KR20220159496A - Devices and methods for sequencing - Google Patents

Abstract

Methods and devices for preparing target molecules (eg, target nucleic acids or target proteins) from biological samples are provided herein. In some embodiments, methods and devices involve sample lysis, sample fragmentation, enrichment of target molecule(s), and/or functionalization of target molecule(s).

Description

Devices and methods for sequencing

Cross-Reference to Related Applications

This application claims USSN 63/014,071 filed on April 22, 2020; USSN 63/014,087 filed on April 22, 2020; USSN 63/014,093 filed on April 22, 2020; USSN 63/014,106 filed on April 22, 2020; USSN 63/041,206 filed on June 19, 2020; USSN 63/139.339 filed January 20, 2021; USSN 63/139,343 filed January 20, 2021; USSN 63/139,346 filed January 20, 2021; and 35 U.S.C. of the filing date of USSN 63/139,348 filed on January 20, 2021. claims priority under § 119(e); The entire contents of each application are incorporated herein by reference.

Proteomics, genomics, and transcriptomics have emerged as important and essential in the study of biological systems. Such analysis of individual organisms or sample types can provide insight into cellular processes and response patterns that lead to improved diagnostic and therapeutic strategies. The complexities surrounding nucleic acid and protein composition and modification present challenges in determining large-scale sequencing information for biological samples.

Summary of Invention

Aspects of the present disclosure include methods, compositions, devices, and/or cartridges for use in a process for preparing a sample for analysis and/or analyzing (eg, analyzing by sequencing) one or more target molecules in a sample. provides In some embodiments, a target molecule is a nucleic acid (eg, DNA or RNA, including without limitation cDNA, genomic DNA, mRNA, and derivatives and fragments thereof). In some embodiments, a target molecule is a protein.

Some aspects of the present disclosure provide an apparatus for preparing a biological sample for sequencing. In some embodiments, the device comprises (i) a dissolution cartridge; (ii) an enrichment cartridge; (iii) a fragmentation cartridge; and (iv) an automation module configured to receive two or more cartridges selected from the group consisting of functionalized cartridges. In some embodiments, the device comprises an automated module configured to take a biological sample comprising one or more microfluidic channels and comprising one or more target molecules. In some embodiments, the device comprises (i) a dissolution cartridge; and (ii) an automation module configured to receive the enrichment cartridge. In some embodiments, the device comprises (i) a dissolution cartridge; and (iii) an automation module configured to receive the fragmentation cartridge. In some embodiments, the device comprises (i) a dissolution cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device comprises (ii) an enrichment cartridge; and (iii) an automation module configured to receive the fragmentation cartridge. In some embodiments, the device comprises (i) an enrichment cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device comprises (i) a fragmentation cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device comprises (i) a fragmentation cartridge; (ii) an enrichment cartridge; and (iii) an automation module configured to receive the fragmentation cartridge. In some embodiments, the device comprises (i) a fragmentation cartridge; (ii) an enrichment cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device comprises (ii) an enrichment cartridge; (iii) a fragmentation cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device comprises (i) a fragmentation cartridge; (ii) an enrichment cartridge; (iii) a fragmentation cartridge; and (iv) an automation module configured to receive the functionalization cartridge. In some embodiments, the device produces nucleic acids with an average read-length greater than the average read-length generated using a control method. A further aspect of the present disclosure is an apparatus for preparing one or more target molecules, configured to perform two or more of the steps selected from (i), (ii), (iii), and (iv), wherein ( i), (ii), (iii), and (iv) provide a device wherein: (i) lysing a biological sample comprising one or more target molecules; (ii) enriching at least one of the one or more target molecules and/or at least one non-target molecule; (iii) fragmenting one or more target molecules; and (iv) functionalizing a terminal moiety of one or more target molecules.

In some embodiments, one or more of the method steps selected from (i), (ii), (iii), and (iv) are performed in a cartridge. In some embodiments, more than one step is performed on the same cartridge. In some embodiments, the cartridge is a single-use cartridge or a multi-use cartridge. In some embodiments, a cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in any one of the automated steps. In some embodiments, a cartridge comprises one or more microfluidic channels configured to contain and/or transport one or more target molecules between any one of the automated steps. In some embodiments, the cartridge includes resin for purification of one or more target molecules between any one of the automated steps. In some embodiments, the resin is Sephadex resin, optionally G-10 Sephadex resin. In some embodiments, a cartridge includes an optional size exclusion medium.

A further aspect of the present disclosure provides methods of making one or more target molecules. In some embodiments, the method of making one or more target molecules comprises two or more of the steps selected from (i), (ii), (iii), and (iv), wherein (i), (ii) , (iii), and (iv) are defined as follows: (i) lysing a biological sample comprising one or more target molecules; (ii) enriching at least one of the one or more target molecules and/or at least one non-target molecule; (iii) fragmenting one or more target molecules; and (iv) functionalizing a terminal moiety of one or more target molecules; wherein at least one of steps (i), (ii), (iii), or (iv) is performed in an automated sample preparation device. In some embodiments, the two steps are performed on an automated sample preparation device. In some embodiments, the three steps are performed on an automated sample preparation device. In some embodiments, the four steps are performed on an automated sample preparation device. In some embodiments, step (i) is performed using a dissolution cartridge. In some embodiments, step (ii) is performed using an enrichment cartridge. In some embodiments, step (iii) is performed using a fragmentation cartridge. In some embodiments, step (iv) is performed using a functionalized cartridge.

A further aspect of the present disclosure provides a cartridge for preparing one or more target molecules. In some embodiments, the cartridge is configured to perform two or more of the steps selected from (i), (ii), (iii), and (iv), wherein (ii), (iii), and (iv) is defined as: (i) lysing a biological sample comprising one or more target molecules; (ii) enriching at least one of the one or more target molecules and/or at least one non-target molecule; (iii) fragmenting one or more target molecules; and (iv) functionalizing a terminal moiety of one or more target molecules. In some embodiments, the cartridge is a single-use cartridge or a multi-use cartridge. In some embodiments, a cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in any one of the automated steps. In some embodiments, a cartridge comprises one or more microfluidic channels configured to contain and/or transport one or more target molecules between any one of the automated steps. In some embodiments, the cartridge includes resin for purification of one or more target molecules between any one of the automated steps. In some embodiments, the resin is a Sephadex resin, optionally a G-10 Sephadex resin.

In some embodiments, a biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, or plant sample. In some embodiments, the biological sample is a blood sample, saliva sample, sputum sample, stool sample, urine sample, buccal swab sample, amnion sample, semen sample, synovial fluid sample, spinal cord sample, or pleural fluid sample. In some embodiments, one or more target molecules are nucleic acids. In some embodiments, one or more target molecules are proteins.

In some embodiments, the device further comprises a peristaltic pump configured to transport one or more fluids into, into, or out of any one of the cartridges received by the device. In some embodiments, the device further comprises a peristaltic pump configured to transport one or more fluids in or through any microfluidic channels of a cartridge received by the device. In some embodiments, the device is configured to transport a fluid with a fluid flow resolution of 1000 microliters or less, 100 microliters or less, 50 microliters or less, or 10 microliters or less. In some embodiments, the device is configured to simultaneously receive two or more cartridges. In some embodiments, the device is configured to simultaneously establish fluid communication between two or more cartridges received by the device. In some embodiments, the device is configured to sequentially receive two or more cartridges.

In some embodiments, the device further comprises a sequencing module. In some embodiments, a device is configured to deliver one or more target molecules to a sequencing module. In some embodiments, a sequencing module performs nucleic acid sequencing. In some embodiments, nucleic acid sequencing comprises single-molecule real-time sequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, and/or Sanger sequencing. In some embodiments, a sequencing module performs protein sequencing. In some embodiments, protein sequencing comprises Edman digestion or mass spectrometry. In some embodiments, a sequencing module performs single-molecule protein sequencing.

In some embodiments, a lysis cartridge comprises one or more microfluidic channels and is configured to take a biological sample comprising one or more target molecules and produce a lysed sample. In some embodiments, an enrichment cartridge comprises one or more microfluidic channels and is configured to enrich for at least one of the one or more target molecules to produce an enriched sample. In some embodiments, a fragmentation cartridge comprises one or more microfluidic channels and is configured to digest or fragment at least one of the one or more target molecules to produce a fragmented sample. In some embodiments, the functionalization cartridge comprises one or more microfluidic channels and is configured to functionalize a terminal moiety of at least one of the one or more target molecules to form a functionalized sample.

In some embodiments, any one cartridge is positioned to receive a sample or target molecule(s) from any other cartridge. In some embodiments, any one cartridge is connected to any other cartridge by one or more microfluidic channels.

In some embodiments, a lysis cartridge includes reagents that lyse the sample but do not degrade or fragment one or more target molecules. In some embodiments, the lysis cartridge comprises reagents that facilitate at least partial isolation or purification of one or more target molecules from non-target molecules in the sample. In some embodiments, reagents include detergents, acids, and/or bases. In some embodiments, reagents include a lysis buffer. In some embodiments, the lysis buffer is selected from the group consisting of RIPA buffer, GCl (guanidine-HCl) buffer, and GlyNP40 buffer. In some embodiments, one or more microfluidic channels in the lysis cartridge promote shearing of cells and/or tissue (eg, shear flow of cells and/or tissue). In some embodiments, the lysis cartridge includes a needle passageway that facilitates mechanical shearing of cells and/or tissue. In some embodiments, the needle passage has an inside diameter of 0.1 to 1 mm. In some embodiments, one or more microfluidic channels in a lysis cartridge comprises an array of posts. In some embodiments, the dissolution cartridge is configured to be heated at an elevated temperature (eg, 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C) . In some embodiments, the device is configured to heat the dissolution cartridge at an elevated temperature (e.g., 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C). It consists of In some embodiments, the device is configured to subject the lysis cartridge to microwave or sonication.

In some embodiments, an enrichment cartridge comprises one or more affinity matrices. In some embodiments, one or more affinity matrices are in a microfluidic channel of an enrichment cartridge. In some embodiments, the one or more target molecules are nucleic acids, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one of the one or more target molecules. In some embodiments, an oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to a target molecule. In some embodiments, the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds at least one of the one or more target molecules. In some embodiments, a protein capture probe is an aptamer or antibody. In some embodiments, the protein capture probe is 10 -9 to 10 -8 M, 10 -8 to 10 -7 M, 10 -7 to 10 -6 M, 10 -6 to 10 -5 M, 10 -5 to 10 It binds to the target protein with a binding affinity of -4 M, 10 -4 to 10 -3 M, or 10 -3 to 10 -2 M. In some embodiments, the one or more target molecules are nucleic acids, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one non-target molecule. In some embodiments, an oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to a non-target molecule. In some embodiments, the oligonucleotide capture probe is not complementary to one or more target molecules. In some embodiments, the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds at least one non-target molecule. In some embodiments, the protein capture probe is 10 -9 to 10 -8 M, 10 -8 to 10 -7 M, 10 -7 to 10 -6 M, 10 -6 to 10 -5 M, 10 -5 to 10 Binds to a non-target protein with a binding affinity of −4 M, 10 −4 to 10 −3 M, or 10 −3 to 10 −2 M. In some embodiments, a protein capture probe does not bind one or more target molecules. In some embodiments, an enrichment cartridge is configured to deplete a sample of non-target molecules.

In some embodiments, a fragmentation cartridge comprises a non-enzymatic reagent that digests or fragments a sample and/or one or more target molecules. In some embodiments, non-enzymatic reagents that digest or fragment a sample and/or one or more target molecules include detergents, acids, and/or bases. In some embodiments, the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules is cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide, hydrochloric acid, BNPS-skatole [2 -(2-nitrophenylsulfenyl)-3-methylindole], and/or 2-nitro-5-thiocyanobenzoic acid. In some embodiments, a fragmentation cartridge comprises one or more enzymatic reagents that digest or fragment at least one of the one or more target molecules. In some embodiments, the one or more enzymatic reagents include one or more proteases. In some embodiments, the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC and ArgC. In some embodiments, the one or more enzymatic reagents include one or more endonucleases or exonucleases. In some embodiments, the fragmentation cartridge may be heated at an elevated temperature (e.g., 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C). . In some embodiments, the device is configured to heat the fragmentation cartridge at an elevated temperature (e.g., 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C). It consists of In some embodiments, the device is configured to subject the fragmentation cartridge to microwave or sonication.

