can be present in a ratio of 8:1 or 16:1 (i.e., 8 or 16 of one primer to 1 of a second primer) to implement asymmetric PCR. In another embodiment, the ratio of PCR primers can be substantially the same as normal PCR. In some embodiments, in the presence of available dNTPs, a thermostable polymerase can synthesize a single-stranded DNA amplicon immobilized on a microbead, which is the capture probe.

[0158] After PCR, the emulsion can be broken and the microbeads with many copies of the capture probe immobilized can be treated under certain conditions (NaOH, low ionic strength or heat treatment, etc.) to obtain the capture probe in the form of single-stranded DNA.

[0159] Sample room design

[0160] The sample chamber can be designed according to the characteristics of the copies of the probe to retain multiple copies of the probe.

[0161] 1. Sample chamber for nanospheres

[0162] The chip of the present disclosure can be designed to retain one nanosphere in each sample chamber having an addressable location. Figure 3 As shown, the chip 200 may include a sample chamber array 202. Figure 3 and Figure 4 As shown, the sample chamber 202 may be shaped as a hollow cylinder having a circular cross-section, an inclined sidewall 204 , and a flat surface at the bottom 206 .

[0163] The distance between the centers of two adjacent sample chambers can be about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, or about 700 nm to about 1300 nm. The diameter of the circular bottom can be about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, or about 300 nm to about 800 nm.

[0164] In addition, if Figure 4 As shown, the sample chamber 202A may have an inclined sidewall 204A and a circular bottom 206A. Each circular bottom 206A may include a circular "landing site" 208A with a diameter of about 250nm to about 300nm. The landing area 208A may be positively charged by chemical modification so that it can retain the nanosphere 212A of the probe with a diameter of about 250nm to about 300nm through charge interaction. It can be noted that, except for the landing site area 208A, the rest of the surface 210A of the hollow cylinder or the top surface of the chip is not positively charged, so the rest of the surface of the hollow cylinder or the top surface of the chip may not form a charge-charge interaction with the nanosphere 212A of the probe. These surface properties of the sample chamber 202A ensure that the landing site 208A can help maintain multiple copies of the probe in the form of nanospheres 212A, and each sample chamber 202A can maintain one such nanosphere 212A. The landing site may occupy the entire bottom surface of the sample chamber 202A. In Figure 4 In another embodiment of the sample chamber 202B shown, the landing site 208B can occupy a portion of the bottom surface 206B of the sample chamber 202B to hold a probe nanosphere 212B, while the top surface of the chip and the remaining surface 210B of the hollow cylinder are not positively charged and cannot hold the probe nanosphere 212B. The landing site can include an area on the vertical wall of the sample chamber.

[0165] In some embodiments, the nanospheres can be attached to a dendritic support or other type of particle, such as a microbead. The support can be porous or partially porous. If the support is porous or partially porous, the size of the pores can be large enough to allow nucleic acids (e.g., DNA), polymerases, dNTPs, and other components that can be used for primer extension sequencing or other appropriate applications to move freely.

[0166] In some embodiments, the nanospheres can be temporarily fixed on a surface, such as the surface of a sample chamber, a sensor, the surface of an electrode, the surface of a carrier (e.g., microbeads), etc. Such a surface can have any shape, such as spherical, flat, rectangular, crystalline, well-shaped, etc. In some embodiments, the substrate material of the surface to which the nanospheres are attached can include, for example, silicon, silicon-based materials, glass, modified functionalized glass, magnetic materials, plastics, metals, ceramics, gels, acrylic resins, biomaterials, etc. The nanospheres can be attached to the surface by any suitable method, non-limiting examples of which include nucleic acid hybridization, biotin-streptavidin binding, thiol binding, photoactivated binding, covalent binding, non-covalent binding, antibody-antigen, physical confinement achieved by hydrogels or other porous polymers, etc., or a combination thereof. In some cases, the nanospheres can be digested with nucleases (e.g., DNA nucleases) to produce smaller nanospheres or fragments of nanospheres.

