Ultra-sensitive biosensor method

Abstract

Provided herein are methods and devices for ultrasensitive detection of target molecules (e.g., target nucleic acids or target proteins) from biological samples. In some embodiments, the methods and devices enable ultrasensitive determination of the concentration of the target molecules.

Description

[Technical Field]

[0001] The present invention relates to an ultrasensitive biosensor method. [Background technology]

[0002] Methods and systems capable of rapidly and accurately detecting, and in some cases quantifying, target analyte molecules in a sample are essential analytical measurements for academic and industrial research, environmental assessment, food safety, medical diagnostics, and the detection of chemical, biological, and / or radiological warfare agents. Most previous techniques for quantifying low levels of analyte molecules in a sample have used amplification procedures to increase the number of reporter molecules so as to provide a measurable signal. For example, these known methods include enzyme-linked immunosorbent assays (ELISAs), which amplify signals in antibody-based assays, and polymerase chain reaction (PCR), which amplify target DNA strands in DNA-based assays.

[0003] These known methods and / or systems are based on an ensemble response in which many analyte molecules cause a measured signal. Most detection schemes require a large number of molecules to be present in the ensemble in order for the aggregate signal to exceed the detection threshold. This requirement limits the sensitivity and dynamic range (e.g., range of detectable concentrations) of most detection techniques. Many known methods further suffer from the problem of nonspecific binding, which can lead to an increased background signal, thereby limiting the minimum concentration that can be accurately or reproducibly detected. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, there is a need for improved methods for detecting and quantifying target molecules in a sample, particularly in samples where such molecules or particles are present in very low concentrations. [Means for solving the problem]

[0005] Embodiments of the present disclosure provide methods, compositions, devices, and / or cartridges or detection chips for use in processes, e.g., for determining the amount (e.g., concentration) of a target molecule in a sample. Some embodiments of the present disclosure provide methods for determining the concentration of a target molecule (e.g., a labeled target molecule) in a sample.

[0006] In some embodiments, a method for determining the concentration of a target molecule in a sample includes: (i) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to produce a plurality of first complexes comprising the target molecule bound to the first affinity agents, wherein at least a portion of the plurality of first affinity agents is immobilized on a surface; (ii) contacting the plurality of first complexes with a plurality of second affinity agents having binding affinity for the first complexes to produce a plurality of second complexes comprising the second affinity agents bound to the first complexes, wherein at least a portion of the second affinity agents is immobilized on a surface. (iii) removing unbound second affinity agent and / or isolating the plurality of second complexes; (iv) optionally isolating at least a segment of each of the labeled molecules from the bound second affinity agent of the plurality of second complexes; (v) combining the segment of the labeled molecule with a reference molecule of known concentration; (vi) determining a ratio of detection events of the labeled molecule to detection events of the reference molecule; and (vii) determining the concentration of the target molecule in the sample based at least in part on the ratio of detection events of the labeled molecule to detection events of the reference molecule.

[0007] In some embodiments, a method for determining the concentration of a target molecule in a sample includes: (i)(a) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to produce a plurality of first complexes comprising the target molecule bound to the first affinity agents; (i)(b) immobilizing at least a portion of the plurality of first affinity agents on a surface; (ii) contacting the plurality of first complexes with a plurality of second affinity agents having binding affinity for the first complexes to produce a plurality of second complexes comprising the second affinity agents bound to the first complexes, wherein at least a portion of the second affinity agents (iii) removing unbound second affinity agent and / or isolating the plurality of second complexes; (iv) optionally isolating at least a segment of each of the labeled molecules from the bound second affinity agent of the plurality of second complexes; (v) combining the isolated segment of the labeled molecule with a reference molecule of known concentration; (vi) determining a ratio of detection events of the labeled molecule to detection events of the reference molecule; and (vii) determining the concentration of the target molecule in the sample based at least in part on the ratio of detection events of the labeled molecule to detection events of the reference molecule.

[0008] In some embodiments, a method for determining the concentration of a labeled target molecule in a sample includes: combining a sample containing the labeled target molecule with a reference molecule of known concentration; determining a ratio of detection events of the labeled target molecule to detection events of the reference molecule; and determining the concentration of the labeled target molecule in the sample based at least in part on the ratio of detection events of the labeled target molecule to detection events of the reference molecule.

[0009] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a single cell, a mammalian cell tissue, an animal sample, a fungal sample, or a plant sample. In some embodiments, the biological sample is a blood sample, a saliva sample, a sputum sample, a fecal sample, a urine sample, a buccal swab sample, an amniotic membrane sample, a semen sample, a synovial membrane sample, a spinal cord sample, or a pleural effusion sample.

[0010] In some embodiments, the target molecule is a protein, a small molecule, or a nucleic acid. In some embodiments, the nucleic acid is a DNA molecule and / or an RNA molecule. In some embodiments, the contacting of (i) and / or (ii) is carried out at a temperature of 4 to 37° C., optionally 4 to 25° C. In some embodiments, the contacting of (i) and / or (ii) is carried out for a period of 5 minutes to 4 hours.

[0011] In some embodiments, the first affinity agent is an antibody or an aptamer, and the target molecule is an antigen, optionally the antigen is a protein, peptide, or polysaccharide. In some embodiments, the first affinity agent is an antibody or an aptamer, and the target molecule is a protein, and the antibody or aptamer specifically binds to an epitope of the target molecule.

[0012] In some embodiments, at least a portion of the plurality of first affinity agents are immobilized on the surface of solid beads, microfluidic channels, nanoapertures, resins, matrices, membranes, polymers, plastics, metals, or glass. In some embodiments, the solid beads are magnetic beads.

[0013] In some embodiments, the second affinity agent is an antibody. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) is linked to at least one fluorophore. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) is linked to two, three, four, or five different fluorophores. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) is linked to two, three, four, or five identical fluorophores.

[0014] In some embodiments, the method further comprises, following (iv), chemically linking at least one fluorophore to each of the labeled molecules (eg, labeled target molecules).

[0015] In some embodiments, each of the labeled molecules (e.g., labeled target molecules) comprises a chemical linker connected to at least one fluorescent dye molecule. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) comprises a biotin-streptavidin conjugate connected to at least one fluorescent dye molecule. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) comprises a biotin-streptavidin conjugate linked to a nucleic acid and at least one fluorescent dye molecule. In some embodiments, each of the labeled molecules (e.g., labeled target molecules) is a labeled nucleic acid, and each of the second affinity agents is linked to a first strand of the labeled nucleic acid, optionally, the first strand being 5 to 50 nucleobases in length.

[0016] In some embodiments, the first strand of the labeled nucleic acid is linked to the second affinity agent via a biotin-streptavidin complex. In some embodiments, the labeled nucleic acid is a single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid includes a region that forms a hairpin loop. In some embodiments, the single-stranded nucleic acid is linked to at least one fluorescent dye molecule.

[0017] In some embodiments, the labeled nucleic acid is a double-stranded nucleic acid comprising a first strand and a second strand comprising a region complementary to the first strand, and in some embodiments, the second strand is linked to at least one fluorophore.

[0018] In some embodiments, the labeled nucleic acid comprises a first strand and a nucleic acid dumbbell, wherein a first region of the nucleic acid dumbbell is complementary to the first strand. In some embodiments, the nucleic acid dumbbell is linked to at least one fluorescent dye molecule. In some embodiments, the labeled nucleic acid further comprises a second single-stranded nucleic acid complementary to a second region of the nucleic acid dumbbell. In some embodiments, the second single-stranded nucleic acid is linked to at least one fluorescent dye molecule.

[0019] In some embodiments, the labeled nucleic acid comprises a first strand and a second single-stranded nucleic acid, the first strand comprising a region that forms a hairpin loop, and the second single-stranded nucleic acid is linked to at least one fluorescent dye molecule.

[0020] In some embodiments, (i) and (ii) occur simultaneously or sequentially. In some embodiments, (iii) comprises removing unbound second affinity agents by washing the sample with a wash buffer. In some embodiments, the wash buffer is phosphate-buffered saline. In some embodiments, (iv) comprises isolating at least a segment of each of the labeled molecules (e.g., labeled target molecules) from the bound second affinity agents by washing the sample with an elution buffer. In some embodiments, the elution buffer is a high-salt buffer. In some embodiments, (iv) comprises isolating at least a segment of each of the labeled molecules (e.g., labeled target molecules) from the bound second affinity agents by changing the temperature of the sample (e.g., increasing the temperature).

[0021] In some embodiments, the labeled nucleic acid is a double-stranded labeled nucleic acid, the second affinity agent is linked to a first strand of the double-stranded labeled nucleic acid, and the isolated segment of the labeled nucleic acid is a second strand of the double-stranded labeled nucleic acid.

[0022] In some embodiments, (iv) comprises isolating the second strand of the double-stranded labeled nucleic acid by washing the sample with an excess of single-stranded nucleic acid that is complementary to the first strand of the double-stranded labeled nucleic acid.

[0023] In some embodiments, each of the reference molecules is linked to at least one fluorophore, and the fluorophore linked to the reference molecule is different from the at least one fluorophore linked to the label molecule. In some embodiments, the at least one fluorophore linked to the reference molecule and the at least one fluorophore linked to the label molecule (e.g., a labeled target molecule) can be excited by the same excitation wavelength. In some embodiments, each of the reference molecules is a reference nucleic acid. In some embodiments, the reference nucleic acid is a single-stranded or double-stranded nucleic acid. In some embodiments, each of the reference molecules is immobilized on the same surface as at least a portion of the plurality of first affinity agents. In some embodiments, each of the reference molecules is immobilized on a surface different from the surface on which at least a portion of the plurality of first affinity agents is immobilized. In some embodiments, each of the reference molecules is linked to a first affinity agent. In some embodiments, each of the reference molecules is a complex comprising a single-stranded nucleic acid immobilized on a surface or linked to a first affinity agent and a dumbbell nucleic acid comprising a region of complementarity to the single-stranded nucleic acid. In some embodiments, the reference molecule is isolated in (iv).

[0024] In some embodiments, the labeled molecule (e.g., labeled target molecule) is a labeled nucleic acid, the reference molecule is a reference nucleic acid, and the labeled and reference nucleic acid are amplified during (iv), optionally using rolling circle amplification.

[0025] In some embodiments, the labeled molecule (e.g., labeled target molecule) (or segment of the labeled molecule) and a reference molecule of known concentration are combined in a detection chip. In some embodiments, the detection chip comprises a regular array of sample wells.

[0026] In some embodiments, the depth of each sample well is between 50 and 500 nm, optionally about 300 nm, hi some embodiments, the diameter of the interior base of each sample well is between 50 and 250 nm, optionally between 75 and 125 nm, and further optionally about 100 nm.

[0027] In some embodiments, the interior base of each sample well is functionalized with a silane-containing compound. In some embodiments, the interior base of each sample well is functionalized with a biotin-streptavidin complex. In some embodiments, the interior base of each sample well is functionalized with a positively charged molecule. In some embodiments, the interior base of each sample well is functionalized with a 1000 nm 2 In some embodiments, the positively charged molecule is a polylysine molecule. In some embodiments, the polylysine molecule comprises 10-200 lysine amino acids, optionally 20-100 lysine amino acids, and further optionally 50 lysine amino acids. In some embodiments, the positively charged molecule is a positively charged terminal silane molecule.

[0028] In some embodiments, the interior base of each sample well is functionalized with a nucleic acid complementary to a labeled nucleic acid and / or a reference nucleic acid. In some embodiments, the ratio of labeled molecules (e.g., labeled target molecules) to reference molecules is determined using fluorescence measurements. In some embodiments, the ratio of labeled molecules (e.g., labeled target molecules) to reference molecules is determined using fluorescence measurements of labeled molecules and reference molecules in the sample well. In some embodiments, the ratio of labeled molecules (e.g., labeled target molecules) to reference molecules is determined based in part on the residence times of labeled molecules and reference molecules in the sample well.

[0029] In some embodiments, the labeled and reference molecules are delivered to and maintained within the sample wells by electrostatic interaction with positively charged molecules at the interior base of each sample well. In some embodiments, the labeled and reference molecules are delivered to and maintained within the sample wells by interaction with nucleic acids at the interior base of each sample well that are complementary to the labeled and / or reference nucleic acids. In some embodiments, the labeled and reference molecules are delivered to and maintained within the sample wells by gravity or a magnetic field. In some embodiments, the labeled and reference molecules are delivered to and maintained within the sample wells using a crowding reagent, optionally a sugar molecule, methylcellulose, polyethylene glycol, dextran, ficoll, bovine serum albumin, or trehalose.

[0030] In some embodiments, the concentration of a target molecule in a sample is determined using a standard curve derived from measurements of standard samples containing known concentrations of a labeled molecule (e.g., a labeled target molecule) and a reference molecule.

[0031] Some aspects of the present disclosure provide a detection chip that includes an array of sample wells, the interior base of each sample well being functionalized with a positively charged molecule. In some embodiments, the array of sample wells is a regular array. In some embodiments, the depth of each sample well is between 50 and 500 nm, optionally about 300 nm. In some embodiments, the diameter of the interior base of each sample well is between 50 and 250 nm, optionally between 75 and 125 nm, and further optionally 100 nm.