In some embodiments, a functionalization cartridge comprises a first chamber comprising a reagent that covalently transforms moiety M0 of one or more target molecules, or of one or more fragments thereof, with modified moiety M1. In some embodiments, reagents are non-enzymatic. In some embodiments, covalent modifications are regiospecific. In some embodiments, a portion of one or more target molecules, or of one or more fragments thereof, is a C-terminal carboxylate group or a C-terminal amino group. In some embodiments, reagents include buffers, salts, organic compounds, acids, and/or bases. In some embodiments, a portion of one or more target molecules, or of one or more fragments thereof, is a C-terminal amino group and the covalent modification is a diazo transfer. In some embodiments, moiety M0 is -NH ₂ and moiety M1 is -N ₃ . In some embodiments, the reagents are imidazole-1-sulfonyl azide and a copper salt (e.g., copper sulfate), and a buffer having a pH of about 9-11 (e.g., carbonic acid having a pH of about 9-11). calcium buffer). In some embodiments, the reagent includes an optional azide delivery agent. In some embodiments, the reagent includes trifluoromethanesulfonyl azide. In some embodiments, the azide delivery agent comprises benzenesulfonyl-azide. In some embodiments, the first chamber is connected to the second chamber through one or more microfluidic channels, and/or optionally a purification chamber. In some embodiments, the second chamber contains reagents that covalently modify moiety M1 to produce a functionalized peptide. In some embodiments, the covalent modification is an electrocyclization click reaction. In some embodiments, the reagent comprises a DBCO-labeled DNA-streptavidin conjugate and a buffer, optionally wherein the DBCO-labeled DNA-streptavidin conjugate is immobilized to the surface of the second chamber. In some embodiments, the functionalized peptide is functionalized with a DBCO-labeled DNA-streptavidin conjugate.

In some embodiments, a purification chamber is located between a first chamber and a second chamber containing a resin that facilitates purification or enrichment of the modified target molecule, or fragments thereof. In some embodiments, the resin is a Sephadex resin, optionally a G-10 Sephadex resin. In some embodiments, the functionalization cartridge can be heated to an elevated temperature (e.g., 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C). have. In some embodiments, the device heats the functionalized cartridge at an elevated temperature (e.g., 20 to 60 °C, 20 to 30 °C, 25 to 40 °C, 30 to 50 °C, 35 to 50 °C, or 50 to 75 °C). is configured to In some embodiments, functionalized cartridges may be subjected to microwave or sonication.

In some embodiments, purification comprises passing the functionalized sample through a size exclusion medium. In some embodiments, the size exclusion medium can be columnar. The column may be a desalting column. In some embodiments, the column is a Zeba column (eg a Zeba 7 kDa or Zeba 40 kDa column). In some embodiments, the size exclusion medium is part of a fluidic device. In some embodiments, the size exclusion medium is part of a system, but is not part of a fluidic device of the system.

In some embodiments, purifying the protein comprises purification via immunoprecipitation. In some embodiments, immunoprecipitation comprises precipitating a target protein from a sample (eg, a sample before or after functionalization) using an antibody that specifically binds the target protein.

In some embodiments, one or more microfluidic channels are configured to contain and/or transport fluid(s) and/or reagent(s).

In some embodiments, any one of the cartridges includes a base layer having a surface that includes channels. In some embodiments, the channels include one or more microfluidic channels. In some embodiments, at least a portion of at least some of the channels have a substantially triangular-shaped cross-section with a single vertex at the base of the channel and two other vertices at the surface of the base layer. In some embodiments, at least a portion of at least a portion of the channels of any one of the cartridges has a surface layer comprising an elastomer configured to substantially seal surface openings of the channels. In some embodiments, the elastomer includes silicone. In some embodiments, at least one portion of at least some of the channels has walls and a base comprising a substantially rigid material that is compatible with the biological material. In some embodiments, any one of the cartridges includes one or more fluid reservoirs. In some embodiments, at least a portion of the channels connect to a reservoir in a temperature zone. In some embodiments, at least a portion of the channel connects to an electrophoretic gel.

1 shows an exemplary method of preparing a target molecule from a biological sample (eg, using an automated sample preparation device or cartridge of the present disclosure).
2 shows an exemplary workflow for sample preparation of a target protein (eg, using an automated sample preparation device or cartridge of the present disclosure).
3 shows an exemplary workflow for sample lysis (eg, using an automated device or cartridge of the present disclosure).
4 shows an exemplary workflow of sample enrichment of a target molecule (eg, using an automated device or cartridge of the present disclosure).
5 shows an exemplary workflow for digestion of a target molecule (eg, using an automated device or cartridge of the present disclosure).
6-7 show an example workflow for C-terminal functionalization of a target protein (eg, using an automated device or cartridge of the present disclosure).
8 shows a schematic diagram of a cross-sectional view of cartridge 100 along the width of channel 102, in accordance with some embodiments.
9A-9B show a top view schematic ( FIG. 9A ) and images of an exemplary cartridge of the present disclosure.
Figures 10A-10B show data from DNA libraries generated with automated end-to-end (DNA extraction-to-finished libraries) sample preparation using the sample preparation apparatus of the present disclosure compared to libraries generated from manually extracted and purified DNA. Indicates sequencing data output.
11A-11D show the results of automated end-to-end (DNA extraction-to-finished library) sample preparation using the sample preparation apparatus of the present disclosure compared to DNA libraries derived from size-selected samples using commercial and manual methods. Sequencing data output from a DNA library is shown.
12 shows an example of a C-terminal carboxylate coupling procedure.
13 shows an example of a C-terminal carboxylate coupling procedure.
14A to 14D show examples of C-terminal coupling procedures. 14A shows representative functionalization of aspartic acid and glutamic acid terminated peptides. 14B shows representative functionalization of lysine and arginine terminated peptides. 14C shows exemplary protection of sulfide moieties prior to functionalization of lysine-terminated peptides (Reaction 1), and competitive intramolecular cyclization that can be overcome using high concentrations of nucleophiles and coupling reagents (Reaction 2). ). 14D shows model functionalization of lysine-terminated peptides (Reaction 3), and model functionalization of arginine-terminated peptides with internal glutamic acid and aspartic acid residues (Reaction 4).
15 shows a model C-terminal lysine coupling procedure.
16A-16C show data for the model C-terminal lysine coupling procedure. 16A and 16B show binding events to the N-terminus of QP126. The red arrow indicates the case where an enzyme (peptidase) is added, after which a change in pulsing behavior is observed due to binding of Clps to a different amino acid. 16C shows the full-length CRP sequence with tagged fragments indicated in bold.
17 shows an example of a C-terminal lysine coupling procedure using a 4-nitrovinyl sulfonamide reagent.
18A-18B show schemes for exemplary C-terminal lysine coupling procedures using diazo delivery chemistries. 18A shows site-selective diazo delivery. 18B shows site-selective diazo delivery using a dipeptide followed by hydrolysis.
19 shows an example of a lysine coupling procedure using diazo delivery.
20 shows representative schemes of solid-phase and solution-phase peptide activation methods.
21 shows an example of a functionalization process using an immobilized carbodiimide reagent.
22 shows an example of peptide surface immobilization.
23A-23B show representative examples of peptide sequencing. 23A shows a representative example of peptide sequencing by iterative cycles of terminal amino acid recognition and cleavage. 23B shows a representative example of dynamic peptide sequencing using labeled amino acid recognition molecules and exopeptidases in a single reaction mixture.
24A-24F show schematic diagrams of exemplary sample preparation devices of the present disclosure.
25-26 show an example workflow for C-terminal functionalization of a target protein (eg, using an automated device or cartridge of the present disclosure).
27A-27D show results of sequencing of peptide samples prepared in an exemplary fluidic device, in accordance with certain embodiments.

sample manufacturing process

In some aspects, the present disclosure provides a process for preparing a sample, eg, for detection and/or analysis. In some embodiments, the processes described herein can be used to ascertain a property or characteristic of a sample, including the identity or sequence (eg, nucleotide sequence or amino acid sequence) of one or more target molecules in the sample. In some embodiments, a process comprises one or more sample transformation steps, such as sample lysis, sample purification, sample fragmentation, purification of fragmented samples, library preparation (e.g., nucleic acid library preparation), purification of library preparation, sample enrichment (e.g. eg, using affinity SCODA), and/or detection/analysis of the target molecule. In some embodiments, a sample can be a purified sample, cell lysate, single-cell, population of cells, or tissue. In some embodiments, a sample is any biological sample. In some embodiments, the sample (eg, biological sample) is a blood, saliva, sputum, stool, urine, or buccal swab sample. In some embodiments, the biological sample is from a human, non-human primate, rodent, dog, cat, horse, or any other mammal. In some embodiments, the biological sample is from a bacterial cell culture (eg, an E. coli bacterial cell culture). A bacterial cell culture may include gram-positive bacterial cells and/or gram-negative bacterial cells. In some embodiments, the sample is a purified sample of nucleic acids or proteins previously extracted from a metagenome sample or environmental sample through a user-developed method. The blood sample may be a freshly drawn blood sample or a dried blood sample (eg, preserved on a solid medium (eg, a Guthrie card)) from a subject (eg, a human subject). can A blood sample may include whole blood, serum, plasma, red blood cells, and/or white blood cells.

In some embodiments, a sample (e.g., a sample comprising cells or tissues) is to be prepared, e.g., lysed (e.g., destroyed, degraded, and/or otherwise) in a process according to the present disclosure. can be digested). In some embodiments, the prepared, e.g., lysed, sample is a cultured cell, biopsy (e.g., a tissue sample from a cancer patient, eg, a tumor biopsy from a human cancer patient), or any other clinical sample. In some embodiments, a sample comprising cells or tissues is lysed using any one of the known physical or chemical methodologies that release a target molecule (eg, a target nucleic acid or target protein) from the cell or tissue. In some embodiments, samples may be lysed using electrolytic methods, enzymatic methods, detergent-based methods, and/or mechanical homogenization. In some embodiments, a sample (eg, complex tissue, gram positive or gram-negative bacteria) may require multiple lysis methods performed in series. In some embodiments, if the sample does not contain cells or tissues (eg, a sample containing purified nucleic acids), the lysis step can be omitted. In some embodiments, lysis of the sample is performed to isolate the target nucleic acid(s). In some embodiments, lysis of the sample is performed to isolate the target protein(s). In some embodiments, the lysis method includes use of a grinder to grind the sample, sonication, surface acoustic wave (SAW), freeze-thaw cycles, heating, addition of detergents, protein denaturing agents (e.g., enzymes such as hydrolases or protease), and/or the addition of a cell wall digestive enzyme (eg, lysozyme or zymolase). Exemplary detergents for dissolution (e.g., non-ionic detergents) include polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, polysorbates, and alkylphenols. oxylates, preferably nonylphenol ethoxylates, alkylglucosides and/or polyoxyethylene alkyl phenyl ethers. In some embodiments, the dissolution method involves placing the sample 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) for at least 1 to 30 minutes, 1 to 25 minutes , heating for 5 to 25 minutes, 5 to 20 minutes, 10 to 30 minutes, 5 to 10 minutes, 10 to 20 minutes, or at least 5 minutes.

In some embodiments, a sample is prepared, eg, lysed, in the presence of a buffer system. This buffer system can be used to suspend the sample during any known dissolution methodology, including those described herein, and/or to prepare a slurry of the sample to stabilize the sample. In some embodiments, the sample is prepared in the presence of RIPA buffer, GCI buffer including guanidine-HCl buffer, Gly-NP40 buffer, TRIS buffer, HEPES buffer, or any other known buffer solution, e.g., lysis do.

Many of the lysis methods described herein allow a sample to be lysed by mechanically homogenizing the sample such that the sample's cell walls are disrupted. For example, methods for inducing lysis by mechanical homogenization include bead-stripping, heating (e.g., a temperature high enough to break the cell wall, e.g., 50°C, 60°C, 70°C, 80°C, 90°C). , or above 95° C.), syringe/needle/microchannel passage (to induce shear), sonication, or maceration with a grinder. In some embodiments, any dissolution methodology can be combined with any other dissolution methodology. For example, any dissolution methodology can be combined with heating and/or sonication and/or syringe/needle/microchannel pathways to speed up dissolution.

In some embodiments, sample preparation includes cell disruption (ie, lysis followed by subsequent removal of unwanted cells and tissue elements). In some embodiments, cell destruction involves protein and/or nucleic acid precipitation. In some embodiments, after settling, the lysed and disrupted sample is subjected to centrifugation. In some embodiments, after centrifugation, the supernatant is discarded. Precipitation is described by Winter, D. and H. Steen (2011). "Optimization of cell lysis and protein digestion protocols for the analysis of HeLa S3 cells by LC-MS/MS." PROTEOMICS 11(24): 4726-4730. In some embodiments, a protein or peptide is immunoprecipitated. In some embodiments, centrifugation of precipitated proteins and/or nucleic acids is followed by discarding of the supernatant and subsequent washing of the pellet fraction (eg, washing with chloroform/methanol or trichloroacetic acid).

In some embodiments, the sample is subjected to lysis in the presence of a lysis buffer (e.g., GCI buffer (6M Guanidine HCl, 0.1 M TEAB, 1% Triton X-100, standard buffer, and 1 mM EDTA/EGTA)). prepared using, by needle shear (e.g., by passing the sample through a 26.5 gauge needle, e.g. at 4°C) is destroyed. In some embodiments, the lysed and disrupted sample is further processed to precipitate proteins and/or nucleic acids (e.g., while vortexing at 4° C. with trichloroacetic acid), optionally followed by centrifugation. In some embodiments, samples are prepared as described in FIG. 3 .