[0167] In one embodiment of preparing a glass slide surface to retain DNA nanospheres, a layer of silicon dioxide can be grown on the surface of a standard silicon wafer (Silicon Quest International, Santa Clara, CA). A titanium layer can be deposited on the silicon dioxide layer, and the titanium layer can be patterned with reference marks using conventional photolithography and dry etching techniques. A hexamethyldisilazane (HMDS) layer (Gelest Inc., Morrisville, Pennsylvania) can be added to the substrate surface by vapor deposition, and then a deep ultraviolet, positive photoresist material can be applied to the surface by centrifugal force. The resulting photoresist surface can be exposed with a selected array pattern and a 248nm photolithography tool. So photoresist is produced, and an array with discrete areas can be produced on HMDS.

[0168] The HMDS layer in the exposed holes can be removed by a plasma etching process, and aminosilanes, such as (3-aminopropyl)-triethoxysilane (APTES), can be vapor deposited in the exposed holes to provide landing sites for DNA nanospheres. A layer of photoresist can be coated on the resulting surface and cut into 75mm x 25mm substrates, and then all the photoresist can be stripped from the substrate surface by ultrasonic treatment. Next, a mixture of approximately 50μm polystyrene beads and polyurethane glue can be applied to the surface of each substrate cut piece in a set of parallel lines, and then a cover glass is pressed into the glue line to form a multi-lane, such as a six-lane, gravity / capillary-driven flow sheet. The aminosilane pattern or patch on the substrate can serve as a landing site for each DNA nanosphere, while HMDS can inhibit the DNA nanosphere from landing between the pattern / patch. DNA nanospheres prepared by the RCA process can be loaded into the flow slide lane by pipetting 2 to 3 times more nanospheres than the number of landing sites available on the slide onto the slide. The loaded slides can be incubated at 23°C for 2 hours in a sealed chamber and then rinsed to neutralize the pH and remove unbound DNA nanospheres.

[0169] In another embodiment, several steps can be added to the above embodiment. First, the silicon wafer can be pre-processed so that patterned microwells of the desired shape (e.g., hollow cylinders) and size can be present on the glass slide before the silicon oxide layer is grown. Second, when the photoresist surface is exposed with a 248 nm photolithography tool in a selected array pattern, the selected array pattern can result in discrete areas of exposed HMDS at the bottom of the microwells.

[0170] 2. Sample Chamber for Bead-emulsion PCR Amplification

[0171] These sample chambers can be made following a procedure similar to that described above for the sample chambers for nanospheres. However, the surface treatment may be different, as a microwell of the appropriate size (to allow access to one microbead, but not two microbeads) may not be surface treated to retain multiple copies of the probe. As shown in the figure. Figure 5 is an example of a sample chamber for beads. Again, the size of the sample chambers may depend on several factors, including: 1) the size of the beads retained by each sample chamber; 2) the amount of solution in the sample chamber to allow for hybridization and wash steps, and 3) the balance between retaining the beads and washing away unbound target nucleic acids after the hybridization step, etc.

[0172] Decoding of probe sequences

[0173] As shown above, nanospheres or microbeads with multiple probe copies can be randomly distributed to sample chambers (microwells). In order to associate the probe with the sample chamber containing the probe, the probe sequence retained in each sample chamber can be identified by decoding the coding sequence CS attached to each probe sequence PS. For example, a decoding program based on high-throughput hybridization, such as the program described by Gunderson et al. in "Decoding Randomly Ordered DNA Arrays", Genome Res., 14(5): 870-77, 2004, can achieve the purpose of decoding. Gunderson's method can provide an algorithm for identifying each member in a collection of a large number of barcode sequence-related objects by using hybridization, and the method can be applied to the manufacture of randomly assembled microbead arrays in wells.