[0032] In some embodiments, positively charged molecules are attached to the interior base of each sample well using a silane-containing compound. In some embodiments, positively charged molecules are attached to the interior base of each sample well using a biotin-streptavidin complex. In some embodiments, the interior base of each sample well is 1000 nm 2 In some embodiments, the positively charged molecule is a polylysine molecule. In some embodiments, the polylysine molecule comprises 10-200 lysine amino acids, optionally 20-100 lysine amino acids, and further optionally 50 lysine amino acids. In some embodiments, the positively charged molecule is a positively charged terminal silane molecule.

[0033] In some embodiments, the labeled target molecule or label molecule further comprises a molecular barcode. Some aspects of the present disclosure provide methods for determining the identity of a target molecule.

[0034] In some embodiments, a method for determining the identity of a target molecule includes: (i) contacting the target molecule with a first affinity agent having binding affinity for the target molecule to produce a first complex comprising the target molecule bound to the first affinity agent, wherein the first affinity agent is immobilized on the surface of a solid phase bead and a labeled molecule is attached to the surface of the solid phase bead; (ii) contacting the first complex with a surface-immobilized second affinity agent having binding affinity for the target molecule to produce a second complex comprising the second affinity agent bound to the first complex; (iii) optionally washing the second complex; (iv) isolating the labeled molecule; (v) contacting the isolated labeled molecule with a detection chip comprising a sample well having a known molecule attached to the sample well; and (vi) determining the identity of the labeled molecule using fluorescence, luminescence, and / or kinetic measurements, thereby identifying the target molecule.

[0035] In some embodiments, a method for determining the identity of a target molecule includes: (i) contacting the target molecule with a first affinity agent having binding affinity for the target molecule to produce a first complex comprising the target molecule bound to the first affinity agent, where the first affinity agent is immobilized on the surface of a solid phase bead; (ii) contacting the first complex with a second affinity agent having binding affinity for the target molecule to produce a second complex comprising the second affinity agent bound to the first complex, where the second affinity agent is attached to a labeled molecule; (iii) optionally washing the second complex; (iv) isolating the labeled molecule; (v) contacting the isolated labeled molecule with a detection chip comprising a sample well with a known molecule attached to the sample well; and (vi) determining the identity of the labeled molecule using fluorescence, luminescence, and / or kinetic measurements, thereby identifying the target molecule.

[0036] In some embodiments, the labeled molecule is a labeled nucleic acid. In some embodiments, the known molecule is a known nucleic acid, optionally, the known nucleic acid is complementary to the labeled nucleic acid.

[0037] In some embodiments, the kinetic measurement comprises the residence time of the labeled molecule within the sample well. In some embodiments, the target molecule is a protein, a small molecule, or a nucleic acid. In some embodiments, the nucleic acid is DNA or RNA.

[0038] In some embodiments, the first affinity agent is an antibody or an aptamer and the target molecule is an antigen, optionally the antigen is a protein, peptide or polysaccharide. In some embodiments, the solid phase beads are plastic, polymeric, glass, or magnetic beads.

[0039] In some embodiments, the second affinity agent is an antibody. In some embodiments, the label molecule is linked to at least one fluorophore.

[0040] In some embodiments, the detection chip comprises a regular array of sample wells, each of which may have a depth of 50-500 nm, optionally about 300 nm, and each of which may have a diameter at its inner base of 50-250 nm, optionally 75-125 nm, and optionally about 100 nm.

[0041] In some embodiments, the labeling molecule further comprises a molecular barcode. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 shows an exemplary workflow of a method of the present disclosure utilizing a single-stranded label and a reference nucleic acid. [Figure 2] FIG. 1 shows an exemplary workflow of a method of the present disclosure utilizing a double-stranded label and a reference nucleic acid. [Figure 3] FIG. 1 shows an exemplary workflow of the disclosed method utilizing dumbbell labels and reference nucleic acids. [Figure 4] FIG. 1 shows an example of the positioning of a reference molecule relative to a labeled molecule (left) and several embodiments of the labeled molecule (right). [Figure 5] FIG. 1 shows exemplary configurations of indicator and reference molecules. [Figure 6] FIG. 1 shows some examples of methods for delivering labeled and reference molecules to sample wells. [Figure 7] FIG. 1 shows an example of surface immobilization of a sample well. [Figure 8] FIG. 1 shows an example of the internal base of a detection chip functionalized with polylysine. [Figure 9] FIG. 1 shows an example of the internal base of a sensing chip functionalized with positively charged silanes. [Figure 10A] 1 illustrates dynamic pulse behavior using the detection chip described herein. [Figure 10B]1 shows dynamic pulse behavior using the detection chip described herein.FIG. 1 shows a histogram of pulse counts (pulse rate) over a 15 minute period. [Figure 10C] Figure 10 shows dynamic pulse behavior using the detection chip described herein, showing cluster separation of two different nucleic acids labeled with fluorophores with characteristic differences in bin ratios for pulses with pulse durations >0.3. [Figure 10D] 1 shows dynamic pulse behavior using the detection chip described herein.FIG. 2 shows a histogram of pulse duration.FIG. [Figure 11A] FIG. 1 shows an exemplary method for modifying an antibody with a cyclooctyne-containing molecule. [Figure 11B] FIG. 1 shows an exemplary method for modifying an antibody with a biotin-streptavidin conjugate. [Figure 12A] FIG. 1 shows an exemplary strategy for eluting a nucleic acid molecule (e.g., a labeled molecule) from a complex (e.g., a sandwiched antibody-antigen complex) comprising a first affinity agent and a second affinity agent. [Figure 12B] FIG. 1 shows an exemplary strategy for eluting a nucleic acid molecule (e.g., a labeled molecule) from a complex (e.g., a sandwiched antibody-antigen complex) comprising a first affinity agent and a second affinity agent. [Figure 12C] FIG. 1 shows an exemplary strategy for eluting a nucleic acid molecule (e.g., a labeled molecule) from a complex (e.g., a sandwiched antibody-antigen complex) comprising a first affinity agent and a second affinity agent. [Figure 12D] FIG. 1 shows an exemplary strategy for eluting a nucleic acid molecule (e.g., a labeled molecule) from a complex (e.g., a sandwiched antibody-antigen complex) comprising a first affinity agent and a second affinity agent. [Figure 13] FIG. 10 shows an exemplary trace illustrating the initiation of the pulse upon addition of labeled dsDNA due to reversible binding of the dsDNA to the PLL-functionalized surface at the bottom of the nanoaperture. [Figure 14]Figure 1 shows measurements of relative signal collected in lifetime sensitivity time bins of a complementary metal-oxide semiconductor (CMOS) sensor, which correspond to the dye fluorescence lifetimes (bin ratios) collected during the detection run of a sample containing 25 pM each of Cy3- and Atto-Rho6G-labeled dsDNA. [Figure 15A] FIG. 1 illustrates an exemplary workflow of the method of the present disclosure utilized. [Figure 15B] FIG. 1 illustrates an exemplary workflow of the method of the present disclosure utilized. DETAILED DESCRIPTION OF THE INVENTION

[0043] In some aspects, the present disclosure provides a method of biosensor detection (e.g., accurately determining the amount, e.g., concentration, of a target molecule). In some embodiments, the method of biosensor detection (e.g., a method of determining the concentration of a labeled target molecule in a sample) includes: combining a sample containing the labeled target molecule with a reference molecule of known concentration; determining a ratio of detection events of the labeled target molecule to detection events of the reference molecule; and determining the concentration of the labeled target molecule in the sample based at least in part on the ratio of detection events of the labeled target molecule to detection events of the reference molecule. In some embodiments, the method of biosensor detection includes determining the identity of the target molecule.

[0044] In some embodiments, a method of biosensor detection (e.g., a method of determining the concentration of a target molecule in a sample) includes: (i) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to produce a plurality of first complexes containing the target molecule bound to the first affinity agents, wherein at least a portion of the plurality of first affinity agents are immobilized on a surface; (ii) contacting the plurality of first complexes with a plurality of second affinity agents having binding affinity for the first complexes to produce a plurality of second complexes comprising second affinity agents bound to the first complexes, wherein at least some of the second affinity agents are linked to labeled molecules; (iii) removing unbound second affinity agent and / or isolating the plurality of second complexes; (iv) optionally isolating at least a segment of each labeled molecule from the bound second affinity agent of the plurality of second complexes; (v) matching a segment of the labeled molecule with a reference molecule of known concentration; (vi) determining the ratio of the detection events of the labeled molecule to the detection events of the reference molecule; and (vii) determining the concentration of the target molecule in the sample based at least in part on the ratio of detection events of the labeled molecule to detection events of the reference molecule;

[0045] In some embodiments, step (i) includes: (i)(a) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to generate a plurality of first complexes containing the target molecule bound to the first affinity agents; and (i)(b) immobilizing at least a portion of the plurality of first affinity agents on a surface.

[0046] target molecule The target molecule can be any protein, small molecule, or nucleic acid. In some embodiments, the target molecule is a naturally occurring molecule. In some embodiments, the target molecule is a synthetic molecule. In some embodiments, the target molecule is derived from or obtained from a biological sample. The target molecule can be an antigen. In some embodiments, the target molecule is an antigen, which is a protein, peptide, or polysaccharide.

[0047] The biological sample can be a single cell, a mammalian cell tissue, an animal sample, a fungal sample, or a plant sample. In some embodiments, the biological sample is a blood sample, a saliva sample, a sputum sample, a fecal sample, a urine sample, a buccal swab sample, an amniotic membrane sample, a semen sample, a synovial membrane sample, a spinal cord sample, or a pleural effusion sample.

[0048] In some embodiments, the sample may be a purified sample, a cell lysate, a single cell, a cell population, or a tissue. In some embodiments, the biological sample is derived from a human, a non-human primate, a rodent, a dog, a cat, a horse, or any other mammal. In some embodiments, the biological sample is derived from a bacterial cell culture (e.g., an Escherichia coli (E. coli) bacterial cell culture). The bacterial cell culture may include gram-positive and / or gram-negative bacterial cells. In some embodiments, the sample is a purified sample of nucleic acids or proteins previously extracted via user-developed methods from a metagenomic sample or an environmental sample. The blood sample may be a freshly drawn blood sample from a subject (e.g., a human subject) or may be a dried blood sample (e.g., stored on a solid medium (e.g., a Guthrie card)). The blood sample may include whole blood, serum, plasma, red blood cells, and / or white blood cells.

[0049] In some embodiments, a sample (e.g., a sample containing cells or tissue) may be prepared, e.g., lysed (e.g., disrupted, disintegrated, and / or otherwise digested) using processes known to those of skill in the art. In some embodiments, the sample prepared, e.g., lysed, includes cultured cells, a tissue sample from a biopsy (e.g., a tumor biopsy from a cancer patient, e.g., a human cancer patient), or any other clinical sample. In some embodiments, a sample containing cells or tissue is lysed using any one of known physical or chemical methodologies to release target molecules (e.g., target nucleic acids or target proteins) from the cells or tissue. In some embodiments, a sample can be lysed using electrolysis, enzymatic methods, detergent-based methods, and / or mechanical homogenization. In some embodiments, samples (e.g., complex tissues, Gram-positive or Gram-negative bacteria) may require multiple lysis methods performed sequentially. In some embodiments, if a sample does not contain cells or tissue (e.g., a sample containing purified nucleic acids), the lysis step may be omitted. In some embodiments, lysis of a sample is performed to isolate a target nucleic acid. In some embodiments, lysis of a sample is performed to isolate a target protein. In some embodiments, lysis methods further include using a mill to pulverize the sample, sonication, surface acoustic waves (SAW), freeze-thaw cycles, heating, addition of detergents, addition of proteolytic agents (e.g., enzymes such as hydrolases or proteases), and / or addition of cell wall digesting enzymes (e.g., lysozyme or zymolase). Exemplary detergents (e.g., non-ionic detergents) for lysis include polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, polysorbates, and alkylphenol ethoxylates, preferably nonylphenol ethoxylate, alkylglucosides, and / or polyoxyethylene alkylphenyl ethers.In some embodiments, the lysis method involves heating the sample for at least 1-30 minutes, 1-25 minutes, 5-25 minutes, 5-20 minutes, 10-30 minutes, 5-10 minutes, 10-20 minutes, or at least 5 minutes at a desired temperature (e.g., at least 60°C, at least 70°C, at least 80°C, at least 90°C, or at least 95°C).