In some embodiments, a sample (eg, a sample comprising a target nucleic acid or target protein) may be purified, eg, after lysis, in a process according to the present disclosure. In some embodiments, a sample may be purified using chromatography (eg, affinity chromatography that selectively binds to the sample) or electrophoresis. In some embodiments, a sample may be purified in the presence of a precipitating agent. In some embodiments, after a purification step or method, the sample may be washed and/or released from the purification matrix (eg, affinity chromatography matrix) using an elution buffer. In some embodiments, a purification step or method may include the use of a reversibly switchable polymer, such as an electroactive polymer. In some embodiments, a sample may be purified by electrophoretic passage of the sample through a porous matrix (eg, cellulose acetate, agarose, acrylamide).

In some embodiments, a sample (eg, a sample comprising a target nucleic acid or target protein) may be fragmented (ie, digested) in a process according to the present disclosure. In some embodiments, a nucleic acid sample may be fragmented to generate small (<1 kilobase) fragments for sequence specific identification to large (up to 10+ kilobase) fragments for long read sequencing applications. Fragmentation of a nucleic acid or protein is, in some embodiments, mechanical (e.g., fluid shear), chemical (eg, iron (Fe+) cleavage) and/or enzymatic (eg, tagging using restriction enzymes, transposase) methods. In some embodiments, a protein sample can be fragmented to generate peptide fragments of any length. Fragmentation of a protein may be achieved, in some embodiments, using chemical and/or enzymatic (eg, proteolytic enzymes such as trypsin) methods. In some embodiments, average fragment length can be controlled by reaction time, temperature, and concentration of sample and/or enzyme (eg, restriction enzyme, transposase). In some embodiments, a nucleic acid can be fragmented by tagging such that the nucleic acid is simultaneously fragmented and labeled with a fluorescent molecule (eg, a fluorophore). In some embodiments, the fragmented sample is purified (e.g., transposase) to remove small and/or undesired fragments as well as residual payloads, chemicals, and/or enzymes (e.g., transposases) used during the fragmentation step. eg, rounds of chromatography or electrophoresis). For example, a fragmented sample (e.g., a sample comprising nucleic acids) can be purified from an enzyme (e.g., a transposase), wherein purification involves denaturing the enzyme (e.g., heat, by a combination of chemical (eg SDS), and enzymatic (eg proteinase K) processes).

In some embodiments, the target molecule(s) are fragmented/digested prior to enrichment. In some embodiments, the target molecule is fragmented/digested after enrichment. In some embodiments, the target molecule(s) are fragmented/digested without any enrichment of the target molecule(s).

Fragmentation/digestion can be performed using any known method, but will typically involve non-enzymatic or enzymatic methods. Non-enzymatic methods typically have advantages as they are related to speed, simplicity, robustness, and ease of automation. These approaches include chemical entities such as cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide-hydrochloric acid, BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], or 2 - acid hydrolysis and/or cleavage with nitro-5-thiocyanobenzoic acid, but is not limited thereto. Non-enzymatic, electro-physical digestion methods including electrochemical oxidation and/or digestion with microwaves have also been employed. Enzymatic methods typically use proteases to fragment proteins into component peptides. These enzymes include trypsin (typically favored for the size of the resulting peptide and for the production of basic residues at the carboxyl terminus of the peptide), chymotrypsin, LysC, LysN, AspN, GluC and/or ArgC.

Enzymatic fragmentation/digestion methods can be optimized for ease of use, speed, automation, and/or effectiveness. In some embodiments, an enzymatic method involves immobilization of an enzyme on a solid substrate. In some embodiments, an enzymatic method is performed in a flow (eg, in a microfluidic channel).

The fragmentation/digestion method can be performed using an automated device or module. Alternatively, or in addition, the fragmentation/digestion method can be performed manually. Enzymatic digestion may utilize any number or combination of enzymes and may further include any known non-enzymatic method.

In some embodiments, the fragmentation/digestion process is as described in FIG. 5 . In some embodiments, a sample comprising the target protein(s) is first denatured and then reduced (e.g., using acetonitrile and TCEP). In some embodiments, the target protein(s) to be fragmented are subjected to capping of amino acid side chains (eg, cysteine blocks) (eg, using an amino acid side chain capping agent). In some embodiments, the target protein(s) are fragmented using a mixture of trypsin and LysC (e.g., for 120 minutes). Enzymatic reactions can be quenched (e.g. using sodium carbonate buffer).

Any suitable reducing agent may be used to reduce the target protein in the sample. In some embodiments, the reducing agent is suitable for reducing disulfide-bonds. In some embodiments, the reducing agent can reversibly reduce disulfide bonds. Suitable reversible reducing agents may include compounds such as dithiothreitol (DTT), β-mercaptoethanol (BME), and/or glutathione (GSH). In some embodiments, a reducing agent can irreversibly reduce disulfide bonds. Suitable irreversible reducing agents may include compounds such as tris(2-carboxyethyl)phosphine (TCEP). In some specific embodiments, the reducing agent includes tris(2-carboxyethyl)phosphine (TCEP).

Any suitable amino acid side chain capping agent can be used to cap amino acid side chains of proteins in a peptide sample. In some embodiments, amino acid side chain capping agents prevent the formation of disulfide bonds. In some embodiments, amino acid side chain capping agents prevent amino acid side chains from undergoing additional reactivity, such as nucleophilic/electrophilic or redox reactivity. In some embodiments, the amino acid side chain capping agent is a cysteine capping agent. In some embodiments, the amino acid side chain capping agent is a sulfhydryl-reactive alkylating reagent (eg a cysteine alkylating agent). For example, in some embodiments, the amino acid side chain capping agent is a haloacetamide (e.g. chloroacetamide, iodoacetamide) or a haloacetate/haloacetic acid (e.g. chloroacetate/chloroacetic acid, iodoacetate /iodoacetic acid). In some embodiments, the amino acid side chain capping agent is an aromatic benzyl halide. Other examples of suitable cysteine alkylating agents include 4-vinylpyridine, acrylamide, and methanethiosulfonate. In some embodiments, the amino acid side chain capping agent comprises iodoacetamide.

In some embodiments, a sample comprising target nucleic acids can be used to generate a nucleic acid library for subsequent analysis (eg, genome sequencing) in a process according to the present disclosure. A nucleic acid library can be a linear library or a circular library. In some embodiments, the nucleic acids of the circular library may include elements that allow for downstream linearization (eg, endonuclease restriction sites, incorporation of uracil). In some embodiments, a nucleic acid library can be purified (eg, by chromatography, eg, affinity chromatography), or using electrophoresis.

In some embodiments, a library of nucleic acids (e.g., linear nucleic acids) comprises an enzyme (e.g., Taq DNA ligase, endonuclease IV, Bst DNA polymerase, Fpg, uracil-DNA glycosylase, T4 endo A process in which a combination of nuclease V and/or endonuclease VIII) extends the 3' end of a nucleic acid, creating a complement to the 5' payload, and repairing any free bases or nicks in the nucleic acid Manufactured using phosphorus end-repair. In some embodiments, libraries of linear nucleic acids are prepared using self-priming hairpin adapters, a process that can eliminate the need to anneal unique sequencing primers to individual nucleic acid fragment primers prior to formation of polymerase complexes. After end-repair, the library of nucleic acids (eg, linear nucleic acids) is solid-phase with subsequent elution into fresh buffer, using passage of the nucleic acids through a size-selective matrix (eg, agarose gel). It can be purified using adsorption. A size-selective matrix can be used to remove nucleic acid fragments that are smaller than the size of the target nucleic acid.

In some embodiments, a sample (eg, a sample comprising a target nucleic acid or target protein) may be enriched for a target molecule in a process according to the present disclosure. Enrichment typically occurs when the complexity of the non-enriched sample exceeds the capacity of the sequencing platform, or when the target molecule is present in low abundance in the sample (e.g., such that it cannot be readily detected by the sequencing platform). used Enrichment involves the use of mechanisms that selectively amplify target molecules. This enrichment is accomplished using antibodies, aptamers, size-based selection, or electrostatic charge-based selection to selectively amplify the target molecule(s) (e.g., target protein(s) or target nucleic acid(s)). may entail

Enrichment can typically be used when the intent of sample preparation is to sequence a specific target molecule. Enrichment can be used to perform or conduct proteome, genomic, or metagenome analysis or investigations where the target molecules are related or homologous to each other.

In some embodiments, a sample is enriched for a target molecule using an electrophoretic method. In some embodiments, a sample is enriched for a target molecule using affinity SCODA. In some embodiments, samples are enriched for target molecules using field inversion gel electrophoresis (FIGE). In some embodiments, samples are enriched for target molecules using pulsed intestinal gel electrophoresis (PFGE). In some embodiments, the matrix (e.g., porous medium, electrophoretic polymer gel) used during enrichment is an immobilized affinity agent (also known as 'immobilized capture probe') that binds to a target molecule present in the sample. includes In some embodiments, a matrix used during enrichment comprises 1, 2, 3, 4, 5, or more unique immobilized capture probes, each of which binds a unique target molecule and/or different Bind to the same target molecule with binding affinity.

In some embodiments, an immobilized capture probe is an oligonucleotide capture probe that hybridizes to a target nucleic acid. In some embodiments, an oligonucleotide capture probe is at least 50%, 60%, 70%, 80%, 90% 95%, or 100% complementary to a target nucleic acid. In some embodiments, a single oligonucleotide capture probe comprises a plurality of related target nucleic acids (e.g., 2, 3 , 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more related target nucleic acids). Enrichment of multiple related target nucleic acids can allow for the creation of metagenome libraries. In some embodiments, oligonucleotide capture probes may enable differential enrichment of related target nucleic acids. In some embodiments, an oligonucleotide capture probe may enable enrichment of a target nucleic acid relative to nucleic acids of the same sequence that differ in their modification state (eg, single nucleotide polymorphism, methylation state, acetylation state). In some embodiments, oligonucleotide capture probes are used to enrich human genomic DNA for a specific gene of interest (eg, HLA). A specific gene of interest may be a gene associated with a specific disease state or disorder. In some embodiments, oligonucleotide capture probes are used to enrich for nucleic acid(s) of a metagenomic sample.

In some embodiments, for the purpose of enriching for nucleic acid target molecules having a length of 0.5 to 2 kilobases, oligonucleotide capture probes may be covalently immobilized to an acrylamide matrix using a 5' acrydite moiety. In some embodiments, for the purpose of enriching for larger nucleic acid target molecules (eg, having a length of >2 kilobases), oligonucleotide capture probes may be immobilized to an agarose matrix. In some embodiments, oligonucleotide capture probes can be immobilized to an agarose matrix using thiol-epoxide chemistry (eg, by covalently attached thiol-modified oligonucleotides to cross-linked agarose beads). . Oligonucleotide capture probes linked to agarose beads can be combined and solidified in a standard agarose matrix (eg, with the same percentage of agarose).

In some embodiments, enrichment of a nucleic acid using a method described herein (eg, enrichment using SCODA) is about 0.5 kilobases (kb), about 1 kb, about 1.5 kb, about 2 kb, about 3 kb, about A nucleic acid target molecule comprising a length of about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 12 kb, about 15 kb, about 20 kb, or more produces In some embodiments, enrichment of a nucleic acid using a method described herein (eg, enrichment using SCODA) is about 0.5 to 2 kb, 0.5 to 5 kb, 1 to 2 kb, 1 to 3 kb, 1 to 4 kb , 1 to 5 kb, 1 to 10 kb, 2 to 10 kb, 2 to 5 kb, 5 to 10 kb, 5 to 15 kb, 5 to 20 kb, 5 to 25 kb, 10 to 15 kb, 10 to 20 kb , or nucleic acid targeting molecules comprising a length of 10 to 25 kb.

In some embodiments, an immobilized capture probe is a protein capture probe (eg, an aptamer or antibody) that binds a target protein or peptide fragment. In some embodiments, the protein capture probe is 10 ⁻⁹ to 10 ⁻⁸ M, 10 ⁻⁸ to 10 ⁻⁷ M, 10 ⁻⁷ to 10 ⁻⁶ M, 10 ⁻⁶ to 10 ⁻⁵ M, 10 ⁻⁵ to 10 It binds to a target protein or peptide fragment with a binding affinity of ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. In some embodiments, the binding affinity is in the picomolar to nanomolar range (eg, about 10 ⁻¹² to about 10 ⁻⁹ M). In some embodiments, the binding affinity is in the nanomolar to micromolar range (eg, about 10 ⁻⁹ to about 10 ⁻⁶ M). In some embodiments, the binding affinity is in the micromolar to millimolar range (eg, about 10 ⁻⁶ to about 10 ⁻³ M). In some embodiments, the binding affinity ranges from picomolar to micromolar (eg, from about 10 ⁻¹² to about 10 ⁻⁶ M). In some embodiments, the binding affinity is in the nanomolar to millimolar range (eg, about 10 ⁻⁹ to about 10 ⁻³ M). In some embodiments, a single protein capture probe can be used to enrich for a plurality of related target proteins that share at least 50%, 60%, 70%, 80%, 90% 95%, or 99% sequence identity. In some embodiments, a single protein capture probe is a plurality of related target proteins (e.g., 2, 3 , 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more related target proteins). Enrichment of a plurality of related target proteins can allow the creation of metaproteomics libraries. In some embodiments, protein capture probes may allow for differential enrichment of related target proteins.