[0174] Detection of hybridization between probe and target

[0175] As described above, in some cases, the DNA sample can be fragmented, polished, ligated with adapters, labeled internally or at the 3' end with biotin or other suitable hapten, including but not limited to digoxigenin, fluorescein, 2,4-dinitrophenyl (DNP), etc.

[0176] like Figure 5 As shown, the probe array can be exposed to a labeled, fragmented target nucleic acid analyte under conditions that allow hybridization between complementary probes and fragmented target nucleic acid analytes. Subsequent washing can remove unbound (unhybridized) fragmented target nucleic acid analytes. The washing conditions can be controlled so that residual microbeads or nanospheres are not washed away, while unbound nucleic acid analytes are washed away. The presence of an analyte hybridized with a probe can be detected by many methods. The following are some non-exclusive illustrative examples:

[0177] 1. ISFET sensor

[0178] In one embodiment, when an ISFET detection method is used, the presence of bound target nucleic acid can be detected by exposing the hybridization product to a hapten binding molecule (e.g., streptavidin, anti-digoxigenin ligand, etc.) linked to a proton signal generating moiety. As described above, the fragmented target nucleic acid is labeled with a suitable hapten. Figure 5As shown, after the probe nanoball 504 is retained in the sample chamber 502, the target nucleic acid 506 and its associated hapten 508 can be hybridized with the nanoball 504. After completing the hybridization step, the hybridization product can be washed at least once to remove the unbound nanoball and target nucleic acid. Then the hapten binding molecule 510 and the proton signal generating portion 512 connected thereto can be reacted with the hapten 508 on the target nucleic acid 506 bound in the sample chamber 502. Then, a substrate 514 that can release at least one proton signal 516 is added to the sample chamber 502. After the proton signal generating portion 512 processes the substrate 514, the released proton signal 516 can be detected by a detector 518 that is addressable and adjacent to the sample chamber 502. It should be noted that in the same sample chamber, there may be multiple target nucleic acids that bind to the same probe nanoball. This may produce a stronger signal than one target nucleic acid binding to the nanoball.

[0179] In the present disclosure, as used herein, the term "proton signal generating moiety" generally refers to an enzyme that produces an acid or base as a byproduct of the catalytic conversion of a substrate or cofactor of the enzyme, thereby causing a measurable pH change in a sample chamber.

[0180] Non-exclusive illustrative examples of proton signal generating moieties include:

[0181] hydrolases, such as esterases (e.g., acetylcholinesterase), lipases, phosphatases and pyrophosphatases (e.g., inorganic pyrophosphatases, ATP hydrolases ("apyrases")), which generate protons as byproducts as a result of biotransformations;

[0182] Phosphotransferases, such as nucleoside kinases, pyruvate kinases, etc., all of which produce protons as byproducts due to biotransformations;

[0183] Dehydrogenases, such as aldehyde dehydrogenase, alcohol dehydrogenase, glucose-t-phosphate dehydrogenase (G6PDH), all of which generate protons as byproducts due to biotransformations;

[0184] Oxidase such as xanthine oxidase, glucose oxidase and peroxidase may also cause pH changes due to the activity of these enzymes;

[0185] • Deaminases such as L-glutaminase and urease may generate ammonia (NH3) as a by-product due to bioconversion.

[0186] In fact, if Figure 4 As shown, after hybridization and washing steps, the proton signal generating moiety conjugated to the hapten binding molecule can be added to the sample chamber, followed by the addition of the substrate required for the proton signal generating moiety. If any, the resulting pH change can be captured and recorded by the ISFET detector at the relevant addressable position.

[0187] 2. Luminescence detector

[0188] In another embodiment, when a luminescent sensor is used, the presence of target nucleic acids hybridized to the probes can be detected by exposing the probe array to a hapten binding molecule (e.g., streptavidin, anti-digoxigenin ligand, etc.) linked to a luminescent signal generating moiety.