[0050] Affinity agents The first affinity agent described herein is a molecule (e.g., an antibody or an aptamer) that has binding affinity for a target molecule. In some embodiments, the first affinity agent is 1×10 -3 M~1×10 -4 M, 1 x 10 -4 M~1×10 -5 M, 1 x 10 -5 M~1×10 -6 M, 1 x 10 -6 M~1×10 -7 M, 1 x 10 -7 M~1×10 -8 M, 1 x 10 -8 M~1×10 -9 M, 1 x 10 -9 M~1×10 -9 M, 1 x 10 -9 M~1×10 -10 M, or 1 x 10 -10 M~1×10 -12 M has a binding affinity for a target molecule. In some embodiments, the first affinity agent specifically binds to a particular epitope of the target molecule (e.g., a target protein). In some embodiments, the first affinity agent is a primary antibody.

[0051] The first affinity agent may be immobilized on a surface. In some embodiments, the first affinity agent is immobilized on a surface before contacting (e.g., binding to) its cognate target molecule for which it has binding affinity. In other embodiments, the first affinity agent is immobilized on a surface after contacting (e.g., binding to) its cognate target molecule for which it has binding affinity, e.g., allowing a complex to form between the first affinity agent and the target molecule. In some embodiments, the first affinity agent is immobilized on solid-phase beads (e.g., magnetic beads, e.g., Dynabead®), microfluidic channels, nanoapertures, resins, matrices, membranes, polymers, plastics, metals, or glass.

[0052] The second affinity agent described herein is generally a molecule (e.g., an antibody or aptamer) that has binding affinity for a complex comprising the first affinity agent bound to its cognate target molecule. In some embodiments, the second affinity agent is 1×10 -3 M~1×10 -4 M, 1 x 10 -4 M~1×10 -5 M, 1 x 10 -5 M~1×10 -6 M, 1 x 10 -6 M~1×10 -7 M, 1 x 10 -7 M~1×10 -8 M, 1 x 10 -8 M~1×10 -9 M, 1 x 10 -9 M~1×10 -9 M, 1 x 10 -9 M~1×10 -10 M, or 1 x 10 -10 M~1×10 -12 M has binding affinity for a complex comprising a first affinity agent bound to its cognate target molecule.

[0053] Labeled and reference molecules In some embodiments, the labeled molecule is a molecule linked to a second affinity agent. In some embodiments of the methods described herein, the labeled molecule is used to represent the total amount or quantity (e.g., concentration) of the target molecule in a sample. The labeled molecule can be detected to accurately determine the total amount or quantity (e.g., concentration) of the target molecule in a sample. The detection of the labeled molecule can be performed using sequencing technology (e.g., protein sequencing or nucleic acid sequencing) or fluorescence measurements.

[0054] The labeled molecule may comprise a nucleic acid. In some embodiments, the labeled molecule is a labeled nucleic acid. In some embodiments, the labeled molecule comprises a biotin-streptavidin complex linked to a nucleic acid and at least one fluorescent dye molecule. In some embodiments, the labeled molecule is a labeled nucleic acid comprising two complementary strands, the first strand of which is linked to a second affinity agent.

[0055] In some embodiments, the labeled nucleic acid is 5 to 200, 5 to 150, 5 to 100, 5 to 50, 5 to 25, 10 to 200, 10 to 100, 10 to 50, 25 to 200, 25 to 100, 25 to 50, 50 to 200, 50 to 100, or 100 to 200 nucleotides in length. The labeled nucleic acid can be single-stranded or double-stranded. The labeled nucleic acid can contain secondary or tertiary structural elements. In some embodiments, the labeled nucleic acid contains a region that forms a hairpin loop. In some embodiments, the labeled nucleic acid is a dumbbell nucleic acid. In some embodiments, the labeled nucleic acid is a double-stranded nucleic acid containing a first strand and a second strand that contains a region complementary to the first strand.

[0056] The labeled molecule may be as shown in Figure 4. For example, in some embodiments, the labeled nucleic acid comprises a first strand and a nucleic acid dumbbell, wherein a first region of the nucleic acid dumbbell is complementary to the first strand. In some embodiments, the labeled nucleic acid comprises a first strand and a nucleic acid dumbbell, wherein the labeled nucleic acid further comprises a second single-stranded nucleic acid that is complementary to a second region of the nucleic acid dumbbell.

[0057] In some embodiments, the labeling molecule is linked to at least one fluorophore. In some embodiments, the labeling molecule is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorophores. In some embodiments, the labeling molecule is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different fluorophores (e.g., each fluorophore is excited by a different excitation wavelength). In some embodiments, the labeling molecule is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 identical fluorophores (e.g., each fluorophore is excited by the same excitation wavelength).

[0058] The fluorophore can be linked (e.g., chemically bound) to the labeled molecule before or after the labeled molecule is linked to the second affinity agent. In some embodiments, the fluorophore is linked to the labeled molecule via a carbon-based chemical bond. In some embodiments, the fluorophore is linked to the labeled molecule via a biotin-streptavidin complex.

[0059] In some embodiments, the reference molecule is a molecule used as an internal standard in the sample (e.g., added to the sample by an operator in a defined, known amount). In some embodiments, the reference molecule is a reference nucleic acid (e.g., a single-stranded or double-stranded nucleic acid). The reference molecule can be immobilized on a surface (e.g., the same surface as the first affinity agent). In some embodiments, the reference molecule is linked to the first affinity agent. In some embodiments, the reference molecule is not immobilized on any surface.

[0060] In some embodiments, the reference molecule is a complex comprising a single-stranded nucleic acid immobilized on a surface or linked to a first affinity agent and a dumbbell nucleic acid comprising a region of complementarity to the single-stranded nucleic acid.

[0061] Determining the ratio of detected events between labeled molecules and reference molecules In the disclosed methods, the amount (e.g., concentration) of a target molecule is determined based on the ratio of detection events of the labeled molecule to detection events of the reference molecule, where the detection events of the labeled molecule represent the presence of the target molecule. In some embodiments, the detection events can be determined using sequencing (e.g., protein sequencing or nucleic acid sequencing) or fluorescence measurements. In the disclosed methods, when determining the ratio of detection events of the labeled molecule to detection events of the reference molecule, the amount (e.g., concentration) of the reference molecule is known. In some embodiments, the ratio of detection events of the labeled molecule to detection events of the reference molecule in a sample (e.g., a biological sample) is plotted against a standard curve (e.g., a standard curve created using a mixture of known amounts of the labeled molecule and the reference molecule). In some embodiments, the standard curve is derived from measurements of standard samples containing known concentrations of the labeled molecule and the reference molecule.

[0062] In some embodiments involving fluorescence measurements, an isolated segment of the labeled molecule and a known concentration of the reference molecule are combined in a detection chip to determine the ratio of detection events of the labeled molecule to detection events of the reference molecule. In some embodiments involving sequencing measurements, an isolated segment of the labeled molecule and a known concentration of the reference molecule are combined with the sample to be sequenced to determine the ratio of detection events of the labeled molecule to detection events of the reference molecule.

[0063] In some embodiments, the ratio of detected events of labeled molecules to detected events of reference molecules is determined using fluorescence measurements of labeled molecules and reference molecules in sample wells of a detection chip.

[0064] The ratio of labeled molecules to reference molecules can be determined, in part, based on the residence time of the labeled molecules and reference molecules in the sample wells of the detection chip. The residence time of the labeled molecules and reference molecules in the detection chip can, in some embodiments, be directly correlated to the concentrations of the labeled molecules and reference molecules in the sample (and sample wells of the detection chip). In some embodiments, the residence time of the molecules can be slowed, for example, to improve signal reliability and lower detection limits. In some embodiments, the labeled molecules and reference molecules are delivered to and maintained within the sample wells by electrostatic interaction with positively charged molecules (e.g., polylysine) at the interior base of each sample well. In some embodiments, the labeled nucleic acids and reference nucleic acids are delivered to and maintained within the sample wells by interaction with nucleic acids at the interior base of each sample well that are complementary to the labeled nucleic acids and / or reference nucleic acids. In some embodiments, the labeled proteins and reference proteins are delivered to and maintained within the sample wells by interaction with antibodies at the interior base of each sample well that have binding affinity for the labeled nucleic acids and / or reference nucleic acids. In some embodiments, the labeled molecule and the reference molecule are delivered to and maintained within the sample well by gravity or a magnetic field (e.g., when the labeled molecule and the reference molecule remain linked to the solid surface of, for example, solid-phase beads, such as magnetic beads). In some embodiments, the labeled molecule and the reference molecule are delivered to and maintained within the sample well using a crowding reagent. Examples of crowding reagents include sugar molecules (e.g., sucrose or trehalose), methylcellulose, polyethylene glycol, dextran, ficoll, or proteins such as bovine serum albumin.

[0065] Residence time measurements can be performed using any suitable buffer. In some embodiments, residence time measurements are performed using 200-350 mM NaCl, 10-30 mM KCl, 3 mM NaHPO, 1 mM KHPO, 5 mM 4-nitrobenzoic acid, 50 mM D-glucose, 0.1% Tween®-20, pH=7.5.

[0066] In some embodiments, fluorescence measurements (e.g., residence time) are analyzed using pulse calling. In some embodiments, reversible binding of labeled and reference molecules to the positively charged surface of the detection chip generates transient pulses, which can then be called and filtered based on their characteristics (e.g., pulse duration, single-frame pulse). In some embodiments, pulses with low single-frame pulses are discarded. In some embodiments, the operator can then generate statistics of qualified pulses, such as histograms of pulse / aperture and pulse duration, and / or histograms of bin ratios, to show the separation of labeled and reference clusters, and based on this, quantification of molecules (e.g., molecular fluorophores) can be achieved.

[0067] method conditions The method can be carried out at any suitable temperature. For example, any step of the method (e.g., contacting the first affinity agent with the sample) can be carried out at a temperature of 4-40°C, 4-37°C, 4-30°C, 4-25°C, 4-15°C, 4-10°C, 10-40°C, 15-37°C, 15-25°C, or room temperature. Similarly, any step of the method can be carried out for any suitable period of time. For example, any step of the method (e.g., contacting the first affinity agent with the sample) can be carried out for 5-60 minutes, 5-300 minutes, 5-200 minutes, 5-100 minutes, 30-180 minutes, 1-4 hours, 1-3 hours, or 1-2 hours.

[0068] In some embodiments, the sample is contacted with the first affinity agent before being contacted with the second affinity agent, hi other embodiments, the sample is contacted with the first affinity agent and the second affinity agent simultaneously.

[0069] The step of removing unbound second affinity agents from the sample is carried out by washing the sample with a wash buffer. The wash buffer may be a high-salt wash buffer, a low-salt wash buffer, or phosphate-buffered saline. The step of isolating the second complex (e.g., the surface-bound second complex) comprising the first affinity agent, the second affinity agent, and the labeled molecule may be carried out by filtering the sample or manually removing the surface (e.g., beads) to which the second complex is bound.

[0070] The step of isolating the labeled molecule from the bound second affinity agent can be carried out by washing the sample with an elution buffer. The elution buffer can be a high-salt buffer, a buffer with a different pH than the sample, or any elution buffer known to those skilled in the art. In some embodiments, the labeled molecule is isolated from the bound second affinity agent by changing the temperature of the sample (e.g., increasing the temperature).

[0071] In some embodiments, labeled nucleic acids are isolated using a process such as that shown in Figures 12A-12D. Figures 12A-12B show that labeled nucleic acids (i.e., reporter strands) can be isolated by washing a sample with an excess of single-stranded nucleic acid (i.e., displacing strand) that is complementary to the reporter strand of a double-stranded labeled nucleic acid. Figure 12C shows that labeled nucleic acids (i.e., reporter strands) can be isolated by washing a sample with an excess of single-stranded nucleic acid (i.e., displacing strand) that is complementary to the first strand (i.e., capture strand) of a double-stranded labeled nucleic acid. Figure 12D shows that labeled nucleic acids (i.e., reporter strands) can be isolated by contacting a sample with a nicking enzyme that cleaves the labeled nucleic acid.

[0072] In some embodiments, if the reference molecule is linked to the surface or otherwise present in the sample before the isolation of the labeled molecule, the reference molecule can be isolated simultaneously with the labeled molecule.In some embodiments, the labeled nucleic acid and the reference nucleic acid are amplified during or after isolation from the sample.Nucleic acid can be amplified using any known amplification technique (for example, rolling circle amplification).

[0073] Cartridge or detection chip In another aspect, a cartridge or detection chip is provided. In some embodiments, the detection chip includes an array of sample wells (e.g., a regular array of sample wells). In some embodiments, the detection chip enables the collection of fluorescence measurements. The detection chip may be functionalized with a positively charged molecule, for example, at the internal base of each sample well (i.e., the bottom of the sample well).

[0074] In some embodiments, the depth of the sample wells in the detection chip is 50-500 nm, 25-250 nm, 50-400 nm, 50-300 nm, 50-200 nm, 50-150 nm, 50-100 nm, 75-150 nm, 100-250 nm, 100-300 nm, 250-500 nm, or 250-350 nm. In some embodiments, the depth of the sample wells in the detection chip is about 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. In some embodiments, the diameter of the inner base of each sample well is 50-250 nm, 50-200 nm, 50-150 nm, 50-100 nm, 75-150 nm, 75-200 nm, 100-150 nm, 100-200 nm, or 150-250 nm. In some embodiments, the diameter of the inner base of each sample well is about 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, or 250 nm.