In some embodiments, multiple capture probes (e.g., multiple capture probe types that bind critical target molecules of, e.g., infectious agents such as adenovirus, Staphylococcus, pneumonia, or tuberculosis) population) can be immobilized on the enrichment matrix. Application of a sample to an enrichment matrix with multiple critical capture probes can result in a diagnosis of a disease or condition (eg, the presence of an infectious agent). In some embodiments, the target molecule or related target molecule may be released from the enrichment matrix after removal of the non-target molecule in a process according to the present disclosure. In some embodiments, the target molecule can be released from the enrichment matrix by increasing the temperature of the enrichment matrix. Adjusting the temperature of the matrix further affects the rate of migration, since increased temperature provides higher capture probe stringency requiring greater binding affinity between the target molecule and the capture probe. In some embodiments, when enriching for a target molecule of interest, the matrix temperature can be gradually increased in a step-wise fashion to release and isolate the target molecule at an ever-increasing stage of homology. In some embodiments, the temperature is increased by about 5%, 10%, 15%, 20%, increased by 25%, 30%, 40%, 50%, or more. In some embodiments, the temperature is 5% to 10%, 5 to 15%, 5% at each step or over a period of time (eg, 1 to 10 minutes, 1 to 5 minutes, or 4 to 8 minutes). to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 5% to 50%, 10% to 25%, 20% to 30%, 30% to 40%, 35% to 50% %, or from 40% to 70%. In some embodiments, the temperature is about 1 °C, 2 °C, 3 °C, 4 °C, increased by 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C. In some embodiments, the temperature is 1 to 10 °C, 1 to 5 °C, 2 to 5 °C at each step or over a period of time (eg, 1 to 10 minutes, 1 to 5 minutes, or 4 to 8 minutes). °C, 2 to 10 °C, 3 to 8 °C, 4 to 9 °C, or 5 to 10 °C. This will allow for the sequencing of target proteins or target nucleic acids increasingly distant with respect to an initial reference target molecule, allowing the discovery of novel proteins (e.g., enzymes) or functions (e.g., enzymatic or gene functions). can In some embodiments, when using multiple capture probes (e.g., multiple deterministic capture probes), the matrix temperature is increased in a stepwise or gradient fashion to allow temperature-dependent release of different target molecules, control and It can result in the creation of a series of barcoded emission bands indicative of the presence or absence of the target molecule.

Enrichment of a sample (eg, a sample comprising a target nucleic acid or target protein) allows for a reduction in the total volume of the sample. For example, in some embodiments, the total volume of the sample after enrichment is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% %, at least 100%, or at least 120%. In some embodiments, the total volume of the sample is from 1 to 20 mL initial volume to 100 to 1000 μL final volume, from 1 to 5 mL initial volume to 100 to 1000 μL final volume, or from 100 to 1000 μL initial volume to 25 μL final volume after enrichment. to 100 μL final volume, from 100 to 500 μL initial volume to 10 to 100 μL final volume, or from 50 to 200 μL initial volume to 1 to 25 μL final volume. For example, in some embodiments, the final volume of the sample after enrichment is between 10 and 100 μL, 10 and 50 μL, 10 and 25 μL, 20 and 100 μL, 20 and 50 μL, 25 and 100 μL, 25 and 250 μL. , 25 to 1000 μL, 100 to 1000 μL, 100 to 500 μL, 100 to 250 μL, 200 to 1000 μL, 200 to 500 μL, 200 to 750 μL, 500 to 1000 μL, 500 to 1500 μL, 500 to 750 μL , 1 to 5 mL, 1 to 10 mL, 1 to 2 mL, 1 to 3 mL, or 1 to 4 mL.

In addition to, or as an alternative to amplification of the target molecule, the sample may contain unwanted non-target molecules (e.g., can be enriched by depletion of high-abundance proteins (e.g. albumin)) (e.g., for low-abundance target molecules). Depletion of unwanted non-target molecules can be accomplished using similar capture strategies as discussed above. When using a depletion strategy, the capture probe will bind unwanted, non-target molecules and allow the target molecules to remain in solution. This strategy equally enables enrichment of the target molecule (ie, increased relative concentration of the target molecule(s)).

For example, the immobilized capture probe used for depletion may be an oligonucleotide capture probe that hybridizes to an unwanted, non-target nucleic acid. In some embodiments, the oligonucleotide capture probe used for depletion is at least 50%, 60%, 70%, 80%, 90% 95%, or 100% complementary to an unwanted, non-target nucleic acid. In some embodiments, a single oligonucleotide capture probe used for depletion comprises a plurality of related target nucleic acids (eg, that share at least 50%, 60%, 70%, 80%, 90% 95%, or 99% sequence identity) , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more relevant target nucleic acids).

In some embodiments, the immobilized capture probe used for depletion is a protein capture probe (eg, an aptamer or antibody) that binds to an unwanted, non-target protein or peptide fragment. In some embodiments, the protein capture probe used for depletion is 10 ⁻⁹ to 10 ⁻⁸ M, 10 ⁻⁸ to 10 ⁻⁷ M, 10 ⁻⁷ to 10 ⁻⁶ M, 10 ⁻⁶ to 10 ⁻⁵ M, 10 Binds to unwanted non-target protein or peptide fragments with a binding affinity of ⁻⁵ to 10 ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. In some embodiments, the binding affinity is in the nanomolar to millimolar range (eg, about 10 ⁻⁹ to about 10 ⁻³ M). In some embodiments, a single protein capture probe used for depletion can be used to deplete a plurality of related target proteins that share at least 50%, 60%, 70%, 80%, 90% 95%, or 99% sequence identity. have. In some embodiments, a single protein capture probe used for depletion has a plurality of related target proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more target proteins). In some embodiments, enrichment involves amplification and depletion of the target molecule(s) (e.g., of high-abundance proteins). In some embodiments, a depletion step is performed prior to amplification and enrichment of the target molecule(s). In some embodiments, to avoid possible contamination of the target molecule(s) by capture elements (e.g., antibodies or aptamers) of the enrichment process, capture elements are depleted (i.e., of the target molecule) from the enriched sample. after enrichment by amplification and/or depletion of unwanted non-target molecules from the original sample).

In some embodiments, the sample is first subjected to a depletion step (e.g., to remove unwanted non-target proteins). In some embodiments, the sample is enriched using amplification or immobilized target capture after the first depletion step (eg, using an antibody that selectively enriches for the target protein). After amplification or immobilized target capture, the sample can then be subjected to a second depletion step (e.g., to remove excess antibody or capture probe). In some embodiments, a sample is enriched, eg, as described in FIG. 4 .

In some embodiments, any number of enrichment steps (e.g., Amplification and/or depletion(s) of the target molecule(s) may be performed by an automated device or module (eg, on a chip or cartridge). In some embodiments, the enrichment step is amenable to automation on a cartridge using capture elements (eg, antibodies) immobilized on a solid phase structure. In some embodiments, any immobilized capture element or probe described herein can be on any solid support structure or surface. The solid support structure or surface may be magnetic and/or may be a frit, filter, chip, or cartridge surface. In some embodiments, capture elements or probes for enrichment may be interchanged (eg, using flow on a chip). In some embodiments, any number of enrichment steps are performed manually. If performed manually, any enriched target molecule can then be placed into an automated sample preparation device described herein.

In some embodiments, the target molecule or target molecules may be detected after enrichment and subsequent release in a process according to the present disclosure to allow analysis of the target molecule(s) and its upstream sample. In some embodiments, a target nucleic acid is determined by gene sequencing, absorbance, fluorescence, electrical conductivity, capacitance, surface plasmon resonance, hybrid capture, antibodies, direct labeling of the nucleic acid (e.g., end-labeling, labeled tagged payload). ), non-specific labeling with an intervening dye (eg, ethidium bromide, SYBR dye), or any other known methodology for nucleic acid detection. In some embodiments, target protein or peptide fragments can be detected using absorbance, fluorescence, mass spectrometry, amino acid sequencing, or any other known methodology for protein or peptide detection.

Sample preparation device and module

A device comprising an instrument, a cartridge (eg, comprising a channel (eg, a microfluidic channel)), and/or a pump (eg, a peristaltic pump) for use in a process for preparing a sample for analysis. or modules are generally provided. Devices that facilitate the capture, enrichment, manipulation, and/or detection of target molecules from a biological sample can be used in accordance with the present disclosure. In some embodiments, apparatus and related methods are provided for automated processing of samples to produce material for next-generation sequencing and/or other downstream analytical techniques. Devices and related methods can be used to perform chemical and/or biological reactions, including reactions for processing nucleic acids and/or proteins according to sample preparation or sample analysis processes described elsewhere herein.

A sample preparation device or module, in some embodiments, can perform any number of the following sample preparation steps:

(1) cell or tissue preparation (eg, lysis); and/or

(2) enrichment of at least one target molecule (eg, at least one target nucleic acid and/or at least one target protein); and/or

(3) at least one target molecule (e.g., digestion or fragmentation of at least one target nucleic acid and/or at least one target protein); and/or

(4) terminal functionalization of at least one target molecule (e.g., C-terminal functionalization of target protein).

In some embodiments, a sample preparation device or module performs a sample preparation step as shown in FIG. 1 . In some embodiments, a sample preparation device or module performs a sample preparation step as shown in FIG. 2 .

In some embodiments, a sample preparation device or module performs all of steps (1) through (4). In some embodiments, a sample preparation device or module performs step (1), and optionally steps (2)-(4). In some embodiments, a sample preparation device or module performs step (1), and optionally steps (2)-(3). In some embodiments, a sample preparation device or module performs step (1) and optionally performs step (2). In some embodiments, a sample preparation device or module performs step (1), and optionally steps (3)-(4). In some embodiments, the sample preparation device or module performs step (1) and optionally performs step (3). In some embodiments, the sample preparation device or module performs step (1) and optionally performs step (4). In some embodiments, the sample preparation device or module does not perform step (1), but merely performs steps (2) through (4). In some embodiments, the sample preparation device or module does not perform step (1), but merely performs steps (3)-(4). In some embodiments, the sample preparation device or module does not perform step (1), but only steps (2) and (4). In some embodiments, the sample preparation device or module does not perform step (1), but only performs one of steps (2), (3), or (4). The order of steps can be changed as needed for the experiment. For example, step (3)—digestion or fragmentation—can precede step (2)—enrichment. In some embodiments, the at least one target molecule can be purified after step (1), and/or step (2), and/or step (3), and/or step 4. In some embodiments, any one of the steps is interspersed with a manual step. This flexibility enables users to handle multiple sample types and sequencing platforms. In some embodiments, a sample preparation device or module is positioned to deliver or transfer a target molecule or plurality of target molecules (eg, a target nucleic acid or target protein) to a sequencing module or device. In some embodiments, a sample preparation device or module is connected directly (eg, physically attached) or indirectly to a sequencing device or module.

In some embodiments, a sample preparation device or module is used to prepare a sample for diagnostic purposes. In some embodiments, a sample preparation device used to prepare a sample for diagnostic purposes delivers or is positioned to deliver a target molecule or plurality of molecules (eg, a target nucleic acid or target protein) to a diagnostic module or diagnostic device. In some embodiments, a sample preparation device or module is directly connected to a diagnostic device (e.g., physically attached) or indirectly connected. In some embodiments, a device includes a cartridge housing configured to receive one or more cartridges (eg, configured to receive one cartridge at a time). 24A shows a schematic diagram of a sample preparation device 300 in accordance with some embodiments. A device (eg, a sample preparation device comprising a cartridge housing) may be configured to receive one or more cartridges (or two or more, or three or more, etc.) sequentially or simultaneously. The sample preparation device 300 is, for example, configured to simultaneously or sequentially receive one or more of the lysis cartridge 301, the enrichment cartridge 302, the fragmentation cartridge 303, and/or the functionalization cartridge 304. It can be. It should be understood that the device need not be configured to receive each of the four cartridges shown in FIG. 4A in all embodiments. For example, in some embodiments sample preparation device 300 is configured to only accept lysis cartridge 301 and enrichment cartridge 302, and fragmentation and functionalization are performed manually rather than in an automated manner.

The sample preparation device may further include a pump configured to transport components (eg, reagents, samples) from (eg, into or into and/or from) a cartridge contained therein. For example, referring to FIG. 24B , the sample preparation device 300 may include components in one or more of the dissolution cartridge 301, the enrichment cartridge 302, the fragmentation cartridge 303, and/or the functionalization cartridge 304. may include a pump 305 configured to transport the In some embodiments, a pump includes an instrument and a contained cartridge, and interaction between the instrument and cartridge of the pump causes fluid flow. For example, pump 305 can be a peristaltic pump, and mechanism 306 can be operatively coupled to a cartridge (e.g., cartridge 301) to cause fluid motion in the cartridge (e.g., cartridge 301). , where the mechanism 306 includes a roller and the cartridge 301 includes a compliant surface deformable by the roller). Additional descriptions of exemplary peristaltic pump methods and devices are described in more detail below.