[0189] The luminescent signal generating part can be an enzyme that produces a fluorescent or chemiluminescent product from the catalytic conversion of a substrate. In one embodiment, an enzyme can convert a substrate into a reaction product that emits fluorescence when excited by light of a specific wavelength. Non-exclusive illustrative examples of enzymes that can produce fluorescent signals include alkaline phosphatase, β-D-galactosidase, and peroxidase when a suitable substrate is present. In another embodiment, the enzyme can convert a substrate into a reaction product that emits photons, rather than showing a visible color. Non-exclusive illustrative examples of enzymes that can produce luminescent signals include alkaline phosphatase, β-D-galactosidase, and peroxidase when a suitable substrate is present.

[0190] Computer Systems

[0191] The present disclosure provides a computer control system programmed to implement the method of the present disclosure. Figure 7 A computer system 700 is shown that is programmed or otherwise configured to perform various functions of the methods and systems of the present disclosure, for example, to perform an amplification reaction, detect and / or monitor the binding of a target substance (e.g., a target nucleic acid) to a probe array, and / or monitor the progress of a hybridization or amplification reaction. The computer system 700 can regulate multiple aspects of simultaneously performing at least one amplification reaction and detecting changes in signals generated by the probe array, such as temperature control, reagent handling, and signal detection. The computer system 700 can be integrated with the system provided in the present disclosure.

[0192] The computer system 700 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 702, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 700 also includes a memory or memory location 704 for communication (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 706 (e.g., a hard disk), a communication interface 708 for communicating with one or more devices (e.g., a network adapter), and peripheral devices 710, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 704, storage unit 706, interface 708, and peripheral devices 710 communicate with the CPU 702 via a communication bus (solid lines) such as a motherboard. The storage unit 706 may be a data storage unit (or data repository) for storing data. The computer system 700 may be operably connected to a computer network ("network") 712 by means of the communication interface 708. The network 712 may be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. In some cases, the network 712 is a telecommunications and / or data network. The network 712 may include one or more computer servers, which may enable distributed computing, such as cloud computing. In some cases, the network 712 may implement a peer-to-peer network with the help of the computer system 700, which may enable devices connected to the computer system 700 to act as clients or servers.

[0193] The CPU 702 may execute a series of machine-readable instructions, which may be embedded in a program or software. The instructions may be stored in a memory location such as the memory 704. The instructions may be directed to the CPU 702, which may then program or otherwise configure the CPU 702 to implement the methods of the present disclosure. Examples of operations performed by the CPU 702 may include fetching, decoding, executing, and writing back.

[0194] CPU 702 may be part of a circuit, such as an integrated circuit. One or more other components of system 700 may be included in a circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0195] Storage unit 706 can store files, such as drivers, libraries, and saved programs. Storage unit 706 can store user data, such as user preferences and user programs. In some cases, computer system 700 can include one or more additional data storage units external to computer system 700, such as located on a remote server that communicates with computer system 700 via an intranet or the Internet.

[0196] Computer system 700 can communicate with one or more remote computer systems via network 712. For example, computer system 700 can communicate with a remote computer system of a user (e.g., a laboratory technician, a physician). Examples of remote computer systems include a personal computer (e.g., a portable PC), a tablet or a computer system (e.g., a iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-supported devices, ) or a personal digital assistant. Users can access the computer system 700 through the network 712.

[0197] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 700, such as the memory 704 or the electronic storage unit 706. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 702. In some cases, the code may be retrieved from the storage unit 706 and stored in the memory 704 for access by the processor 1905 at any time. In some cases, the electronic storage unit 706 may be excluded and the machine executable instructions may be stored in the memory 704.

[0198] The code can be precompiled and configured for use on a machine with a processor suitable for executing the code, or it can be compiled at run time. The code can be executed in a precompiled or as-compiled manner by choosing the programming language in which it is provided.