[0075] In some embodiments, the detection chip includes a positively charged molecule attached to the interior base of the sample well. In some embodiments, the positively charged molecule is attached using a silane-containing compound or a biotin-streptavidin complex. In some embodiments, the interior base of the sample well is 1000 nm 2 functionalized with 20-500, 30-400, 50-350, 50-300, 50-250, 50-200, 50-100, 100-400, or 100-200 positive charges per molecule.

[0076] In some embodiments, the positively charged molecule is a polylysine molecule. The polylysine molecule can comprise a linear or branched chain of lysine amino acids. In some embodiments, the polylysine molecule comprises 10-200, 10-150, 10-100, 25-200, 25-150, 25-100, 20-100, 20-75, 25-50, or 50-100 lysine amino acids. In some embodiments, the polylysine molecule comprises approximately 10, 25, 50, 75, 100, 125, 150, 175, 200, or 225 lysine amino acids. In some embodiments, the polylysine molecule is as shown in FIG. 8.

[0077] In some embodiments, the positively charged molecule is a positively charged terminal silane molecule. In some embodiments, the positively charged terminal silane molecule is as shown in FIG.

[0078] In some embodiments, the cartridge or detection chip includes a base layer having a surface including channels, at least a portion of at least some of the channels (1) having a substantially triangular cross-section with a single apex at the base of the channel and two other apexes at the surface of the base layer, and (2) having a surface layer including an elastomer configured to substantially seal the surface opening of the channel.

[0079] In some embodiments, the cartridge or detection chip comprises a base layer. In some embodiments, the base layer has a surface comprising one or more channels. As used herein, the term "channel" is known to those skilled in the art and can refer to a structure configured to accommodate and / or transport a fluid. A channel generally comprises a wall; a base (e.g., a base connected to and / or formed from a wall); and a surface opening that may be open, covered, and / or sealed in one or more portions of the channel.

[0080] As used herein, the term "microchannel" refers to a channel that includes at least one dimension that is 1000 microns (1000 μm) or less in size. For example, a microchannel can include at least one dimension (e.g., width, height) that is 1000 microns (1000 μm) or less in size (e.g., 100 microns (100 μm) or less, 10 microns (10 μm) or less, 5 microns (5 μm) or less). In some embodiments, a microchannel includes at least one dimension that is 1 micron (1 μm) or more (e.g., 2 microns (2 μm) or more, 10 microns (10 μm) or more). Combinations of the above-referenced ranges are also possible (e.g., 1 micron (1 μm) or more and 1000 microns (1000 μm) or less, 10 microns (10 μm) or more and 100 microns (100 μm) or less). Other ranges are also possible. In some embodiments, a microchannel has a hydraulic diameter of 1000 microns (1000 μm) or less. As used herein, the term "hydraulic diameter" (DH) is known to those skilled in the art and may be determined as follows: DH=4A / P, where A is the cross-sectional area of ​​fluid flow through the channel and P is the wetted perimeter of the cross section (the perimeter of the cross section of the channel that is in contact with the fluid).

[0081] In some embodiments, at least a portion of at least some of the channels have a substantially triangular cross-section. In some embodiments, at least a portion of at least some of the channels have a substantially triangular cross-section with a single apex at the base of the channel and two other apexes at the surface of the base layer. Referring again to Figure 24, in some embodiments, at least a portion of at least some of the channels 102 have a substantially triangular cross-section with a single apex at the base of the channel and two other apexes at the surface of the base layer.

[0082] As used herein, the term "triangle" is used to refer to shapes that can be inscribed or circumscribed to approximate or equal actual shapes, and is not limited to purely triangular shapes. For example, the cross section of a triangle may include non-zero curvature in one or more portions.

[0083] The triangular cross section may comprise a wedge. As used herein, the term "wedge" is known to those skilled in the art and refers to a shape having a thick end and tapering to a thin end. In some embodiments, the wedge has an axis of symmetry from the thick end to the thin end. For example, the wedge may have a thick end (e.g., a surface opening of a channel) and taper to a thin end (e.g., a base of a channel), and may have an axis of symmetry from the thick end to the thin end.

[0084] Furthermore, in certain embodiments, the substantially triangular cross-section (i.e., "V-groove") may have various aspect ratios. As used herein, the term "aspect ratio" of a V-groove refers to the ratio of height to width. For example, in some embodiments, the V-groove may have an aspect ratio of 2 or less, 1 or less, or 0.5 or less, and / or 0.1 or more, 0.2 or more, or 0.3 or more. Combinations of the above-referenced ranges are also possible (e.g., between or equal to 0.1 and 2, between or equal to 0.2 and 1). Other ranges are also possible.

[0085] In some embodiments, at least a portion of at least some channels has a cross-section including a substantially triangular portion and a second portion that opens into the substantially triangular portion and extends below the substantially triangular portion relative to the surface of the channel. In some embodiments, the second portion has a diameter (e.g., an average diameter) significantly smaller than the average diameter of the substantially triangular portion. In some embodiments, a portion along the length of the channel may have both a substantially triangular portion and a second portion (a "deep section"), while a different portion along the length of the channel has only a substantially triangular portion. In some such embodiments, when a device (e.g., a roller) engages a portion having both a substantially triangular portion and a second portion (a deep section), a seal with the surface layer is not achieved, and therefore pumping action is not initiated. However, because the device engages along the length of the channel, if the device deforms a surface layer where a portion of the channel has only a substantially triangular section, the second portion (a deep section) of that portion may be missing, creating a seal (and consequently a pressure differential), thereby initiating pumping action. Thus, in some cases, the presence or absence of a deep section along the length of the channel of a cartridge or detection chip may allow control of which portions of the channel can undergo pumping action upon engagement with the device.

[0086] The inclusion of such a "deep section" as the second portion of at least some of the channels of a cartridge or detection chip may contribute to any of a variety of potential advantages. For example, such a deep section may, in some cases, contribute to a reduction in pump volume during peristaltic pumping. In some such cases, the pump volume can be reduced by a factor of two or more to obtain higher volumetric resolution. In some cases, such a deep section may provide a well-defined starting point for the pump volume that is not determined by where the rollers land on the channel. For example, the interface between a portion of a channel having both a substantially triangular portion and a second portion (the deep section) and a portion of a channel having only a substantially triangular portion can be used as a well-defined starting point for the pump volume because it can pump only the fluid occupying the volume of the latter channel portion. In some cases, there may be some error in where the rollers land on the channel depending on various factors, such as the alignment of the cartridge or detection chip. The inclusion of a deep section may, in some cases, reduce or eliminate the variation in pump volume associated with such error.

[0087] As used herein, the average diameter of the substantially triangular portion of a channel may be measured as the average across the z-axis from the apex of the substantially triangular portion to the surface of the channel.

[0088] Devices and Modules Devices or modules are generally provided that include an apparatus, cartridge, or detection chip (e.g., including a channel (e.g., a microfluidic channel)), and / or a pump (e.g., a peristaltic pump) for use in the methods described herein. The devices can be used in accordance with the present disclosure to facilitate accurate determination of the amount (e.g., concentration) of a target molecule from a biological sample. The devices and associated methods can be used to perform chemical and / or biological reactions, including reactions for processing nucleic acids and / or proteins according to any step of the biosensor detection methods described elsewhere herein.

[0089] In some embodiments, the devices of the present disclosure can perform any number of the following steps: combining a sample containing labeled target molecules with a known concentration of a reference molecule; determining a ratio of detection events of the labeled target molecule to detection events of the reference molecule; and determining the concentration of the labeled target molecule in the sample based at least in part on the ratio of detection events of the labeled target molecule to detection events of the reference molecule.

[0090] The device of the present disclosure, in some embodiments, comprises the steps of: (i) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to produce a plurality of first complexes containing the target molecule bound to the first affinity agents, wherein at least a portion of the plurality of first affinity agents are immobilized on a surface; (ii) contacting the plurality of first complexes with a plurality of second affinity agents having binding affinity for the first complexes to produce a plurality of second complexes comprising second affinity agents bound to the first complexes, wherein at least some of the second affinity agents are linked to labeled molecules; (iii) removing unbound second affinity agent and / or isolating the plurality of second complexes; (iv) optionally isolating at least a segment of each labeled molecule from the bound second affinity agent of the plurality of second complexes; (v) matching a segment of the labeled molecule with a reference molecule of known concentration; (vi) determining the ratio of the detection events of the labeled molecule to the detection events of the reference molecule; and (vii) determining the concentration of the target molecule in the sample based at least in part on the ratio of detection events of the labeled molecule to detection events of the reference molecule.

[0091] In some embodiments, step (i) includes: (i)(a) contacting a sample containing the target molecule with a plurality of first affinity agents having binding affinity for the target molecule to generate a plurality of first complexes containing the target molecule bound to the first affinity agents; and (i)(b) immobilizing at least a portion of the plurality of first affinity agents on a surface.

[0092] In some embodiments, all of steps (i) through (vii) are performed by a device of the present disclosure. In some embodiments, step (i) is performed by a device of the present disclosure, and optionally any number of steps (ii) through (vii). In some embodiments, step (ii) is performed by a device of the present disclosure, and optionally any number of steps (i) and / or (iii) through (vii). In some embodiments, step (iii) is performed by a device of the present disclosure, and optionally any number of steps (i), (ii), and / or (iv) through (vii). In some embodiments, step (iv) is performed by a device of the present disclosure, and optionally any number of steps (i) through (iii) and / or (v) through (vii). In some embodiments, step (v) is performed by a device of the present disclosure, and optionally any number of steps (i) through (iv), (vi), or (vii). In some embodiments, a device of the present disclosure performs step (vi), optionally performing any number of steps (i)-(v), (vii), and / or (viii). In some embodiments, a device of the present disclosure performs step (vii), optionally performing any number of steps (i)-(vi), and / or (viii). In some embodiments, a device of the present disclosure performs step (viii), optionally performing any number of steps (i)-(vii). The order of the steps can be changed depending on the needs of the experiment. In some embodiments, any one of the steps incorporates a manual step. This flexibility allows the user to accommodate multiple sample types and detection platforms.

[0093] In some embodiments, a device of the present disclosure is configured to deliver or transfer a labeled molecule and / or a reference molecule to a detection module or device. In some embodiments, a device of the present disclosure is directly connected (e.g., physically attached) to a detection device or module, or indirectly connected to a detection device or module.

[0094] In some embodiments, the cartridge or detection chip includes one or more reservoirs or reaction vessels configured to receive fluid and / or contain one or more reagents used in the biosensor method. In some embodiments, the cartridge or detection chip includes one or more channels (e.g., microfluidic channels) configured to accommodate and / or transport fluid (e.g., fluid containing one or more reagents) used in the biosensor method. Reagents include buffers, enzyme reagents, polymer matrices, capture reagents, size-specific selection reagents, sequence-specific selection reagents, and / or purification reagents. Additional reagents for use in biosensor methods are known to those of skill in the art.

[0095] In some embodiments, the cartridge or detection chip contains one or more stored reagents (e.g., in liquid or lyophilized form suitable for reconstitution into a liquid form). The stored reagents of the cartridge or detection chip include reagents suitable for performing a desired step and / or for processing a desired sample type. In some embodiments, the cartridge or detection chip is a single-use cartridge or detection chip (e.g., a disposable cartridge or detection chip) or a multi-use cartridge or detection chip (e.g., a reusable cartridge or detection chip). In some embodiments, the cartridge or detection chip is configured to receive a user-supplied sample. The user-supplied sample can be added to the cartridge or detection chip before or after the device receives the cartridge or detection chip, e.g., manually by a user or by an automated process.

[0096] Devices according to the present disclosure generally include mechanical, electronic, and / or optical components that can be used to operate the cartridges or detection chips described herein. In some embodiments, device components operate to achieve and maintain a specific temperature on the cartridge or detection chip, or on specific regions of the cartridge or detection chip. In some embodiments, device components operate to apply a specific voltage to electrodes on the cartridge or detection chip for a specific period of time. In some embodiments, device components operate to move liquids to, from, or between reservoirs and / or reaction vessels on the cartridge or detection chip. In some embodiments, device components operate to move liquids through channels on the cartridge or detection chip, e.g., to, from, or between reservoirs and / or reaction vessels on the cartridge or detection chip. In some embodiments, device components move liquids via a peristaltic pumping mechanism (e.g., an apparatus) that interacts with elastomeric, reagent-specific reservoirs or reaction vessels on the cartridge or detection chip. In some embodiments, the device components move fluids via a peristaltic pumping mechanism (e.g., an apparatus) configured to interact with an elastomeric component (e.g., a surface layer comprising an elastomer) associated with the channel of the cartridge or detection chip to pump fluid through the channel. The device components can include, for example, computational resources for driving a user interface that allows for input of sample information, selection of specific steps, and reporting of run results.