As noted elsewhere, the prepared sample from the sample preparation device may be transported (directly or indirectly) to a downstream detection module (eg, sequencing module, diagnostic module). For example, FIG. 24C shows an embodiment in which conduit 308 connects sample preparation device 300 and detection module 307 (eg, a sequencing module). Sample preparation device 300 and detection module 307 may be directly coupled (eg, physically attached) or indirectly coupled (eg, via one or more intervening modules).

While in some embodiments the various steps of the process are performed in separate cartridges (e.g., lysis step in a lysis cartridge, enrichment step in an enrichment cartridge, fragmentation step in a fragmentation cartridge, functionalization step in a functionalization cartridge), in other embodiments Two or more (or all) of these steps may be performed on a single cartridge. For example, a cartridge may include different regions for different steps of the overall process (each region including various reservoirs, channels, and/or microchannels for performing each step). 24D shows a schematic illustration of such an embodiment insofar as the cartridge 401 includes a dissolution region 402 , an enrichment region 403 , a fragmentation region 404 , and a functionalization region 405 . Cartridge 401 shows areas for four such steps, but the illustration is purely illustrative, and more or fewer areas for more or fewer steps may exist in a given cartridge (e.g., It should be understood that a cartridge may contain only a lysis zone and an enrichment zone, or various other combinations. Sample preparation device 400 may be configured to receive a cartridge 401 as shown in FIG. 24D , according to certain embodiments. As in the embodiment described in FIGS. 24B-24C , sample preparation device 400 is operatively coupled to cartridge 407 (eg, to transport a component such as a fluid), as shown in FIG. 24E . and a pump 406 comprising a ringing mechanism 407. Additionally, as shown in FIG. 24F , conduit 408 can connect sample preparation device 400 to downstream detection module 409 (eg, sequencing module, diagnostic module), according to certain embodiments. . This connection may allow transport of the prepared sample from the sample preparation device 400 to the detection module 409, either directly or indirectly, according to certain embodiments.

In some embodiments, a cartridge includes one or more reservoirs or reaction vessels configured to contain a fluid and/or contain one or more reagents used in a sample preparation process. In some embodiments, the cartridge is a fluid used in a sample preparation process (e.g., one or more channels (eg, microfluidic channels) configured to contain and/or transport a fluid comprising one or more reagents). Reagents include buffers, enzymatic reagents, polymer matrices, capture reagents, size-specific selection reagents, sequence-specific selection reagents, and/or purification reagents. Additional reagents for use in the sample preparation process are described elsewhere herein.

In some embodiments, the cartridge contains one or more stored reagents (eg, in liquid or lyophilized form suitable for reconstitution into liquid form). The stored reagents of the cartridge include reagents suitable for carrying out the desired process and/or suitable for processing the desired sample type. In some embodiments, the cartridge is a single-use cartridge (eg, a disposable cartridge) or a multi-use cartridge (eg, a reusable cartridge). In some embodiments, a cartridge is configured to receive a user-supplied sample. The user-supplied sample may be added to the cartridge before or after the cartridge is received by the device, eg, manually by a user or in an automated process. In some embodiments, the cartridge is a sample preparation cartridge. In some embodiments, a sample preparation cartridge is capable of isolating or purifying a target molecule (eg, a target nucleic acid or target protein) from a sample (eg, a biological sample).

9A shows a top view schematic diagram of one embodiment of a cartridge 200, according to certain embodiments. Cartridge 200 is configured to perform one or more of the various processes described in this disclosure, such as lysis, enrichment, depletion, fragmentation, and/or terminal functionalization of a target molecule from a fluid sample (eg, a biological sample). It can be. The configuration of a cartridge for any of these processes may be determined, for example, by the presence of reagents selected for the process in the cartridge (eg, in a reservoir, reaction vessel, or channel of the cartridge). For example, the cartridge 200 in FIG. 9A includes a first reagent reservoir 201 that contains or may contain reagents for the first step of the process (eg, purification/size selection reagents), the first step of the process A second reagent reservoir 202 that contains or may contain reagents for step 2 (eg, target molecule extraction reagents), and reagents for step 3 of the process (eg, library preparation reagents). A third reagent reservoir 203, which may or may not be included. Some such reagents may be stored in a reservoir or channel of a cartridge (eg, a packaged consumable cartridge), or the reagent may be introduced into a reservoir or channel of a cartridge before or during any of the processes described. A sample (eg, biological sample) may be introduced into the sample, for example, through a sample inlet or port. For example, FIG. 8 shows a sample inlet 206 through which a biological sample may be introduced into a network of channels 205 (eg, in the form of microchannels) of the cartridge 200 . Reagents from any reservoir (e.g., first reagent reservoir 201, etc.) are passed through channels 205 to perform the desired step of the process (e.g., lysis, enrichment, fragmentation, functionalization). It can be made to flow to the desired area of the cartridge 200. For example, reagents for purification/size selection can be flowed from the first reagent reservoir 201 to the fourth reservoir 204, and the sample can be flowed from the sample inlet 206 to the fourth reservoir 204. Upon interaction (eg, via mixing), a purification process of the sample may proceed (eg, via purification/size selection) in the fourth reservoir 204 . Samples and reagents can be made to flow in the cartridge (eg, through channels) through any of a variety of techniques. One such technique is to induce flow through peristaltic pumping. Additional descriptions of exemplary peristaltic pumping are described below. Other areas of the cartridge, such as, for example, fifth reservoir 205, which according to some embodiments may be configured to perform library retrieval, may be configured for other stages of the process. 9B shows an image of an example cartridge that can be configured to perform one or more processes described herein. It should be understood that cartridge configurations other than those shown in FIG. 9B are possible, and FIG. 9B is shown for illustrative purposes.

In some embodiments, a cartridge comprises an affinity matrix for enrichment as described herein. In some embodiments, the cartridge comprises an affinity matrix for enrichment using affinity SCODA, FIGE, or PFGE. In some embodiments, the cartridge comprises an affinity matrix comprising an immobilized affinity agent having binding affinity for a target nucleic acid or target protein.

In some embodiments, a sample preparation device of the present disclosure is generated using a control method (eg, the Sage BluePippin method, a manual method (eg, a manual bead-based size selection method)) Produce target nucleic acids with an average read-length for downstream sequencing applications that are longer than the average read-length (eg, enrichment or purification). In some embodiments, the sample preparation device is configured to have a voltage of at least 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2400, 2500, 2600; Produce target nucleic acids with average read-lengths for sequencing that include lengths of 2700, 2800, 2900, or 3000 nucleotides. In some embodiments, the sample preparation device has a range of 700 to 3000, 1000 to 3000, 1000 to 2500, 1000 to 2400, 1000 to 2300, 1000 to 2200, 1000 to 2100, 1000 to 2000, 1000 to 1900, 1000 to 1800 to 1700, 1000 to 1600, 1000 to 1500, 1000 to 1400, 1000 to 1300, 1000 to 1200, 1500 to 3000, 1500 to 2500, 1500 to 2000, or 2000 to 3000 nucleotides in length. A target nucleic acid having read-length is produced.

Devices according to the present disclosure generally contain mechanical and electronic and/or optical components that can be used to operate a cartridge as described herein. In some embodiments, device components operate to achieve and maintain specific temperatures for a cartridge or for specific regions of a cartridge. In some embodiments, a device component operates to apply a specific voltage to an electrode of a cartridge for a specific duration of time. In some embodiments, device components operate to transfer liquid to, from, or between the cartridge's reservoir and/or reaction vessel. In some embodiments, device components operate to move liquid through the channel(s) of the cartridge, eg, into, from, or between a reservoir and/or reaction vessel of the cartridge. In some embodiments, a device component moves liquid through a peristaltic pumping mechanism (eg, instrument) that interacts with an elastomeric, reagent-specific reservoir or reaction vessel in a cartridge. In some embodiments, a device component is a peristaltic pumping mechanism (e.g., a device) configured to interact with an elastomeric component (e.g., a surface layer comprising an elastomer) associated with a channel of a cartridge to pump fluid through the channel. moves the liquid through Device components may include, for example, computer resources for running a user interface into which sample information may be entered, specific processes may be selected, and execution results may be reported.

In some embodiments, the cartridge handles small-volume fluid (e.g., 1 to 10 μL, 2 to 10 μL, 4 to 10 μL, 5 to 10 μL, 1 to 8 μL, or 1 to 6 μL of fluid) can do. In some embodiments, a sequencing cartridge is physically embedded or associated with a sample preparation device or module (eg, to allow a prepared sample to be transferred to a reaction mixture for sequencing). In some embodiments, a sequencing cartridge physically embedded or associated with a sample preparation device or module includes a microfluidic channel having a fluidic interface in the form of a face seal gasket or conical press fit (eg, a Luer fitting). do. In some embodiments, the fluid interface can then be broken after delivery of the prepared sample to physically separate the sequencing cartridge from the sample preparation device or module.

The following non-limiting examples are meant to illustrate aspects of the devices, methods, and compositions described herein. Use of a sample preparation device or module according to the present disclosure may proceed with one or more of the steps described below. The user can open the lid of the device and insert the cartridge supporting the desired process. The user can then add a sample that can be combined with a specific lysis solution to the sample port on the cartridge. The user then closes the device lid, enters any sample-specific information through the touch screen interface on the device, and sets any process-specific parameters (e.g., range of desired size selection, homology to capture target molecule). degree desired, etc.), and the execution of the sample preparation process can be initiated. After a run, the user can view relevant run data (e.g., confirmation of successful completion of the run, run-specific metrics, etc.), as well as process-specific information (e.g., amount of sample produced, specific target sequence presence or absence, etc.) can be received. The data generated by the run can be processed into subsequent bioinformatics analyzes that can be local or cloud-based. Depending on the process, the finished sample can be extracted from the cartridge for subsequent use (eg, genome sequencing, qPCR quantification, cloning, etc.). The device can then be opened and the cartridge can then be removed.

In some embodiments, the sample preparation 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 handling described herein. For example, a pump may include an instrument and/or a cartridge. In some embodiments, the mechanism of the pump includes rollers, cranks, and rockers. In some such embodiments, the crank and rocker are configured as a crank-and-rocker mechanism connected to rollers. Coupling of the crank-and-rocker mechanism with the rollers of the instrument may in some cases allow certain advantages described herein to be achieved (e.g., easy release of the instrument from the cartridge, well-metered stroke volume). . In certain embodiments, a cartridge of a pump includes a channel (eg, a microfluidic channel). In some embodiments, at least a portion of a channel of a cartridge has a specific cross-sectional shape and/or surface layer that can contribute to any of a number of the advantages described herein.

One non-limiting aspect of some cartridges that may provide particular benefit in some cases is the inclusion of channels in the cartridge with a particular cross-sectional shape. For example, in some embodiments, a cartridge includes v-shaped channels. One potentially convenient, but non-limiting way to form such a v-shaped channel is by molding or machining a v-shaped groove into the cartridge. A recognized advantage of including v-shaped channels (also referred to herein as v-grooves or channels having a substantially triangular-shaped cross-section) is that, in certain embodiments, the rollers of the instrument are engaged with the cartridge to allow fluid flow from the channels. is to cause For example, in some cases, the v-shaped channels are dimensionally insensitive to the rollers. In other words, in some cases, there is no single dimension that the rollers of the instrument (eg wedge-shaped rollers) must adhere to in order to properly engage the v-shaped channels. In contrast, certain conventional cross-sectional shapes of channels, such as semi-circular, require rollers to have specific dimensions (e.g., to create fluid seals that cause differential pressure in a peristaltic pumping process) to properly engage the channels ( eg radius). In some embodiments, the inclusion of dimensionally insensitive channels to the rollers may result in simpler and less expensive fabrication and increased configurability/flexibility of the hardware component.

In certain aspects, the cartridge includes a surface layer (eg, a flat surface layer). One exemplary aspect is by layering a film (also referred to herein as a surface layer) comprising (eg, consisting essentially of) an elastomer (eg, silicone) over the v-groove, so that, in effect, a flexible It relates to potentially advantageous embodiments that involve producing half of the tubes. 24 depicts an exemplary cartridge 100 according to certain such embodiments, described in more detail below. Then, in some embodiments, by deforming the surface layer comprising the elastomer into a channel to form a pinch, and then converting the pinch, negative pressure can be created on the trailing edge of the pinch that creates suction, and positive pressure Pressure can be created on the leading edge of the pinch to pump fluid in the direction of the leading edge of the pinch. In certain embodiments, it pumps by interfacing a cartridge (comprising a channel having a surface layer) with a device that includes a roller, which engages the roller with a portion of the surface layer to cause the portion of the surface layer to be coupled to the associated channel's wall and and/or pinch with the base and/or translate the roller in a rolling motion along the wall and/or base of the associated channel to translate the pinch of the surface layer relative to the wall and/or base, and/or move the roller along the wall and/or base of the associated channel It is configured to perform a motion of the roller including disengaging with the part. In certain embodiments, a crank-and-rocker mechanism is incorporated into the mechanism that performs this motion of the rollers.