[0199] The systems and methods provided herein, such as computer system 700, can be specifically embodied in programming. Various aspects of the technology can be viewed as a "product" or "manufactured product" in the form of machine (or processor) executable code and / or associated data carried or embodied on some type of machine-readable medium. The machine executable code can be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of a computer, processor, etc., or its related modules, such as various semiconductor memories, tape drives, disk drives, etc. that can provide non-temporary storage for software programming at any time. All or part of the software can sometimes communicate via the Internet or various other telecommunications networks. For example, such communication can load software from one computer or processor to another computer or processor, such as a computer platform loaded from a management server or host to an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as light waves used across physical interfaces between local devices through wired and optical landline networks and through various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0200] Thus, machine-readable media such as computer executable code may take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., such as may be used to implement the databases shown in the accompanying drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables. Copper wires and optical fibers, including the wires that make up a bus within a computer system. Carrier transmission media may take the form of electrical or electromagnetic signals or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include: floppy disks, diskettes, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch card tapes, any other physical storage media with patterns of holes, RAM, ROMs, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves for transporting data or instructions, cables or links for transporting such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0201] The computer system 700 may include or communicate with an electronic display 714 that includes a user interface (UI) 716 for providing, for example, cycle times, temperature values, temperature controls, detector data, and reagent handling. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0202] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 702. The algorithms may, for example, control the temperature of each addressable location, assign a barcode of a probe to each addressable location, collect signals, and analyze the collected data.

[0203] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided by way of example only. It is not intended to limit the present invention by the specific examples provided in the patent specification. Although the present invention has been described according to the aforementioned patent specification, the description and illustration of the embodiments herein are not meant to be interpreted in a restrictive sense. Without departing from the present invention, many variations, changes and substitutions will occur to those skilled in the art. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportion set forth herein, which depends on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to implement the present invention. Therefore, it is contemplated that the present invention will also cover any such substitution, modification, variation or equivalent. The intent of the appended claims is to define the scope of the present invention, and thus covers methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for analyzing a sample for the presence of a target nucleic acid, comprising: (a) providing a chip, the chip comprising a sensor adjacent to a sample chamber, wherein: (i) the sample chamber is configured to retain multiple copies of a capture probe, wherein the capture probe is part of a pooled library of a plurality of capture probes, (ii) the capture probe comprises a nucleic acid fragment sequence complementary to at least a portion of the target nucleic acid, and a barcode sequence attached to a first end of the nucleic acid fragment sequence, (iii) the capture probe is selectively coupled to the target nucleic acid, (iv) the plurality of copies of the capture probe are not covalently attached to the surface of the sample chamber, and (v) the sensor detecting at least one signal indicating the presence or absence of the target nucleic acid in the sample chamber; (b) providing the sample in the sample chamber under conditions that allow the capture probe to selectively couple to the target nucleic acid; (c) measuring the at least one signal; (d) determining the presence or absence of the target nucleic acid in the sample; and The method described herein is not intended to obtain direct diagnostic results.

2. The method of claim 1, wherein the sensor is in an array of multiple sensors in the chip, wherein the sample chamber is in an array of multiple sample chambers in the chip, and wherein each of the multiple sample chambers is adjacent to at least one sensor in the array of multiple sensors. The method of claim 1 , wherein the target nucleic acid is a fragment of a first nucleic acid.

4. The method according to claim 1, further comprising: Before (b), (a1) capture probe template of circularized coupled adapter; and (a2) amplifying the adapter-coupled capture probe template to form a linearly amplified concatemer molecule comprising multiple copies of the capture probe. The method of claim 1 , wherein the barcode sequence is 3 to 30 nucleotides in length.