[0097] In some embodiments, a device or module (e.g., a sample preparation device; a sequencing device; a combined sample preparation and sequencing device) is configured to precisely transport small volumes of fluid with a well-defined fluid flow resolution and, in some cases, a well-defined flow rate. In some embodiments, a device or module is configured to transport fluid at a flow rate of 0.1 μL / sec or greater, 0.5 μL / sec or greater, 1 μL / sec or greater, 2 μL / sec or greater, 5 μL / sec or greater, or higher. In some embodiments, a device or module herein is configured to transport fluid at a flow rate of 100 μL / sec or less, 75 μL / sec or less, 50 μL / sec or less, 30 μL / sec or less, 20 μL / sec or less, 15 μL / sec or less, or lower. Combinations of these ranges are also possible. For example, in some embodiments, a device or module herein is configured to transport fluid at a flow rate of 0.1 μL / sec or greater to 100 μL / sec or greater, or 5 μL / sec or greater to 15 μL / sec. For example, in certain embodiments, the systems, devices, and modules herein have fluid flow resolution on the order of tens or hundreds of microliters. Further discussion of fluid flow resolution is provided elsewhere herein. In certain embodiments, the systems, devices, and modules are configured to transport small volumes of fluid through at least a portion of a cartridge or detection chip.

[0098] In some embodiments, the cartridge or detection chip can handle small volumes of fluid (e.g., 1-10 μL, 2-10 μL, 4-10 μL, 5-10 μL, 1-8 μL, or 1-6 μL of fluid). In some embodiments, the sequencing cartridge or detection chip is physically embedded in or associated with a device of the present disclosure (e.g., to allow for delivery of prepared samples to a reaction mixture for sequencing). In some embodiments, a sequencing cartridge or detection chip physically embedded in or associated with a device of the present disclosure comprises a microfluidic channel with a fluid interface in the form of a face-seal gasket or a conical press-fit (e.g., a Luer fitting). In some embodiments, the fluid interface can then be disrupted to physically separate the sequencing cartridge or detection chip from a device of the present disclosure after delivery of the prepared sample.

[0099] The following non-limiting examples are intended to illustrate aspects of the devices, methods, and compositions described herein. Use of the disclosed devices in accordance with the present disclosure may proceed through one or more of the steps described below. The user can open the device lid and insert a cartridge or detection chip supporting the desired step. The user can then add a sample, which can be combined with a specific lysis solution, to a sample port on the cartridge or detection chip. The user can then close the device lid, enter any sample-specific information via the device's touchscreen interface, select any process-specific parameters (e.g., desired size selection range, desired degree of homology for target molecule capture, etc.), and begin running the biosensor method. After the run, the user can receive relevant run data (e.g., confirmation that the run was successfully completed, run-specific metrics, etc.), as well as process-specific information (e.g., amount of sample generated, presence or absence of a specific target sequence, etc.). Data generated by the run can undergo subsequent bioinformatics analysis, either locally or cloud-based. Depending on the step, the completed sample can be extracted from the cartridge or detection chip for subsequent use (e.g., genome sequencing, qPCR quantification, cloning, etc.). The device can then be opened and the cartridge or detection chip removed.

[0100] In some embodiments, the biosensor detection module includes a pump. In some embodiments, the pump is a peristaltic pump. Some such pumps include one or more of the inventive components for fluid processing described herein. For example, the pump may include a device and / or a cartridge or a detection chip. In some embodiments, the pump device includes a roller, a crank, and a rocker. In some such embodiments, the crank and rocker are configured as a crank-rocker mechanism connected to the roller. Coupling the crank-rocker mechanism with the roller of the device can, in some cases, achieve some of the advantages described herein (e.g., easy removal of the device from the cartridge or detection chip, good measurement of stroke volume). In certain embodiments, the pump cartridge or detection chip includes a channel (e.g., a microfluidic channel). In some embodiments, at least a portion of the channel of the cartridge or detection chip has a particular cross-sectional shape and / or surface layer that can contribute to any of the advantages described herein.

[0101] One non-limiting aspect of some cartridges or detection chips that may provide certain advantages in some cases is the inclusion of a channel having a particular cross-sectional shape within the cartridge or detection chip. For example, in some embodiments, the cartridge or detection chip includes a V-shaped channel. One potentially convenient, but non-limiting, way of forming such a V-shaped channel is by molding or machining a V-shaped groove into the cartridge or detection chip. In certain embodiments in which rollers of a device engage with the cartridge or detection chip to induce fluid flow through the channel, the inclusion of a V-shaped channel (also referred to herein as a V-groove or a channel having a substantially triangular cross-section) has been recognized as an advantage. For example, in some cases, the V-shaped channel is dimensionally independent of the rollers. In other words, in some cases, there is no single dimension that a roller (e.g., a wedge-shaped roller) of a device must adhere to in order to properly engage with the V-shaped channel. In contrast, certain conventional cross-sectional shapes of channels, such as semicircular, may require the rollers to have a particular dimension (e.g., radius) in order to properly engage with the channel (e.g., to create a fluid seal and induce a pressure differential in a peristaltic pumping process). In some embodiments, the inclusion of channels that are dimensionally independent of the rollers may result in easier and cheaper manufacturing of hardware components and increased configurability / flexibility.

[0102] In certain embodiments, the cartridge or detection chip includes a surface layer (e.g., a flat surface layer). One exemplary embodiment relates to a potentially advantageous embodiment involving laminating a membrane (also referred to herein as a surface layer) comprising (e.g., consisting essentially of) an elastomer (e.g., silicone) over a V-groove to create substantially half of a flexible tube. In some embodiments, the elastomer-containing surface layer is then deformed within the channel to form a pinch, and the pinch can then be moved to generate negative pressure at the trailing edge of the pinch, which creates suction, and fluid can be pumped toward the leading edge of the pinch, generating positive pressure at the leading edge of the pinch. In certain embodiments, this pumping is accomplished by interfacing the cartridge or detection chip (including a channel with a surface layer) with a roller-containing device configured to perform a motion of the roller, including engaging the roller with a portion of the surface layer to pinch the portion of the surface layer against the wall and / or base of the associated channel, moving the roller in a rotational motion along the wall and / or base of the associated channel to move the pinch of the surface layer against the wall and / or base, and / or disengaging the roller from a second portion of the surface layer. In certain embodiments, a crank rocker mechanism is incorporated into the device to perform this motion of the roller.

[0103] Fluorescence detection Excitation light is provided to the sequencing device or module from one or more light sources external to the sequencing device or module. Optical components of the sequencing device or module can receive the excitation light from the light source and direct the light, e.g., via a waveguide, to an array of sample wells in the sequencing device or module to illuminate an illumination region within the sample well. In some embodiments, the sample wells can have a configuration that allows a target molecule or sample containing multiple molecules to be held in close proximity to the surface of the sample well, thereby facilitating delivery of excitation light to the sample well and detection of emission light from the target molecule or sample containing multiple molecules. A target molecule or sample containing multiple molecules disposed within the illumination region can emit emission light in response to illumination with the excitation light. For example, a nucleic acid or protein (or a plurality thereof) can be labeled with a fluorescent marker that emits light in response to achieving an excited state by illumination with the excitation light. The emission light emitted by the target molecule or sample containing multiple molecules can then be detected by one or more photodetectors in pixels corresponding to the sample well with the target molecule or sample containing the multiple molecules being analyzed. According to some embodiments, multiple sample wells can be analyzed in parallel when performed across an array of sample wells, which may range in number between approximately 10,000 pixels and 10,000,000 pixels.

[0104] The sequencing device or module can include an optical system for receiving excitation light and directing the excitation light to the sample well array. The optical system can include one or more grating couplers configured to couple the excitation light into the sequencing device or module and direct the excitation light to other optical components. The optical system can include optical components that direct the excitation light from the grating coupler to the sample well array. Such optical components can include an optical splitter, an optical combiner, and a waveguide. In some embodiments, one or more optical splitters can couple the excitation light from the grating coupler and deliver the excitation light to at least one waveguide. According to some embodiments, the optical splitter can have a configuration that enables substantially uniform delivery of excitation light across all waveguides, such that each of the waveguides receives substantially the same amount of excitation light. Such embodiments can improve the performance of the sequencing device or module by improving the uniformity of the excitation light received by the sample wells of the sequencing device or module.Examples of suitable components for inclusion in a sequencing device or module for coupling excitation light to a sample well and / or directing emission light to a photodetector are described in U.S. patent application Ser. No. 14 / 821,688, filed August 7, 2015, and entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," U.S. patent application Ser. No. 14 / 543,865, filed November 17, 2014, and entitled "INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES," and U.S. patent application Ser. No. 14 / 543,865, filed June 26, 2020, and entitled "OPTICAL AND ELECTRICAL SECONDARY PATH ELIMINATION." No. PCT / US2020 / 039868, entitled "Optical Coupler and Waveguide System," filed December 15, 2017, both of which are incorporated herein by reference. Examples of suitable grating couplers and waveguides that may be implemented in a sequencing device or module are described in U.S. patent application Ser. No. 15 / 844,403, entitled "Optical Coupler and Waveguide System," filed December 15, 2017, which is incorporated herein by reference in its entirety.

[0105] Additional photonic structures may be positioned between the sample well and the photodetector and configured to reduce or prevent excitation light from reaching the photodetector, which would otherwise contribute to signal noise when detecting emission light. In some embodiments, the metal layer, which may act as circuitry for the sequencing device or module, may also act as a spatial filter. Examples of suitable photonic structures may include spectral filters, polarizing filters, and spatial filters, and are described in U.S. Patent Application No. 16 / 042,968, filed July 23, 2018, and entitled "Optical Rejection Photonic Structures," which is incorporated herein by reference in its entirety.

[0106] Components located remotely from the sequencing device or module can be used to position and align the excitation source with the sequencing device or module. Such components may include optical components, including lenses, mirrors, prisms, windows, apertures, attenuators, and / or optical fibers. Additional mechanical components can be included in the instrument to enable control of one or more alignment components. Such mechanical components may include actuators, stepper motors, and / or knobs. Examples of suitable excitation sources and alignment mechanisms are described in U.S. patent application Ser. No. 15 / 161,088, filed May 20, 2016, and entitled "PULSED LASER AND SYSTEM," which is incorporated herein by reference in its entirety. Another example of a beam steering module is described in U.S. patent application Ser. No. 15 / 842,720, filed December 14, 2017, and entitled "COMPACT BEAM SHAPING AND STEERING ASSEMBLY," which is incorporated herein by reference in its entirety. Further examples of suitable excitation sources are described in U.S. patent application Ser. No. 14 / 821,688, filed Aug. 7, 2015, and entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," which is incorporated herein by reference in its entirety.

[0107] Photodetectors associated with individual pixels of a sequencing device or module can be configured and arranged to detect light emitted from the pixel's corresponding sample well. Further examples of suitable photodetectors are described in U.S. Patent Application No. 14 / 821,688, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," which is incorporated herein by reference in its entirety. In some embodiments, the sample wells and their respective photodetectors can be aligned along a common axis. In this way, the photodetectors can sufficiently overlap the sample within the pixel.

[0108] Characteristics of the detected emission light can provide an indication for identifying a marker associated with the emission light. Such characteristics can include any suitable type of characteristics, including the arrival time of photons detected by the photodetector, the amount of photons accumulated over time by the photodetector, and / or the distribution of photons across two or more photodetectors. In some embodiments, the photodetector can have a configuration that allows detection of one or more timing characteristics (e.g., luminescence lifetime) associated with the sample's emission light. The photodetector can detect a distribution of photon arrival times after a pulse of excitation light propagates through a sequencing device or module, and the distribution of arrival times can provide an indication of a timing characteristic (e.g., a proxy for the luminescence lifetime) of the sample's emission light. In some embodiments, one or more photodetectors provide an indication of the probability of emission light being emitted by a marker (e.g., luminescence intensity). In some embodiments, multiple photodetectors can be sized and positioned to capture the spatial distribution of the emission light. Output signals from one or more photodetectors can then be used to distinguish one marker from multiple markers, and multiple markers can be used to identify one marker within a sample. In some embodiments, the sample may be excited with multiple excitation energies, and the emission light and / or timing characteristics of the emission light emitted by the sample in response to the multiple excitation energies may distinguish one marker from multiple markers.

[0109] During operation, parallel analysis of samples in the sample wells is performed by exciting some or all of the samples in the wells with excitation light and detecting signals from the sample emissions with photodetectors. The emitted light from the samples can be detected by corresponding photodetectors and converted into at least one electrical signal. The electrical signal can be transmitted along conductors within the circuitry of the sequencing device or module, which can be connected to an instrument connected to the sequencing device or module. The electrical signal can then be processed and / or analyzed. The processing and / or analysis of the electrical signal can be performed on a suitable computing device located inside or outside the instrument.