Conventional peristaltic pumps usually involve tubing inserted into an appliance that includes rollers on a rotating carriage so that the tubing is always engaged with the rest of the appliance when the pump is functioning. In contrast, in certain embodiments, the channels in the cartridge herein are linear or include at least one linear segment such that the roller engages a horizontal surface. In certain embodiments, the rollers are connected to small spring-loaded roller arms such that the rollers can track a horizontal surface while continuously pinching portions of the surface layer. Spring loading the mechanism (eg, the roller arm of the mechanism) can in some cases help regulate the force applied by the mechanism (eg, the roller) to the surface layer and channels of the cartridge.

In certain embodiments, each rotation of a crank in a crank-and-rocker mechanism connected to a roller provides a distinct pumping volume. In certain embodiments, it is simple to park the instrument in a disengaged position, where the roller is disengaged from any cartridge. In certain embodiments, the forward and backward pumping motions are fairly symmetrical, as provided by the instruments described herein, such that a similar amount of force (torque) (eg, within 10%) is required for the forward and backward pumping motions. .

In certain embodiments, for certain sizes of instruments, it may be advantageous to have relatively high crank radii (eg, 2 mm or greater, optionally including associated connections). Consequently, in certain embodiments, it may also be advantageous to have a relatively high stroke length (eg, 10 mm or more) that engages an associated cartridge. Having a relatively high crank radius and stroke length ensures, in certain embodiments, that there is no mechanical interference between the instrument and the cartridge when moving components of the instrument relative to the cartridge.

In certain embodiments, having a v-shaped groove advantageously allows for use with rollers of various sizes having wedge-shaped edges. In contrast, for example, having a rectangular channel rather than a v-groove requires that the width of the roller associated with the rectangular channel be more controlled and accurate with respect to the width of the rectangular channel, and the force applied to the rectangular channel will be more accurate. make it necessary Similarly, channel(s) with semi-circular cross-sections may also require more controlled and precise dimensions for the width of the associated rollers.

In certain embodiments, an instrument described herein may include a multi-axis system (eg, a robot) configured to move at least a portion of the instrument in multiple dimensions (eg, two dimensions, three dimensions). . For example, a multi-axis system can be configured to move at least a portion of an instrument to any pumping lane position among associated cartridge(s). For example, in certain embodiments, carriages of the present disclosure may be functionally connected to a multi-axis system. In certain embodiments, the rollers may be functionally coupled indirectly to a multi-axis system. In certain embodiments, an instrument portion comprising a crank-and-rocker mechanism connected to rollers may be functionally connected to a multi-axis system. In certain embodiments, each pumping lane is addressed by location and can be accessed by instruments described herein using a multi-axis system.

Nucleic Acid Sequencing Process

Some aspects of the disclosure further involve sequencing the nucleic acid (eg, deoxyribonucleic acid or ribonucleic acid). In some aspects, the compositions, devices, systems, and techniques described herein can be used to identify a series of nucleotides incorporated into a nucleic acid (eg, by detecting the time-course of incorporation of a series of labeled nucleotides). In some embodiments, the compositions, devices, systems, and techniques described herein are polymerase enzymes (e.g., It can be used to identify a series of nucleotides incorporated into template-dependent nucleic acid sequencing reaction products synthesized by RNA polymerase).

Accordingly, also provided herein are methods of determining the sequence of a target nucleic acid. In some embodiments, a target nucleic acid is enriched prior to determining the sequence of the target nucleic acid (e.g., enriched using an electrophoretic method, eg, affinity SCODA). In some embodiments, a sample (e.g., A plurality of target nucleic acids (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 50) present in a purified sample, cell lysate, single-cell, population of cells, or tissue , or more) are provided herein. In some embodiments, a sample is prepared as described herein (e.g., lysed, purified, fragmented, and/or , enriched for target nucleic acids). In some embodiments, a target nucleic acid is an enriched target nucleic acid (eg, enriched using an electrophoretic method, eg, affinity SCODA).

In some embodiments, a method of sequencing comprises (i) exposing a complex in a target volume to one or more labeled nucleotides, wherein the complex comprises a target nucleic acid or a plurality of nucleic acids present in a sample, at least one primer, and polymerization comprising an enzyme; (ii) directing one or more excitation energies, or a series of pulses of one or more excitation energies, towards the vicinity of the target volume; (iii) detecting a plurality of emitted photons from one or more labeled nucleotides during sequential incorporation into a nucleic acid comprising one of the at least one primers; and (iv) identifying the sequence of the incorporated nucleotide by determining one or more characteristics of the emitted photon.

In another aspect, the present disclosure provides a method of sequencing a target nucleic acid or a plurality of target nucleic acids present in a sample by sequencing a plurality of nucleic acid fragments, wherein the target nucleic acid(s) comprises fragments. . In certain embodiments, a method comprises combining a plurality of fragment sequences to provide a sequence or partial sequence for a parent nucleic acid (eg, a parent target nucleic acid). In some embodiments, the step of combining is performed by computer hardware and software. The methods described herein may allow a set of related nucleic acids (eg, two or more nucleic acids present in a sample), such as an entire chromosome or genome, to be sequenced. In some embodiments, a primer is a sequencing primer. In some embodiments, sequencing primers are nucleic acids that may or may not be immobilized to a solid support (e.g., target nucleic acid). A solid support can include, for example, sample wells (eg, nanopores, reaction chambers) on a chip or cartridge used for nucleic acid sequencing. In some embodiments, sequencing primers can be immobilized to a solid support and nucleic acids (e.g., Hybridization of the target nucleic acid) further immobilizes the nucleic acid molecule to the solid support. In some embodiments, a polymerase (e.g., RNA polymerase) is immobilized on a solid support, and soluble sequencing primers and nucleic acids are contacted to the polymerase. In some embodiments, a polymerase, a nucleic acid (e.g., A complex comprising a target nucleic acid) and a primer is formed in solution, and the complex is immobilized to a solid support (eg, via immobilization of a polymerase, a primer, and/or a target nucleic acid). In some embodiments, none of the components are immobilized to a solid support. For example, in some embodiments, a complex comprising a polymerase, a target nucleic acid, and a sequencing primer is formed in situ and the complex is not immobilized to a solid support. In some embodiments, a sequencing-by-synthesis method involves the presence of a population of target nucleic acid molecules (e.g., copies of a target nucleic acid) and/or amplification of a target nucleic acid to achieve a population of target nucleic acids (e.g., polymerase chain reaction (PCR)). However, in some embodiments, sequencing by synthesis is used to determine the sequence of a single nucleic acid molecule in any one reaction being evaluated, and nucleic acid amplification may not be required to prepare a target nucleic acid. In some embodiments, a plurality of single molecule sequencing reactions are performed in parallel (eg, on a single chip or cartridge) according to aspects of the present disclosure. For example, in some embodiments, multiple single molecule sequencing reactions are each performed in separate sample wells (eg, nanopores, reaction chambers) on a single chip or cartridge.

In some embodiments, sequencing of a target nucleic acid molecule comprises at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more) nucleotides. In some embodiments, at least 2 nucleotides are contiguous nucleotides. In some embodiments, at least 2 amino acids are non-contiguous nucleotides. In some embodiments, sequencing of a target nucleic acid comprises less than 100% (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, 70%) of all nucleotides in the target nucleic acid. less than, 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). For example, in some embodiments, sequencing of a target nucleic acid comprises identifying less than 100% of one type of nucleotide in the target nucleic acid. In some embodiments, sequencing the target nucleic acid comprises identifying less than 100% of each type of nucleotide in the target nucleic acid.

terminal functionalization

A target molecule can be functionalized at the distal end or position. For example, a target protein can be functionalized at its N-terminal end or at its C-terminal end. A target nucleic acid can be functionalized at either its 5' end or its 3' end. Nucleobases (eg guanidine) or sugar moieties (eg ribose or deoxyribose) may be functionalized.

C-terminal carboxylate functionalization

In one aspect, the disclosure provides

a. reacting a plurality of peptides of formula (I) or salts thereof with a compound of formula (II) to obtain a plurality of compounds of formula (III) or salts thereof; and

b. reacting a plurality of compounds of formula (III) , or salts thereof, with a compound of formula (IV) to obtain a plurality of compounds of formula (V)

Provides a method of selective C-terminal functionalization of a peptide comprising; Here, m, n, P, R(CO ₂ H) _n , HX, X, L ₁ , L ₂ , R ₁ , R ₂ , Y and Z are defined as follows.

m is an integer from 1 to 25 (inclusive). In certain embodiments, m is from 1 to 10 (inclusive). In certain embodiments, m is 5 to 10 (inclusive). In certain embodiments, m is 1 to 5 (inclusive). In certain embodiments, m is 1, 2, 3, 4, 5, 6, 7 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, or 25.

n is 1 or 2; In certain embodiments, n is 1. In certain embodiments, n is 2.

Each P is independently a peptide. In certain embodiments, P has 2 to 100 amino acid residues. In certain embodiments, P has 2 to 30 amino acid residues.

Each R(CO ₂ H) _n is independently an amino acid residue having n carboxylate moieties. n is 1 or 2; In certain embodiments, n is 1. When n is 1, R(CO ₂ H) _n is lysine or arginine. In certain embodiments, R(CO ₂ H) _n is lysine. In another specific embodiment, R(CO ₂ H) _n is arginine. In certain embodiments, n is 2. When n is 2, R(CO ₂ H) _n is glutamic acid or aspartic acid. In certain embodiments, R(CO ₂ H) _n is glutamic acid. In another specific embodiment, R(CO ₂ H) _n is aspartic acid.

HX is a nucleophilic moiety that can be acylated, where H is a proton. X is one or more heteroatoms. In certain embodiments, X is O, S, or NH, or NO.

L ₁ is a linker. In certain embodiments, L ₁ is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently, replaced by a heteroatom, aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L ₁ is polyethylene glycol (PEG). In other embodiments, L ₁ is a peptide, or oligonucleotide. In certain embodiments, L ₁ is less than 5 nm. In certain embodiments L ₁ is less than 1 nm.

L ₂ is a linker or absent. In certain embodiments, L ₂ is absent. In certain embodiments, L ₂ is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently, replaced by a heteroatom, aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L ₂ is polyethylene glycol (PEG). In other embodiments, L ₂ is a peptide, or oligonucleotide. In certain embodiments L ₂ is between 5 and 20 nm (inclusive).

R ₁ is a moiety comprising a click chemistry handle. In certain embodiments, R ₁ is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne, or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (eg, mono- or polycyclic) alkyne (eg, diarylcyclooctyne, or bicyclic[6.1.0]nonine). In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, R ₁ is a moiety comprising an azide. In certain embodiments, the tetrazine comprises the structure:

R ₂ is a moiety comprising a click chemistry handle complementary to R ₁ . The click chemistry handle of R ₂ can undergo a click reaction with R ₁ (ie, an electrocyclization reaction to form a 5-membered heterocyclic ring). For example, when R ₁ includes an azide, nitrile oxide, or tetrazine, R ₂ can include an alkyne or strained alkene. Conversely, when R ₁ includes an alkyne or strained alkene, R ₂ can include an azide, nitrile oxide, or tetrazine. In certain embodiments, R ₂ is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (eg, mono- or polycyclic) alkyne (eg, diarylcyclooctyne, or bicyclic[6.1.0]nonine). In certain particular embodiments, R ₂ comprises BCN. In certain other embodiments, R ₂ includes DBCO. In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, the tetrazine comprises the structure:

Y is a moiety resulting from the click reaction of R ₁ and R ₂ . Y is a 5-membered heterocyclic ring resulting from an electrocyclization reaction (eg, 3+2 cycloaddition, or 4+2 cycloaddition) between the reactive click chemistry handles of R ₁ and R ₂ . In certain embodiments, Y is 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4- It is a diradical containing a dihydropyridazyl moiety.

Z is a water soluble moiety. In certain embodiments, Z imparts water solubility to the compound to which it is attached. In certain embodiments, Z includes polyethylene glycol (PEG). In certain embodiments, Z comprises single-stranded DNA. In certain particular embodiments, Z comprises Q24. In certain embodiments, Z comprises double-stranded DNA. In certain embodiments (eg, a compound of Formula (V) ), Z further comprises biotin (eg, bisbiotin). When Z comprises biotin (eg, bisbiotin), Z may further comprise streptavidin. In certain embodiments, Z comprises double-stranded DNA. In some embodiments, a moiety of Z may be bound intramolecularly to another molecule or surface, for example anchoring a compound comprising Z to a molecule or surface.

In certain embodiments, the compound of formula (II) is of formula (IIa) :

In certain embodiments, formula (III) is of formula (IIIa) :

In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, m is 1. In certain embodiments, m is 5.

In certain embodiments, Formula (IV) includes TCO, and single-stranded DNA. In certain embodiments, formula (IV) further comprises biotin (eg, bisbiotin). In certain embodiments, formula (IV) is Q24-BisBt-BCN. In certain embodiments, formula (IV) is Q24-BisBt-DBCO. In certain embodiments, formula (IV) is Q24-BisBt-TCO. In general, formula (IV) can include a branching moiety (eg, a 1, 3, 5-tricarboxylate moiety), wherein the two branches are direct or indirect attachments to a biotin moiety and , the third branch is attachment to a water soluble moiety (eg, a polynucleotide such as Q24). 18B and 20 , in certain embodiments, formula (IV) is (i) a bisbiotin-azide functionalized linker and (ii) an alkyne (eg, BCN)-functionalized polynucleotide (eg Q24). Click-coupled products can be derivatized to introduce additional click handles R ₂ , such as BCN or DBCO.