6. The method according to claim 1, further comprising: Before (b), (a1) delivering a single copy of the capture probe template, or a single copy of a double-stranded nucleic acid containing the capture probe, to an aqueous microreactor in an oil-in-water emulsion, wherein the microreactor comprises a plurality of primers capable of annealing to the capture probe, a single microbead capable of binding to the capture probe template and amplifying a first copy of the capture probe attached to the surface of the microbead, and an amplification reaction solution containing reagents sufficient to perform a nucleic acid amplification reaction; (a2) subjecting the microreactor to a nucleic acid amplification reaction under conditions that can produce a first copy of the capture probe; (a3) repeating step (a2) multiple times; and (a4) disrupting the water-in-oil emulsion and generating multiple copies of the capture probe attached to the microbeads. The method of claim 1 , wherein the sample comprises a plurality of target nucleic acid molecules comprising the target nucleic acid.

8. The method of claim 7, wherein the sensor is part of an array of a plurality of sensors, wherein each of the plurality of sensors detects the presence or absence of at least one of the plurality of target nucleic acid molecules, and wherein each sensor in the array of the plurality of sensors is individually addressable.

9. The method of claim 8, wherein each capture probe comprises: 1) a sequence of a nucleic acid fragment complementary to a portion of one of the target nucleic acids; and 2) a barcode sequence attached to the first end of the nucleic acid fragment sequence; And wherein the method further comprises in (d): (d1) decoding the barcode sequence retained on the capture probes in the corresponding sample chambers connected to the individually addressable sensors.

10. The method of claim 1, wherein the chip comprises an additional sensor, wherein the sample comprises the target nucleic acid and an additional target nucleic acid, and wherein the additional sensor detects the additional target nucleic acid.

11. The method of claim 10, wherein the additional sensor is adjacent to an additional sample chamber, wherein the additional sample chamber is configured to retain multiple copies of the additional capture probe, wherein the additional capture probe selectively couples to the additional target nucleic acid.

12. The method of claim 1, wherein the sensor comprises an ion sensitive field effect transistor.

13. The method of claim 1, wherein the sensor comprises a chemically sensitive field effect transistor.

14. The method of claim 1, wherein the sensor comprises an optical sensor.

15. The method of claim 1, wherein the at least one signal comprises a change in pH.

16. A system for analyzing a sample for the presence of a target nucleic acid, the system comprising: (a) A chip comprising a sensor adjacent to a sample chamber, wherein: (i) the sample chamber is configured to retain the sample having the target nucleic acid and a plurality of copies of a capture probe, wherein the capture probe is part of a pooled library of a plurality of capture probes, (ii) the capture probe comprises a nucleic acid fragment sequence complementary to at least a portion of the target nucleic acid, and a barcode sequence attached to a first end of the nucleic acid fragment sequence, (iii) the capture probe is selectively coupled to the target nucleic acid, (iv) the plurality of copies of the capture probe are not covalently attached to the surface of the sample chamber, and (v) the sensor detecting at least one signal from the sample, the at least one signal indicating the presence or absence of the target nucleic acid; and (b) a computer processor coupled to the chip, programmed to (i) measure the at least one signal while contacting the chip with the sample; and (ii) determine the presence or absence of the target nucleic acid in the sample.

17. The system of claim 16, wherein the sensor is in an array of multiple sensors in the chip, wherein the sample chamber is in an array of multiple sample chambers in the chip, wherein each of the multiple sample chambers is adjacent to at least one sensor in the array of multiple sensors, and wherein each sensor is individually addressable.

18. The system of claim 16, wherein the sensor is an ion-sensitive field effect transistor, a chemical-sensitive field effect transistor, or an optical sensor.

19. The system of claim 17, wherein the computer processor is further programmed to (iii) map the array of individually addressable sensors; and (iv) when the capture probe comprises a barcode sequence, associate the barcode sequence with the corresponding individually addressable sensor.

20. The system of claim 16, wherein the chip comprises additional sensors, and wherein the sample comprises additional target nucleic acids.

21. The system of claim 20, wherein the additional sensor detects at least one additional signal indicative of the presence or absence of the additional target nucleic acid.

22. The system of claim 17, wherein the array of multiple sensors comprises at least 96 sensors.

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