[0110] The instrument may include a user interface for controlling the operation of the instrument and / or the sequencing device or module. The user interface may be configured to allow a user to input information into the instrument, such as commands and / or settings used to control the instrument's functions. In some embodiments, the user interface may include buttons, switches, dials, and / or a microphone for voice commands. The user interface may allow a user to receive feedback regarding the performance of the instrument and / or the sequencing device or module, such as information obtained by proper alignment and / or readout signals from photodetectors on the sequencing device or module. In some embodiments, the user interface may provide feedback using a speaker to provide audible feedback. In some embodiments, the user interface may include indicator lights and / or a display screen to provide visual feedback to the user.

[0111] In some embodiments, the instruments or devices described herein may include a computer interface configured to connect to a computing device. The computer interface may be a USB interface, a FireWire interface, or any other suitable computer interface. The computing device may be any general-purpose computer, such as a laptop or desktop computer. In some embodiments, the computing device may be a server (e.g., a cloud-based server) accessible over a wireless network via a suitable computer interface. The computer interface may facilitate communication of information between the instrument and the computing device. Input information for controlling and / or configuring the instrument may be provided to the computing device and transmitted to the instrument via the computer interface. Output information generated by the instrument may be received by the computing device via the computer interface. The output information may include feedback regarding instrument performance, sequencing device or module performance, and / or data generated from the photodetector readout signal.

[0112] In some embodiments, the instrument may include a processing device configured to analyze data received from one or more photodetectors of the sequencing device or module and / or send control signals to the excitation source. In some embodiments, the processing device may include a general-purpose processor and / or a specially adapted processor (e.g., one or more central processing units (CPUs), such as microprocessors or microcontroller cores, field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), custom integrated circuits, digital signal processors (DSPs), or combinations thereof). In some embodiments, processing of data from one or more photodetectors may be performed by both the instrument's processing device and an external computing device. In other embodiments, the external computing device may be omitted and processing of data from one or more photodetectors may be performed solely by the sequencing device's or module's processing device.

[0113] According to some embodiments, an instrument configured to analyze a target molecule or sample containing multiple molecules based on its luminescence emission characteristics can detect differences in luminescence lifetimes and / or intensities between different luminescent molecules and / or differences in the lifetimes and / or intensities of the same luminescent molecule in different environments. The inventors have recognized and appreciated that differences in luminescence emission lifetimes can be used to distinguish between the presence or absence of different luminescent molecules and / or to distinguish between different environments or conditions to which the luminescent molecules are exposed. In some cases, aspects of the system can be simplified by distinguishing luminescent molecules based on lifetime (e.g., rather than emission wavelength). As an example, wavelength-discriminating optics (e.g., wavelength filters, dedicated detectors for each wavelength, dedicated pulsed light sources at different wavelengths, and / or diffractive optics) may be reduced in number or eliminated when distinguishing luminescent molecules based on lifetime. In some cases, a single pulsed light source operating at a single characteristic wavelength can be used to excite different luminescent molecules that emit light within the same wavelength region of the optical spectrum but have measurably different lifetimes. Analytical systems that use a single pulsed light source, rather than multiple light sources operating at different wavelengths, to excite and distinguish different luminescent molecules that emit in the same wavelength region are less complex to operate and maintain, more compact, and can be manufactured at lower cost.

[0114] While analytical systems based on luminescence lifetime analysis may have certain advantages, the amount of information obtained by the analytical system and / or detection accuracy may be increased by enabling additional detection techniques. For example, some embodiments of the system may be further configured to identify one or more characteristics of a sample based on emission wavelength and / or emission intensity. In some implementations, emission intensity may additionally or alternatively be used to distinguish between different luminescent labels. For example, some luminescent labels may emit at significantly different intensities or have significant differences in excitation probability (e.g., at least about 35% difference) even if their decay rates are similar. By referencing binned signals to the measured excitation light, it may be possible to distinguish between different luminescent labels based on intensity levels.

[0115] According to some embodiments, different luminescence lifetimes can be distinguished by a photodetector configured to time-bin luminescence emission events following excitation of the luminescent labels. Time binning can occur during a single charge accumulation cycle for the photodetector. A charge accumulation cycle is the interval between readout events during which photogenerated carriers accumulate within the bins of the time-binning photodetector. Examples of time-binning photodetectors are described in U.S. patent application Ser. No. 14 / 821,656, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," which is incorporated herein by reference in its entirety. In some embodiments, the time-binning photodetector can generate charge carriers within a photon absorption / carrier generation region and directly transfer the charge carriers to charge carrier storage bins within a charge carrier storage region. In such embodiments, the time-binning photodetector may not include a carrier transfer / capture region. Such time-binning photodetectors are sometimes referred to as "direct binning pixels." An example of a time-binning photodetector including a direct binning pixel is described in U.S. patent application Ser. No. 15 / 852,571, filed Dec. 22, 2017, entitled "Integrated photodetector with direct binning pixel," which is incorporated herein by reference in its entirety.

[0116] In some embodiments, different numbers of the same type of fluorophore can be linked to different components of a target molecule (e.g., a target nucleic acid or a target protein) or to multiple molecules present in a sample (e.g., multiple nucleic acids or multiple proteins), allowing each individual molecule to be identified based on its emission intensity. For example, two fluorophores can be linked to a first labeled molecule, and four or more fluorophores can be linked to a second labeled molecule. Due to the different numbers of fluorophores, there can be different excitation and emission probabilities associated with different molecules. For example, there can be more emission events for the second labeled molecule during a signal accumulation interval, resulting in a significantly higher apparent intensity for the bin than for the first labeled molecule.

[0117] The inventors have recognized and appreciated that distinguishing nucleic acids or proteins based on fluorophore decay rates and / or fluorophore intensity can allow for simplification of optical excitation and detection systems. For example, optical excitation can be performed using a single wavelength source (e.g., a light source that produces one characteristic wavelength rather than multiple light sources, or a light source that operates at multiple different characteristic wavelengths). Furthermore, wavelength-discriminating optics and filters may not be required in the detection system. Also, a single photodetector may be used in each sample well to detect emissions from different fluorophores. The phrase "characteristic wavelength" or "wavelength" is used to refer to a central or dominant wavelength within a limited emission bandwidth. For example, limited bandwidth emission can include a central or peak wavelength within a 20 nm bandwidth output by a pulsed light source. In some cases, "characteristic wavelength" or "wavelength" can be used to refer to a peak wavelength within the full bandwidth of the emission output by a source.

[0118] Sequencing Device or Module In some embodiments, nucleic acid or protein sequencing according to the present disclosure can be performed using a system that enables single molecule analysis. The system can include a sequencing device or module and an instrument configured to interface with the sequencing device or module. The sequencing device or module can include an array of pixels, each pixel including a sample well and at least one photodetector. The sample well of the sequencing device or module can be formed on or through the surface of the sequencing device or module and can be configured to receive a sample placed on the surface of the sequencing device or module.

[0119] In some embodiments, the sample wells are components of a cartridge or detection chip (e.g., a disposable or single-use cartridge or detection chip) that can be inserted into the device. Collectively, the sample wells can be considered an array of sample wells. The multiple sample wells can have an appropriate size and shape so that at least some of the sample wells receive a sample containing a single target molecule or multiple molecules (e.g., a target nucleic acid or target protein). In some embodiments, the number of molecules in the sample wells can be distributed among the sample wells of a sequencing device or module such that some sample wells contain one molecule (e.g., a target nucleic acid or target protein) and other sample wells contain zero, two, or multiple molecules.

[0120] Molecular barcoding In some embodiments, the methods provided herein include contacting a molecular barcode with a barcode recognition molecule that binds to one or more sites on the molecular barcode. In some embodiments, the barcode recognition molecule binds to one or more sites on multiple molecular barcodes. Thus, in some embodiments, the barcode recognition molecule can be used to decode barcode content from multiple different single molecules in a mixture (e.g., different analytes containing the same or different molecular barcodes). As an illustrative and non-limiting example, a multiplexed mixture can contain multiple analytes attached to molecular barcodes. Some of these molecular barcodes can include a sample index that indicates the sample origin of the analyte attached thereto, and the barcode recognition molecule that binds to the sample index can be used to determine which analyte originated from the corresponding sample.

[0121] In some embodiments, the single molecule construct comprises a molecular barcode (e.g., a kinetic barcode). In some embodiments, the molecular barcode of the present disclosure is a nucleic acid barcode (e.g., a single-stranded nucleic acid). In some embodiments, the nucleic acid barcode comprises DNA, RNA, PNA, and / or LNA. In some embodiments, the molecular barcode is a polypeptide barcode.

[0122] In some embodiments, the molecular barcode comprises a series of index sequences. For example, in some embodiments, the molecular barcode is a nucleic acid barcode comprising a series of index sequences. In some embodiments, each index sequence is different from any other index sequence in the series of index sequences. In some embodiments, at least two index sequences in the series of index sequences are the same. In some embodiments, the series of index sequences corresponds to a series of barcode recognition molecule binding sites. In some embodiments, the barcode recognition molecule binds to a site on the molecular barcode that comprises two index sequences in the series of index sequences. In some embodiments, each index sequence provides different information about the barcode content.

[0123] Further, in some embodiments, the molecular barcode is attached to the analyte (e.g., payload molecule, detection molecule). In some embodiments, the analyte is derived from a biological or synthetic source. In some embodiments, the analyte is derived from a serum sample, a blood sample, a tissue sample, or a single cell. In some embodiments, the analyte is a biomolecule. In some embodiments, the analyte is a nucleic acid or a polypeptide. In some embodiments, the analyte is a nucleic acid aptamer, a protein, or a protein fragment. In some embodiments, the analyte is a small molecule, a metabolite, or an antibody. In some embodiments, the molecular barcode is attached to the analyte via a linker. In some embodiments, the linker comprises a cleavage site (e.g., a photocleavable site). Thus, in some embodiments, a single-molecule construct comprising a cleavage sequence allows for removal of the analyte, simplifying loading and / or analysis on a substrate surface (e.g., a chip).

[0124] Also, in some embodiments, the molecular barcode comprises an attachment molecule. In some embodiments, the attachment molecule is any moiety or linking group suitable for surface immobilization of the molecular barcode. In some embodiments, the attachment molecule comprises a covalent or non-covalent linking group. In some embodiments, the attachment molecule comprises a biotin moiety. In some embodiments, the attachment molecule comprises a bis-biotin moiety. Linking groups and other compositions and methods useful for surface immobilization are described in more detail elsewhere herein and are known in the art.

[0125] In some embodiments, the cleavage site is an optional component that may not be incorporated into the single molecule construct depending on the desired implementation. In some embodiments, the attachment molecule may be adjacent to the analyte such that the molecular barcode can be attached to the surface via the analyte. Examples of other configurations and linkage strategies for single molecule constructs are provided elsewhere herein.

[0126] In some embodiments, the methods of the present disclosure relate to a barcode deconvolution approach, which involves deciphering the identity of a molecule, the origin of a sample, and / or the location of a single molecule on an array. In some embodiments, the methods provided herein are advantageously used to deconvolute molecular barcode information in multiplexed samples. For example, the methods of the present disclosure can be applied to techniques for single-cell polypeptide sequencing. In some embodiments, the resulting single-molecule constructs can be analyzed by polypeptide sequencing (e.g., dynamic peptide sequencing) and barcode recognition according to the present disclosure.

[0127] In some embodiments, a labeling molecule described herein can be attached or linked to a barcode (e.g., a molecular barcode) described herein. In some embodiments, a reference molecule described herein can be attached or linked to a barcode (e.g., a molecular barcode) described herein. In some embodiments, an affinity agent described herein can be attached or linked to a barcode (e.g., a molecular barcode) described herein.

[0128] In some embodiments, the method for detecting a target molecule involves using the workflow described in FIG. 15A. For example, as shown in FIG. 15A, in the first step, solid-phase beads (e.g., magnetic beads) can be initially loaded with a primary antibody (e.g., a 1° antibody) and a surface-associated labeling molecule (reporter). The labeling molecule can be a labeled nucleic acid. The labeling molecule can be attached or linked to a barcode (e.g., a molecular barcode) described herein. In the second step, a sample containing the target molecule (e.g., when the target molecule is an antigen) can be added to beads containing a surface-immobilized antibody. In some embodiments, the sample contains an antigen that binds (e.g., specifically binds) to the surface-immobilized antibody. In the third step, beads can be added to a surface containing a surface-immobilized secondary antibody (2° antibody) to generate a complex containing the bound target molecule between the surface-immobilized secondary antibody and the primary antibody attached to the bead, the bead being linked to the surface-associated labeling molecule. In the fourth step, the complex is washed with a wash solution or buffer. In the fifth step, the labeled molecule (reporter) is removed (or cleaved) and isolated from the bead. In the sixth step, the labeled molecule is added to a detection chip (e.g., containing multiple sample wells), which contains a known molecule (e.g., a known nucleic acid or a "known readout oligo") attached to the chip. The labeled molecule has binding affinity for the known molecule. The association and kinetics of binding between the labeled molecule and the known molecule provide for the determination of the identity of the labeled molecule. Such determination of the identity of the labeled molecule allows for the identification of a soluble antibody (e.g., a secondary antibody (2° antibody)) associated with the labeled molecule, which can then allow for the identification of an antigen that can bind to the soluble antibody.