In certain embodiments, formula (V) is of formula (Va) :

where m and n are 1 or 2; L ₂ , Y, and Z are as defined above. In certain particular embodiments, n is 1. In certain particular embodiments, n is 2. In certain particular embodiments, m is 1. In certain particular embodiments, m is 5. In certain particular embodiments, L ₂ is absent. In certain embodiments, Y is 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, and 1,4- and a moiety selected from dihydropyridazyl. In certain embodiments, Z comprises single-stranded DNA. In certain embodiments, Z comprises double-stranded DNA. In certain embodiments, Z includes biotin (eg, bisbiotin). In certain embodiments, Z further comprises streptavidin.

In certain embodiments, the reaction of step (a) is performed in the presence of a carbodiimide reagent. In certain embodiments, the carbodiimide reagent is water soluble. In certain embodiments, the carbodiimide reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In certain embodiments, the reaction of step (a) is conducted at a pH ranging from 3 to 5. In certain embodiments (eg, for total peptide concentrations of less than 1 mM), the concentration of EDC is about 10 mM and the concentration of the compound of formula (II) is about 20 mM. In certain embodiments (e.g., with respect to trypsin/LysC digestion, as described below) the concentration of compound of formula (II) can be about 50 mM and the concentration of EDC is to prevent C-terminal intramolecular cyclization. may be about 25 mM to inhibit.

In certain embodiments of step (a), the plurality of compounds of formula (III) are prepared prior to step (b), for example by passing the compounds through a G10 Sephadex column and/or passing the compounds through a C18 resin column. enriched The use of C18 resin-based enrichment is particularly useful when the compound of formula (II) is greater than about 200 g/mol. If G-10 Sephadex is used for enrichment, the elution buffer can be 0.5x PBS (pH 7.0). If C18 resin is used for enrichment, the elution buffer may be 0.1% formic acid with 80% acetonitrile in water. The C18 eluate can be dried and the residue re-suspended in 0.5x PBS before step (b).

In certain embodiments, the reaction of step (a) is performed in the presence of an immobilized carbodiimide reagent. For example, a carbodiimide reagent can be covalently attached to a moiety that is immobilized and/or insoluble in the reaction solvent, thereby facilitating separation of excess reagent and/or reaction by-products and/or unreacted peptides. . For example, see FIG. 20 . In certain embodiments, the immobilized carbodiimide reagent comprises a carbodiimide moiety covalently attached to a resin, such as polystyrene (PS). In certain embodiments, the PS-immobilized carbodiimide reagent is of the formula:

In certain embodiments, when the reaction of step (a) is performed in the presence of an immobilized carbodiimide reagent, e.g., a PS-immobilized reagent as described herein, the reaction is carried out at a pH ranging from 4 to 5. and/or at ambient temperature and/or for about 20 minutes.

In certain embodiments, carrying out the reaction of step (a) in the presence of an immobilized carbodiimide reagent, e.g., a PS-immobilized reagent as described herein, results in all unreacted (i.e., non-acylated) ) facilitates the removal of peptides, since unreacted peptides remain covalently bound to the immobilized carbodiimide reagent.

An exemplary process using an immobilized carbodiimide reagent is shown in FIG. 21 . An exemplary flow diagram for an automated conformance process is shown in FIG. 7 .

In certain embodiments of step (b), the click reaction between the plurality of compounds of formula (III) and the compound of formula (IV) is uncatalyzed. In certain embodiments, the click reaction is catalyzed using, for example, a copper salt (eg, a Cu ⁺ salt, or a Cu ²⁺ salt that is reduced to a Cu ⁺ salt in situ). Suitable Cu ²⁺ salts include CuSO ₄ . In certain embodiments, the reaction of step (b) comprises heating the reaction mixture.

In certain embodiments, the compound of formula (IV) is added to a plurality of compounds of formula (III) . In certain embodiments, the total concentration of the compound of formula (IV) and the plurality of compounds of formula (III) is maintained in the range of 10 μM to 1 mM.

In certain embodiments of step (b), when Z comprises single-stranded DNA, the method further comprises hybridizing a complementary DNA strand to the single-stranded DNA to obtain a compound wherein Z comprises double-stranded DNA. to include In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is Cy3B.

In certain embodiments of step (b), when Z comprises biotin (eg bisbiotin), the method comprises contacting biotin (eg bisbiotin) with streptavidin so that Z is biotin (eg bisbiotin) eg, bisbiotin) and streptavidin.

In certain embodiments, a plurality of peptides of formula (I) , or salts thereof, are obtained by subjecting proteins to enzymatic digestion to obtain a digestive mixture comprising a plurality of peptides of formula (I) , or salts thereof. In certain embodiments, enzymatic digestion comprises cleaving the C-terminal linkage of aspartic acid and/or glutamic acid residues of the protein. In certain specific embodiments, the enzymatic digestion is Glu-C digestion.

In certain embodiments, after digestion of 20 μg protein, the total concentration of the plurality of peptides of formula (I) , or salts thereof, is less than 100 μM.

In certain embodiments, enzymatic digestion is performed in phosphate buffer (pH 7.8) or ammonium bicarbonate buffer (pH 4.0).

In certain embodiments, enzymatic digestion involves cleaving the C-terminal bonds of lysine and/or arginine residues of the protein. In certain specific embodiments, the enzymatic digestion is trypsin+Lys-C digestion.

In certain embodiments, the carboxylic acid moiety of the protein, if present, is protected prior to enzymatic digestion. For example, carboxylic acid moieties of the protein, if present, can be esterified prior to enzymatic digestion. In certain specific embodiments, the esterified carboxylic acid is a methyl ester.

In certain embodiments, the sulfide moiety of the protein is protected prior to enzymatic digestion. In certain specific embodiments, the sulfide moiety is protected by exposing the protein to tris(carboxyethyl)phosphine (TCEP) and iodoacetamide (ICM), or maleimide.

In certain embodiments, the method further comprises enriching the digestive mixture prior to step (a).

C-terminal amine functionalization

In another aspect, the present disclosure provides

a. reacting a plurality of peptides of formula (VI) or salts thereof with a compound of formula (VII) to obtain a plurality of compounds of formula (VIII) , or salts thereof; and

b. reacting a plurality of compounds of formula (VIII) , or salts thereof, with a compound of formula (IX) to obtain a plurality of compounds of formula (X)

Provides a method of selective C-terminal amine functionalization of peptides comprising; wherein P, L ₃ , L ₄ , R ₃ , R ₄ , Y ₁ , and Z ₁ are as defined below.

Each P is independently a peptide. In certain embodiments, P has 2 to 100 amino acid residues. In certain embodiments, P has 2 to 30 amino acid residues.

L ₃ is a linker. In certain embodiments, L ₃ is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently, replaced by a heteroatom, aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L ₃ is polyethylene glycol (PEG). In other embodiments, L ₃ is a peptide, or oligonucleotide.

L ₄ is a linker or absent. In certain embodiments, L ₄ is absent. In certain embodiments, L ₄ is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally, independently, replaced by a heteroatom, aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L ₄ is polyethylene glycol (PEG). In other embodiments, L ₄ is a peptide, or oligonucleotide.

R ₃ is a moiety containing a click chemistry handle. In certain embodiments, R ₃ is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (eg, mono- or polycyclic) alkyne (eg, diarylcyclooctyne, or bicyclic[6.1.0]nonine). In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, R ₁ is a moiety comprising an azide. In certain embodiments, the tetrazine comprises the structure:

R ₄ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In certain embodiments, R ₄ is substituted or unsubstituted phenyl. In certain particular embodiments, R ₄ is phenyl. In certain particular embodiments, R ₄ is 4-nitrophenyl.

R ₅ is a moiety comprising a click chemistry handle complementary to R ₃ . The click chemistry handle of R ₅ can undergo a click reaction with R ₃ (ie, an electrocyclization reaction to form a 5-membered heterocyclic ring). For example, when R ₃ includes an azide, nitrile oxide, or tetrazine, R ₅ can include an alkyne or strained alkene. Conversely, when R ₃ includes an alkyne or strained alkene, R ₅ can include an azide, nitrile oxide, or tetrazine. In certain embodiments, R ₅ is a moiety comprising an azide, tetrazine, nitrile oxide, alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (eg, mono- or polycyclic) alkyne (eg, diarylcyclooctyne, or bicyclic[6.1.0]nonine). In certain particular embodiments, R ₅ comprises BCN. In certain other embodiments, R ₅ includes DBCO. In certain embodiments, the strained alkene is trans-cyclooctene. In certain embodiments, the tetrazine comprises the structure:

Y ₁ is a moiety resulting from the click reaction of R ₃ and R ₅ . Y ₁ is a 5-membered heterocyclic ring resulting from an electrocyclization reaction (eg, 3+2 cycloaddition, or 4+2 cycloaddition) between the reactive click chemistry handles of R ₃ and R ₅ . In certain embodiments, Y ₁ is 1,2,3-triazolyl, 4,5-dihydro-1,2,3-triazolyl, isoxazolyl, 4,5-dihydroisoxazolyl, or 1,4 - is a diradical containing a dihydropyridazyl moiety.

Z ₁ is a water soluble moiety. In certain embodiments, Z ₁ confers water-solubility to the compound to which it is attached. In certain embodiments, Z ₁ comprises polyethylene glycol (PEG). In certain embodiments, Z ₁ comprises single-stranded DNA. In certain particular embodiments, Z1 includes Q24. In certain embodiments, Z1 comprises single-stranded DNA. In certain embodiments (eg, a compound of Formula (V) ), Z ₁ further comprises biotin (eg, bisbiotin). When Z ₁ includes biotin (eg, bis-biotin), Z ₁ may further include streptavidin. In certain embodiments, Z ₁ comprises double-stranded DNA. In some embodiments, a moiety of Z ₁ may be bound intramolecularly to another molecule or surface, eg anchoring a compound comprising Z ₁ to a molecule or surface.

In certain embodiments, the compound of formula (VII)

is selected from

In certain embodiments, formula (VIII) is of formula (VIIIa) or formula (VIIIb) :

.

.

In certain embodiments, Formula (IX) includes TCO, single-stranded DNA, and biotin (eg, bisbiotin). In certain embodiments, formula (IX) is Q24-BisBt-BCN. In certain embodiments, Formula (IX) is Q24-BisBt-DBCO. In certain embodiments, Formula (IX) is Q24-BisBt-TCO. In general, formula (IX) can include a branching moiety (eg, a 1, 3, 5-tricarboxylate moiety), wherein the two branches are direct or indirect attachments to a biotin moiety and , the third branch is attachment to a water soluble moiety (eg, a polynucleotide such as Q24). In certain embodiments, formula (IX) is a fragment comprising (i) a bisbiotin-azide functionalized linker and (ii) an alkyne (eg, BCN)-functionalized polynucleotide (eg, Q24). It contains a triazole moiety derived from click-coupling. Click-coupled products can be derivatized to introduce additional click handles R ₅ , such as BCN or DBCO.

In certain embodiments, the reaction of step (a) comprises a buffer having a concentration in the range of about 20 mM to 500 mM and a pH in the range of about 9 to 11, and acetonitrile in the range of about 20 to 70% of the total volume. performed in the presence of In certain embodiments, the reaction of step (a) is performed at approximately 37° C. in pH 9.5 buffer/acetonitrile (1:3 v/v). In certain embodiments, the reaction of step (a) is performed using a concentration of the compound of formula (VII) between about 500 μM and 50 mM.

In certain embodiments, the plurality of compounds of formula (VIII) are enriched prior to step (b). In certain embodiments, enrichment includes ethyl acetate/hexane extraction. A suitable range for ethyl acetate/hexanes includes, but is not limited to, 20 to 100 volume % ethyl acetate in hexanes. In certain embodiments, the volume of organic solvent used for extraction is about 10x the volume of the aqueous layer. Other water immiscible organic solvents such as diethyl ether, dichloromethane, chloroform, benzene, toluene, and n-1-butanol can be used for extraction.

In certain embodiments, the reaction of step (b) comprises reacting a compound of formula (VIII) with about 1 equivalent of a compound of formula (IX) . In certain embodiments, the reaction of step (b) comprises heating the reaction mixture.

In certain embodiments of step (b), when Z ₁ comprises single-stranded DNA, the method comprises hybridizing a complementary DNA strand to the single-stranded DNA to obtain a compound wherein Z ₁ comprises double-stranded DNA. additionally include In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is Cy3B.

In certain embodiments of step (b), when Z ₁ comprises biotin (eg, bisbiotin), the method comprises contacting biotin (eg, bisbiotin) with streptavidin so that Z ₁ is biotin ( eg, bisbiotin) and streptavidin.