[0129] In some embodiments, the method for detecting a target molecule involves using the workflow described in FIG. 15B. For example, as shown in FIG. 15B, in the first step, magnetic solid-phase beads can be first loaded with a surface-immobilized antibody (e.g., a primary antibody (1° antibody)). In the second step, a sample containing a target molecule (e.g., when the target molecule is an antigen) can be added to the beads containing the surface-immobilized antibody. In some embodiments, the sample contains an antigen that binds (e.g., specifically binds) to the surface-immobilized antibody. In the third step, a soluble antibody (e.g., a secondary antibody (2° antibody)) linked to a labeled molecule (reporter) can be added to the beads to generate a complex containing the target molecule bound between the surface-immobilized antibody and the soluble antibody linked to the labeled molecule, where the labeled molecule is optionally a nucleic acid. In the fourth step, the beads are washed with a wash solution or buffer. In the fifth step, the beads are isolated using a magnet, and the labeled molecule (reporter) is removed (or cleaved) from the soluble antibody. The labeled molecule is then isolated. In the sixth step, the labeled molecule is added to a detection chip (e.g., containing multiple sample wells), which contains a known molecule (e.g., a known nucleic acid or a "known readout oligo") attached to the chip. The labeled molecule has binding affinity for the known molecule. The association and kinetics of binding between the labeled molecule and the known molecule provide for determining the identity of the labeled molecule. Such determination of the identity of the labeled molecule allows for the identification of a soluble antibody (e.g., a secondary antibody (2° antibody)) associated with the labeled molecule, which can then allow for the identification of an antigen that can bind to the soluble antibody. In some embodiments, the labeled molecule is attached or linked to a barcode (e.g., a molecular barcode) described herein.

[0130] Aspects of the present disclosure relate to identifying the content of molecular barcodes. As used herein, terms such as "identifying," "recognizing," and "recognition" with respect to molecular barcodes include determining the partial identity (e.g., partial sequence information) as well as the complete identity (e.g., complete sequence information) of a molecular barcode. In some embodiments, the term includes determining or inferring the nucleotide sequence of at least a portion of a molecular barcode (e.g., based on complementarity with an oligonucleotide probe). In still other embodiments, the term includes determining or inferring a particular characteristic of a molecular barcode, such as the presence or absence of a particular index sequence at one or more sites on the molecular barcode. Thus, in some embodiments, the terms "barcode content," "barcode identity," and similar terms used herein may refer to qualitative information about a molecular barcode and are not limited to the specific sequence information (e.g., the nucleotide sequence of an index) that biochemically characterizes the molecular barcode.

[0131] In some embodiments, barcode recognition is performed by observing distinct association events between the barcode recognition molecule and the molecular barcode, with each association event generating a change in signal magnitude that persists over a duration. In some embodiments, these magnitude changes are detected as a series of signal pulses, or a series of pulses in a signal trace output. As described herein, the signal pulse information can be used to identify barcode content based on a barcode-specific pattern within the series of signal pulses. In some embodiments, the barcode-specific pattern includes multiple signal pulses, each signal pulse including a pulse duration. In some embodiments, the multiple signal pulses can be characterized by a summary statistic (e.g., mean, median, time decay constant) of the distribution of pulse durations in the barcode-specific pattern. In some embodiments, the average pulse duration of the barcode-specific pattern can be between about 1 ms and about 10 seconds (e.g., between about 1 ms and about 1 s, between about 1 ms and about 100 ms, between about 1 ms and about 10 ms, between about 100 ms and about 10 s, between about 1 s and about 10 s, between about 10 ms and about 100 ms, or between about 100 ms and about 500 ms). In some embodiments, the average pulse duration is between about 50 ms and about 2 seconds, between about 50 ms and about 500 ms, or between about 500 ms and about 2 seconds.

[0132] In some embodiments, different barcode-specific patterns corresponding to different barcode contents may be distinguished from one another based on statistically significant differences in summary statistics. For example, in some embodiments, one barcode-specific pattern may be distinguishable from another barcode-specific pattern based on a difference in mean pulse duration of at least 10 milliseconds (e.g., about 10 ms to about 10 s, about 10 ms to about 1 s, about 10 ms to about 100 ms, about 100 ms to about 10 s, about 1 s to about 10 s, or about 100 ms to about 1 s). In some embodiments, the difference in mean pulse duration is at least 50 ms, at least 100 ms, at least 250 ms, at least 500 ms, or more. In some embodiments, the difference in mean pulse duration is about 50 ms to about 1 s, about 50 ms to about 500 ms, about 50 ms to about 250 ms, about 100 ms to about 500 ms, about 250 ms to about 500 ms, or about 500 ms to about 1 s. In some embodiments, the mean pulse duration of one barcode-specific pattern differs from the mean pulse duration of another barcode-specific pattern by about 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, or more. It should be understood that in some embodiments, the smaller the difference in mean pulse duration between different barcode-specific patterns, the more pulse duration may be required within each barcode-specific pattern to distinguish them from each other with statistical confidence.

[0133] In some embodiments, a barcode-specific pattern generally refers to multiple association (e.g., binding) events between a barcode recognition molecule and a molecular barcode. In some embodiments, a barcode-specific pattern comprises at least 10 association events (e.g., at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1,000, or more association events). In some embodiments, a barcode-specific pattern comprises about 10 to about 1,000 association events (e.g., about 10 to about 500 association events, about 10 to about 250 association events, about 10 to about 100 association events, or about 50 to about 500 association events). In some embodiments, multiple association events are detected as multiple signal pulses.

[0134] In some embodiments, a barcode-specific pattern refers to a plurality of signal pulses that can be characterized by summary statistics as described herein. In some embodiments, a barcode-specific pattern includes at least 10 signal pulses (e.g., at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1,000, or more signal pulses). In some embodiments, a barcode-specific pattern includes about 10 to about 1,000 signal pulses (e.g., about 10 to about 500 signal pulses, about 10 to about 250 signal pulses, about 10 to about 100 signal pulses, or about 50 to about 500 signal pulses).

[0135] In some embodiments, a barcode-specific pattern refers to multiple association (e.g., binding) events between barcode recognition molecules and molecular barcodes that occur over a time interval. In some embodiments, barcode recognition can be performed by repeated wash cycles in which the molecular barcode is exposed to different sets of barcode recognition molecules for different durations. In some embodiments, the time interval for a barcode-specific pattern is from about 1 minute to about 30 minutes (e.g., from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 5 minutes to about 20 minutes, from about 5 minutes to about 15 minutes, or from about 5 minutes to about 10 minutes).

[0136] In some embodiments, experimental conditions can be configured to achieve a time interval that allows for sufficient association events to provide a desired level of confidence with the barcode-specific pattern (e.g., before a given set of barcode recognition molecules is removed during a wash cycle). This can be achieved by configuring reaction conditions based on various characteristics, including, for example, reagent concentration, molar ratio of one reagent to another (e.g., ratio of barcode recognition molecules to molecular barcodes, ratio of one barcode recognition molecule to another), number of different reagent types (e.g., number of different types of barcode recognition molecules), binding characteristics (e.g., kinetic and / or thermodynamic binding parameters for barcode recognition molecule binding), reagent modifications (e.g., polyols and other protein modifications that may alter interaction kinetics), reaction mixture components (e.g., one or more components such as pH, buffers, salts, divalent cations, surfactants, and other reaction mixture components described herein), reaction temperature, and various other parameters, as well as combinations thereof, that will be apparent to one of skill in the art. The reaction conditions can be configured based on one or more embodiments described herein, including, for example, signal pulse information (e.g., pulse duration, inter-pulse duration, magnitude change), labeling strategy (e.g., fluorophores, number and / or type of linking groups), surface modification (e.g., modification of sample well surface including molecular barcode immobilization), sample preparation (e.g., analyte size, molecular barcode modification for immobilization), and other embodiments described herein.

[0137] Barcode recognition molecule In some embodiments, the present disclosure provides barcode recognition molecules and methods for using the same.In some embodiments, barcode recognition molecules can be selected or engineered based on the desired binding kinetics for barcode regions.For example, in some embodiments, the methods described herein can be carried out in a multiplexed format, where multiple sites must be distinguished from each other based on the binding interaction at each site.Therefore, the binding interaction at one site should be sufficiently different from the binding interaction at another site so that different sites can be distinguished with higher confidence based on signal pulse information.

[0138] Without wishing to be bound by theory, the barcode recognition molecule may have an association rate or "on" rate of binding (k on ) and the bond dissociation or "off" rate (k off ) and the binding affinity (K D ) binds to the barcode region according to the rate constant k off and k on are critical determinants of the pulse duration (e.g., the time corresponding to a detectable association event) and the inter-pulse duration (e.g., the time between detectable association events), respectively. In some embodiments, these kinetic rate constants can be engineered to achieve the pulse duration and pulse rate (e.g., signal pulse frequency) that provides the best precision.

[0139] In some embodiments, barcode recognition molecules can be engineered by one of skill in the art using conventionally known techniques. In some embodiments, desirable properties include low to moderate affinity (e.g., K of about 50 nM or greater, e.g., about 50 nM to about 50 μM, about 100 nM to about 10 μM, about 500 nM to about 50 μM) for one or more sites on the molecular barcode. DFor example, in some embodiments, the present disclosure provides methods of barcode recognition by detecting reversible binding interactions, where barcode recognition molecules that reversibly bind to molecular barcodes with low to moderate affinity advantageously provide more informative binding data with greater certainty than high affinity binding interactions.

[0140] In some embodiments, the barcode recognition molecule binds to about 10 -6 Less than M (e.g., about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Under M, 10 -16 Dissociation constant (K D ) binds to one or more sites on the molecular barcode. In some embodiments, the barcode recognition molecule has a K of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. D In some embodiments, the barcode recognition molecule binds to one or more sites on the molecular barcode with a K of about 50 nM to about 50 μM (e.g., about 50 nM to about 500 nM, about 50 nM to about 5 μM, about 500 nM to about 50 μM, about 5 μM to about 50 μM, or about 10 μM to about 50 μM). D In some embodiments, the barcode recognition molecule binds to one or more sites on the molecular barcode with a K D binds to one or more sites on the molecular barcode.

[0141] In some embodiments, the barcode recognition molecule is at least 0.1 s -1 Dissociation rate (k off ) binds to one or more sites on the molecular barcode. In some embodiments, the dissociation rate is about 0.1 s -1 ~about 1,000s -1 (For example, about 0.5 seconds -1 ~about 500s -1, about 0.1 seconds -1 ~approx. 100s -1 , about 1 s -1 ~approx. 100s -1 , or about 0.5 seconds -1 ~about 50s -1 In some embodiments, the dissociation rate is about 0.5 s -1 ~approx. 20 seconds -1 In some embodiments, the dissociation rate is about 2 s -1 ~approx. 20 seconds -1 In some embodiments, the dissociation rate is about 0.5 s -1 ~about 2s -1 is.

[0142] In some embodiments, K D or k off The value of K can be a known literature value, or the value can be determined empirically. D or k off The value of k can be measured in a single molecule assay or an ensemble assay. off The value of k can be empirically determined based on signal pulse information obtained in single molecule assays as described elsewhere herein. For example, k off The value of K can be approximated by the reciprocal of the average pulse duration. In some embodiments, the barcode recognition molecule has a different K for each of the two or more sites. D or k off In some embodiments, a first K D or k off is the second K for the second site D or k off In some embodiments, K differs by at least 10% (e.g., at least 25%, at least 50%, at least 100%, or more) from D or k offThe first and second values ​​differ by about 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, for example, about 2-fold, 3-fold, 4-fold, 5-fold, or more.

[0143] As described herein, a barcode recognition molecule may be any biomolecule that is capable of binding one or more sites on a molecular barcode rather than other barcode regions. Recognition molecules include, for example, oligonucleotides, nucleic acids, and proteins, any of which may be synthetic or recombinant.

[0144] In some embodiments, the barcode recognition molecule is an oligonucleotide (e.g., an oligonucleotide probe). In some embodiments, the methods provided herein can be performed by contacting a nucleic acid barcode with an oligonucleotide probe that binds to one or more sites on the nucleic acid barcode. In some embodiments, binding between the oligonucleotide probe and the nucleic acid barcode occurs via hybridization or annealing. Beyond certain experimental conditions (e.g., concentration, temperature), binding characteristics are driven in large part by the length and content of the oligonucleotide probe and its degree of complementarity with the site on the nucleic acid barcode to which it binds (e.g., hybridizes or anneals). Thus, in some embodiments, the oligonucleotide probe offers a variety of adjustable features for adjusting signal pulse characteristics, including, but not limited to, length, nucleotide content (e.g., G / C content, nucleotide analogs with different binding properties (e.g., LNA or PNA analogs)), degree of complementarity, and experimental factors (e.g., concentration, temperature, buffer conditions (e.g., pH, salt, magnesium), and DNA denaturing or stabilizing solvents).

[0145] In some embodiments, the oligonucleotide probe is at least 4 nucleotides in length. In some embodiments, the oligonucleotide probe is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length. In some embodiments, the oligonucleotide probe is less than 30 nucleotides in length (e.g., less than 25 nucleotides in length, less than 20 nucleotides in length, less than 15 nucleotides in length, less than 12 nucleotides in length, or less than 10 nucleotides in length). In some embodiments, the oligonucleotide probe is about 3 to about 30 nucleotides in length (e.g., about 3 to about 10, about 3 to about 8, about 5 to about 25, about 5 to about 15, or about 5 to 10 nucleotides in length). In some embodiments, the oligonucleotide probes are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0146] In some embodiments, the oligonucleotide probes can bind to one or more barcode regions that are not completely complementary to the oligonucleotide probe and provide barcode content information. For example, in some embodiments, the oligonucleotide probes bind to one or more barcode regions having sequences that are less than 100% complementary to the oligonucleotide (e.g., less than 99%, less than 98%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1% or less).

[0147] In addition to oligonucleotides, nucleic acid aptamers can be used as barcode recognition molecules according to the present disclosure.Nucleic acid aptamers are nucleic acid molecules that are engineered to bind to targets with desired affinity and selectivity.Therefore, nucleic acid aptamers can be engineered to bind to desired barcode regions using selection and / or enrichment techniques known in the art.In some embodiments, barcode recognition molecules include nucleic acid aptamers, such as DNA aptamers or RNA aptamers.

[0148] In some embodiments, the barcode recognition molecule is a protein or polypeptide. In some embodiments, the recognition molecule is an antibody or antigen-binding portion of an antibody, an SH2 domain-containing protein or fragment thereof, or an inactivating enzyme biomolecule, such as a peptidase, aminotransferase, ribozyme, aptazyme, or tRNA synthetase (including the aminoacyl-tRNA synthetases and related molecules described in U.S. Patent Application No. 15 / 255,433, filed September 2, 2016, entitled "MOLECULES AND METHODS FOR ITERATIVE POLYPEPTIDE ANALYSIS AND PROCESSING").

[0149] In some embodiments, the barcode recognition molecule is an amino acid recognition molecule. For example, in some embodiments, the molecular barcode comprises a polypeptide barcode, and the amino acid recognition molecule can be used to decode the barcode content from the polypeptide. In some embodiments, the amino acid recognition molecule binds to one or more types of terminal amino acids with different kinetic binding properties. In some embodiments, the amino acid recognition molecule binds to different segments of a polypeptide with different kinetic binding properties. For example, in some embodiments, the amino acid recognition molecule binds to polypeptide segments that contain the same type of amino acid at the N-terminus or C-terminus, but differ in amino acid content at the penultimate (e.g., n+1) and / or subsequent positions relative to the terminal amino acid (e.g., one or more different amino acid types at the second, third, fourth, fifth, or higher positions). These concepts (e.g., differential binding kinetics based on differences in amino acid content only at the penultimate and higher positions) and further examples of amino acid recognition molecules are described in more detail in PCT International Publication No. WO2020102741A1, filed November 15, 2019, entitled "METHODS AND COMPOSITIONS FOR PROTEIN SEQUENCING," which is incorporated by reference in its entirety.

[0150] In some embodiments, the methods provided herein include contacting a molecular barcode with one or more barcode recognition molecules. For purposes of this discussion, one or more barcode recognition molecules in the context of the methods described herein may alternatively be referred to as a set of barcode recognition molecules. In some embodiments, the set of barcode recognition molecules includes at least 2 and up to 20 (e.g., 2-15, 2-10, 5-10, 10-20) barcode recognition molecules. In some embodiments, the set of barcode recognition molecules includes more than 20 (e.g., 20-25, 20-30) barcode recognition molecules. However, it should be understood that any number of barcode recognition molecules can be used in accordance with the methods of the present disclosure to accommodate a desired application.

[0151] According to the present disclosure, in some embodiments, molecular barcode content can be identified by detecting emission from a label attached to the barcode recognition molecule. In some embodiments, the labeled barcode recognition molecule includes a barcode recognition molecule that binds to at least one molecular barcode and a luminescent label having an emission associated with the barcode recognition molecule. In this manner, emission (e.g., emission lifetime, emission intensity, and other emission characteristics described elsewhere herein, including emission-based kinetic binding data) can be correlated with binding of the barcode recognition molecule to identify at least one molecular barcode. In some embodiments, multiple types of labeled barcode recognition molecules can be used in methods according to the present disclosure, each type including a luminescent label having an emission that is uniquely identifiable among the multiple types. Suitable luminescent labels may include luminescent molecules such as fluorescent dyes and are described elsewhere herein.

[0152] In some embodiments, the barcode recognition molecule includes a label with binding-induced emission. For example, in some embodiments, the labeled aptamer can include a donor label and an acceptor label. As a free and unbound molecule, the labeled aptamer adopts a conformation in which the donor and acceptor labels are separated by a distance (e.g., about 10 nm or more) that limits detectable FRET between the labels. Upon binding to the barcode region, the labeled aptamer adopts a conformation in which the donor and acceptor labels are within a distance (e.g., about 10 nm or less) that promotes detectable FRET between the labels. In yet other embodiments, the labeled aptamer can include a quenching moiety and function similarly to a molecular beacon, where emission is internally quenched as a free molecule and restored upon binding to the barcode region (e.g., Hamaguchi et al. (2001), Analytical Biochemistry 294, 126-131). Similar and alternative labeling strategies, such as using FRET between the labeled aptamer and the labeled molecular barcode, will be apparent to those skilled in the art. Without wishing to be bound by theory, it is believed that these and other types of mechanisms for binding-induced luminescence may advantageously reduce or eliminate background luminescence, increasing the overall sensitivity and accuracy of the methods described herein.

[0153] In some embodiments, the molecular barcode content can be identified by detecting one or more electrical properties of a labeled barcode recognition molecule. In some embodiments, the labeled barcode recognition molecule includes a barcode recognition molecule that binds to at least one molecular barcode and a conductive label associated with the barcode recognition molecule. In this manner, one or more electrical properties (e.g., charge, current oscillation color, and other electrical properties including conductivity-based kinetic binding data) can be associated with the binding of the barcode recognition molecule to identify at least one molecular barcode. In some embodiments, multiple types of labeled barcode recognition molecules may be used in the methods according to the present disclosure, each type including a conductive label that generates a uniquely distinguishable electrical signal change (e.g., a change in conductance, such as a change in conductivity amplitude and conductivity transition in a barcode-specific pattern) from among the multiple types. In some embodiments, each of the multiple types of labeled barcode recognition molecules includes a conductive label with a different number of charged groups (e.g., a different number of negatively and / or positively charged groups). Thus, in some embodiments, the conductive label is a charged label. Examples of charged labels include dendrimers, nanoparticles, nucleic acids, and other polymers with multiple charged groups. In some embodiments, a conductive label is uniquely identifiable by its net charge (eg, a net positive charge or a net negative charge), by its charge density, and / or by its number of charged groups.

[0154] Example Embodiments of the present disclosure are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature. [Example]

[0155] Preparation of streptavidin-immobilized PLL surface and detection of binding events of dye-labeled dsDNA molecules at low concentrations The detection chip shown in FIG. 8 (e.g., containing positively charged molecules functionalized on the interior base of each sample well) was fabricated as follows: 1. A complementary metal oxide semiconductor (CMOS) chip containing nanoapertures functionalized on the bottom surface with biotin-PEG-silane was wetted three times with 70% isopropanol, 0.1% Tween®-20, and three times with 1× binding buffer (50 mM MOPS, 75 mM KOAc, 10 mM DTT, 0.03% Tween®-20, pH 7.5).

[0156] 2. The chip was incubated with 20 nM streptavidin in 1× binding buffer for 30 minutes at room temperature. 3. The chip was washed five times with SC-6 buffer (NaCl 200-350 mM, KCl 10-30 mM, NaHPO 3 mM, KHPO 1 mM, 4-nitrobenzoic acid 5 mM, D-glucose 50 mM, 0.1% Tween®-20, pH=7.5).

[0157] 4. The chip was functionalized by adding 100 nM biotin-polylysine (PLL) to SC-6, incubating at room temperature for 30 minutes, and washing with SC-6 five times. The detection chip produced by this method was 1000 nm 2 The subsequent attachment of polylysine provides an average of 2-3 biotins per molecule, with a maximum length of 1000 nm depending on the length of polylysine used. 2 Approximately 40 to 300 positive charges were provided per molecule.

[0158] 5. Reaction buffer (SC-6 buffer containing an oxygen scavenging system) was added to the chip and the detection procedure was initiated. 6. After 10 min of detection, 25 bp Atto-Rho6G labeled dsDNA molecules were added to the reaction buffer at a final concentration of 25 pM, and detection was continued for another 50 min.

[0159] 7. The traces collected from the detection procedure showed a clear pattern of pulse behavior initiated upon addition of dye-labeled dsDNA molecules, demonstrating that the chip prepared in this manner successfully detected the reversible binding event of a single dye-labeled DNA molecule at very low concentrations.

[0160] An exemplary trace collected from the detection run is shown in Figure 13. This exemplary trace (divided into two time bins generated by the fluorescence lifetime sensitivity operation of the CMOS chip) shows the initiation of a pulse upon addition of labeled dsDNA due to the reversible binding of dsDNA to the PLL-functionalized surface at the bottom of the nanoaperture. [Example]

[0161] Preparation of streptavidin-immobilized PLL surfaces, detection of differently labeled dsDNA molecules at low concentrations, and determination of dye ratios. Using the detection chip manufactured by the method of Example 1, the detection of the ratio of dye-labeled dsDNA molecules at very low concentrations was demonstrated as follows.

[0162] 1. Two types of dsDNA molecules (20-40 bp) containing distinguishable fluorescent dye molecules were separately prepared (one containing Cy3 and the other containing Atto-Rho6G, which can be distinguished by their fluorescence lifetime on a Quantum-Si CMOS chip). The two types of labeled dsDNA molecules were mixed in a 1:1 ratio and diluted with reaction buffer (SC-6 buffer containing an oxygen scavenging system) to a final concentration of 25 pM each.

[0163] 2. The sample was added to a CMOS chip prepared as in Example 1, and the detection procedure was carried out for 1 hour. 3. The data collected from the detection run consisted of traces containing signal pulses corresponding to the reversible binding events of labeled dsDNA molecules to the positively charged PLL surface. The signal pulses were identified and assigned to Cy3 or Atto-Rho6G based on the fluorescence lifetime information detected by the CMOS chip during the pulse.

[0164] Consistent with a 1:1 mixture ratio of the two types of labeled dsDNA molecules, Cy3 and Atto-Rho6G pulses were determined to be present in approximately equal amounts (52% vs. 48%), as shown in Figure 14. The "bin ratio" (a measure of the relative signal collected in the lifetime-sensitive time bins of the CMOS sensor, corresponding to the fluorescence lifetime of the dye) was determined for each pulse, and a Gaussian mixture model applied to the bin ratio distribution was used to classify each pulse as a Cy3 or Atto-Rho6G detection event. [Example]

[0165] Detection of reversible binding events of dye-labeled dsDNA molecules on chips containing surfaces directly coupled with positively charged amine-terminated silanes. A detection chip containing positively charged amine-terminated silanes was prepared as follows: 1. CMOS chips were prepared by silane surface passivation chemistry using amine-terminated silane molecules as shown in Figure 9. This procedure resulted in the direct coupling of positively charged amine-terminated molecules to the glass surface located at the bottom of the nanoapertures (also shown in Figure 9).

[0166] 2. The chip was wetted with 70% isopropanol (3 times), 0.1% Tween®-20 (3 times) and washed 5 times with buffer SC-6. 3. Two types of dsDNA molecules (20-40 bp) containing distinguishable fluorescent dye molecules were prepared separately (one containing Cy3 and the other containing Atto-Rho6G, which can be distinguished by their fluorescence lifetime on a Quantum-Si CMOS chip). The two types of labeled dsDNA molecules were mixed in a 1:1 ratio and diluted with reaction buffer (SC-6 buffer containing an oxygen scavenging system) to a final concentration of 25 pM each.

[0167] 4. The sample was added to the CMOS chip and the detection procedure was carried out for 1 hour. 5. Successful dynamic pulse behavior was observed. See Figures 10A-10D for representative data collected using these detection chips.

[0168] The detection chip fabricated by this above method provides a charge density limited by the available aperture opening area, with an estimated charge density of 1000 nm 2 The number of counts per person was 80 to 120.

[0169] Further aspects of the disclosure The aspects of the above exemplary embodiments and examples can be combined in various combinations and subcombinations to create further embodiments of the present disclosure. Unless the aspects of the above exemplary embodiments and examples are mutually exclusive, all such combinations and subcombinations are intended to be within the scope of the present disclosure. It will be apparent to those skilled in the art that the embodiments of the present disclosure include many aspects. Therefore, the scope of the claims should not be limited by the preferred embodiments described in the description and examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

[Claim 1] The invention described in this specification.