In certain embodiments, a plurality of peptides of formula (VI) , or salts thereof, are obtained by subjecting proteins to enzymatic digestion to obtain a digestive mixture comprising a plurality of peptides of formula (VI) , or salts thereof. Enzymatic digestion involves cleaving the C-terminal linkages of lysine and/or arginine residues of proteins. In certain embodiments, enzymatic digestion is performed using trypsin, Lys-C, or a combination thereof. In certain embodiments, enzymatic digestion involves reacting the protein with trypsin and Lys-C in Tris-HCl buffer (pH 8.5). In certain embodiments, after digestion of 20 μg protein, the total concentration of the plurality of peptides of formula (VI) , or salts thereof, is less than 100 μM.

In certain embodiments, the sulfide moiety of the protein is protected prior to enzymatic digestion. In certain specific embodiments, the sulfide moiety is protected by exposing the protein to tris(carboxyethyl)phosphine (TCEP) and iodoacetamide (ICM), or maleimide.

In certain embodiments, the method further comprises enriching the digestive mixture prior to step (a). In certain embodiments, the digestive mixture is used in a method of selective C-terminal amine functionalization of peptides without enrichment or purification.

Selective Amine Functionalization via Diazo Transfer

Prior to sequencing, digested peptides must be functionalized with moieties capable of immobilizing the peptides onto the sequencing substrate. Accordingly, the present disclosure provides the reaction of a plurality of peptides of Formula (XI) , or salts thereof, with a compound of Formula (XII) under conditions comprising Cu ²⁺ , or a precursor thereof, and a buffer having a pH of about 10 to 11 to obtain a plurality of ε-azido compounds of formula (XIII) , or salts thereof.

wherein each P is independently a peptide with an N-terminal amine.

Each P is independently a peptide with an N-terminal amine. In certain embodiments, P has 2 to 100 amino acid residues. In certain embodiments, P has 2 to 30 amino acid residues. In some embodiments, the concentration of peptide in a reaction is any conceivable concentration required.

In certain embodiments, the Cu ²⁺ salt is CuCl ₂ , CuBr ₂ , Cu(OH) ₂ , or CuSO ₄ . In certain embodiments, the Cu ²⁺ salt is CuSO ₄ . In certain embodiments, the molar amount of the Cu ²⁺ salt is about 2.5 times the molar amount of the compound of formula (XI) . In certain particular embodiments, the concentration of the Cu ²⁺ salt is about 250 μM. In some embodiments, the concentration of the Cu ²⁺ salt is between 1 and 5 mM or between 100 and 1000 μM.

In certain embodiments, the conditions are about 20 to 30 °C, e.g., 20 to 25 °C, 22 to 27 °C, 25 to 30 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, further comprising a reaction at 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

In certain embodiments, the conditions react for about 30 to 60 minutes, e.g., 30 to 35 minutes, 35 to 40 minutes, 40 to 45 minutes, 45 to 50 minutes, 50 to 55 minutes, or 55 to 60 minutes. additionally includes

In certain embodiments, the buffer has a pH of about 10.5. In certain embodiments, the buffering agent comprises a bicarbonate, such as sodium bicarbonate. In certain embodiments, the buffering agent comprises a carbonate, such as potassium carbonate. In certain embodiments, the buffering agent comprises a phosphate, such as potassium phosphate. In some embodiments, the buffering agent does not include amino groups. In some embodiments, the buffer is a good buffer (eg, HEPES, TRIS). In certain embodiments, the buffer has a concentration in the range of 10 mM to 1 M, eg, 10 to 100 mM, 50 to 500 mM, 50 to 100 mM, or 100 mM.

In certain embodiments, the concentration of the compound of Formula (XI) is about 100 μM. In some embodiments, the concentration of the compound of Formula (XI) is about 50 μM. In some embodiments, the concentration of the compound of Formula (XI) is between 1 nM and 1 mM.

In certain embodiments, the amount of compound of formula (XII) used in the reaction is from 10 to 30 molar equivalents, for example about 20 molar equivalents, relative to the amount of compound of formula (XI) used in the reaction. In certain embodiments, the concentration of the compound of Formula (XII) is between about 1 and 3 mM, for example about 2 mM.

In certain embodiments, the N-terminal:ε selectivity of the diazo transfer reaction is at least about 90%.

In some embodiments, the method further comprises enriching the plurality of compounds of Formula (XIII) , or salts thereof. In certain embodiments, excess compound of formula (XII) is removed from the reaction mixture using a purification cartridge, such as a G-10 Sephadex column. In certain embodiments, removal of excess Formula (XIII) using a G-10 Sephadex column comprises buffer exchange with 25 mM HEPES, 25 mM KOAc, pH 7.8.

In some embodiments, a plurality of peptides of formula (XI) , or salts thereof, are subjected to enzymatic digestion as described herein to obtain a digestive mixture comprising a plurality of peptides of formula (XI) , or salts thereof. obtained by obtaining Enzymatic digestion involves cleaving the C-terminal linkage of aspartic acid and/or glutamic acid residues of a protein.

In some embodiments, the enzymatic digestion is trypsin+Lys-C digestion. In some embodiments, trypsin+Lys-C digestion comprises reacting the protein with trypsin and Lys-C in pH 9.5 buffer at room temperature.

In some embodiments, the method reacts a plurality of compounds of formula (XIII), or salts thereof, with a DBCO-labeled DNA-streptavidin conjugate, such that the azide moiety of the compound of formula (XIII) , or salts thereof, is further comprising undergoing an electrocyclization reaction with the alkyne moiety of DBCO (diarylcyclooctyne) to form a plurality of peptide-DNA-streptavidin conjugates.

In some embodiments, the DBCO-labeled DNA-streptavidin is of formula (XIV) :

wherein R ₆ is DBCO; L ₅ is a linker or absent; Z ₂ is a dsDNA-streptavidin conjugate;

A plurality of peptide-DNA-streptavidin conjugates are of formula (XV) , or a salt thereof:

wherein Y ₂ is a moiety resulting from a click reaction with an azide moiety of formula (XIIIb) and R ₆ .

R ₆ is a moiety comprising a click chemistry handle complementary to the azide moiety of formula (XIIIb) . The click chemistry handle of R ₆ can undergo a click reaction (ie, an electrocyclization reaction to form a 5-membered heterocyclic ring) with an azide moiety of formula (XIIIb) . In certain embodiments, R ₆ comprises an alkyne or strained alkene. In certain embodiments, the alkyne is a primary alkyne. In certain embodiments, the alkyne is a cyclic (eg, mono- or polycyclic) alkyne (eg, diarylcyclooctyne, or bicyclic[6.1.0]nonine). In certain particular embodiments, R ₆ comprises BCN. In certain other embodiments, R ₆ includes DBCO. In certain embodiments, the strained alkene is trans-cyclooctene.

In certain embodiments, L ₅ is absent. In certain embodiments, L ₅ is a substituted or unsubstituted aliphatic chain, wherein one or more carbon atoms are optionally replaced by a heteroatom, aryl, heteroaryl, cycloalkyl, or heterocyclyl moiety. In certain embodiments, L ₅ is polyethylene glycol (PEG). In other embodiments, L ₅ is a peptide, or oligonucleotide.

In certain embodiments, Z ₂ is prepared from a bis-biotin tag that specifically binds to the cis form of streptavidin, leaving the other cis-binding site free for surface immobilization.

In certain embodiments, Z ₂ comprises PEG. In certain embodiments, Z ₂ further comprises biotin (eg, bisbiotin). In certain embodiments, when Z ₂ comprises single-stranded DNA, the method further comprises hybridizing the complementary DNA strand to the single-stranded DNA to obtain a compound in which Z ₂ comprises double-stranded DNA . In certain embodiments, the single-stranded DNA is Q24 and the complementary DNA strand is Cy3B.

In certain embodiments, formula (XIV) is Q24-BisBt-BCN. In certain embodiments, Formula (XIV) is Q24-BisBt-DBCO. In certain embodiments, Formula (XIV) is Q24-BisBt-TCO. In general, Formula (XIV) can include a branching moiety (eg, a 1, 3, 5-tricarboxylate moiety), wherein the two branches are direct or indirect attachments to a biotin moiety and , the third branch is attachment to a water soluble moiety (eg, a polynucleotide such as Q24). In certain embodiments, formula (XIV) is a fragment comprising (i) a bisbiotin-azide functionalized linker and (ii) an alkyne (eg, BCN)-functionalized polynucleotide (eg, Q24). It contains a triazole moiety derived from click-coupling. Click-coupled products can be derivatized to introduce additional click handles R ₆ , such as BCN or DBCO.

In certain embodiments, when Z ₂ comprises biotin (eg, bisbiotin), the method comprises contacting biotin (eg, bisbiotin) with streptavidin so that Z ₂ is biotin (eg, bisbiotin). biotin) and streptavidin.

In certain embodiments, the method of selective N-functionalization of a peptide is performed according to one or more steps as shown in FIG. 6 .

click chemistry

In certain embodiments, the reaction used to conjure the host to the tag is a “click chemistry” reaction (eg, Huisgen alkyne-azide cycloaddition). It should be understood that any "click chemistry" reaction known in the art can be used for this purpose. Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry adapted to rapidly and reliably create materials by linking small units together. See, eg, Kolb, Finn and Sharpless, Angewandte Chemie International Edition (2001) 40: 2004-2021; See Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions (some of which may be classified as "click chemistry") include formation of esters, thioesters, amides from activated acids or acyl halides (eg, such as peptide coupling); nucleophilic shift reactions (eg, such as nucleophilic shifts of halides or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-phosphorus addition; immigration formation; Michael addition (e.g. maleimide addition); and Diels-Alder reactions (eg, tetrazine [4 + 2] cycloaddition).

The term “click chemistry” describes chemistry adapted to rapidly and reliably create covalent bonds by linking together small units containing reactive groups, K. of The Scripps Research Institute. Refers to a chemical synthesis technique introduced by K. Barry Sharpless. See, eg, Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; See Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary reactions include azide-alkyne Huisgen cycloaddition; and Diels-Alder reactions (eg, tetrazine [4 + 2] cycloaddition). In some embodiments, a click chemistry reaction is modular, broad in scope, provides high chemical yield, produces harmless by-products, stereospecific, and/or a single reaction product. and/or can be carried out under physiological conditions. In some embodiments, a click chemistry reaction exhibits high atom economics, can be performed under simple reaction conditions, uses readily available starting materials and reagents, and/or does not use toxic solvents or uses benign or Uses easily removable solvents (preferably water) and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).

As used herein, the term "click chemistry handle" refers to a reactant, or reactive group, capable of participating in a click chemistry reaction. For example, strained alkynes, such as cyclooctyne, are click chemical handles because they can participate in strain-facilitated cycloadditions (see, eg, Table 1). Generally, click chemistry reactions require at least two molecules containing click chemistry handles that can react with each other. Pairs of such click chemistry handles that are reactive with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemical handle to cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to some aspects of the present invention are set forth herein, for example in Tables 1 and 2. Other suitable click chemistry handles are known to those skilled in the art.

Table 1 : Exemplary click chemistry handles and reactions.

In some embodiments, a click chemistry handle is used that can react to form covalent bonds in the presence of a metal catalyst, such as copper (II). In some embodiments, click chemistry handles are used that can react to form covalent bonds in the absence of a metal catalyst. Such click chemistry handles are well known to those skilled in the art and described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900 - 4908. Includes click chemistry handle.

Table 2 : Exemplary click chemistry handles and reactions.

From Becer, Hoogenboom, and Schubert, Click Chemistry Beyond Metal-Catalyzed Cycloaddition , Angewandte Chemie International Edition (2009) 48: 4900 - 4908.

Additional click chemistry handles suitable for use in the methods of conjugation described herein are well known to those skilled in the art, and such click chemistry handles include the click chemistry reaction partners described in PCT/US2012/044584 and references therein. , groups, and handles, which references are incorporated herein by reference for click chemistry handles and methodologies.

compound

In certain aspects, the present disclosure provides, in various embodiments, Formulas (II), (IIa), (III), (IIIa), (IV), (V), (Va), (VII), (VIII), (VIIIa), (VIIIb), (XIV), (X), (XI), (XII), (XIIIa), (XIIIb), (XV), and salts thereof.

In certain embodiments, the compound is water soluble.

In certain embodiments, the compounds are useful for applications involving the analysis of proteins and peptides, such as peptide sequencing. For example, in certain embodiments, compounds of Formulas (V), (X), (XV) , and salts thereof may be covalently or non-covalently attached to a surface.

Justice

In the following description, certain specific details are set forth to provide a thorough understanding of the various embodiments of the present invention. However, one skilled in the relevant art will understand that the present invention may be practiced without these details.

Throughout this specification and claims, unless the context requires otherwise, the word “comprise” and its derivatives, such as “comprises” and “comprising,” are used in an open, inclusive sense ( ie, "including but not limited to").

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise.

The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms (“C _1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C _1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C _1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C _1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C _1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C _1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C _1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C _1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C _1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C _1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C ₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C _2-6 alkyl”). Examples of C _1-6 alkyl groups include methyl (C ₁ ), ethyl (C ₂ ), propyl (C ₃ ) (eg n-propyl, isopropyl), butyl (C ₄ ) (eg n -butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C ₅ ) (eg n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C ₆ ) (eg, n-hexyl). Further examples of alkyl groups include n-heptyl (C ₇ ), n-octyl (C ₈ ), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (eg, halogen, such as F) ("substituted alkyl") . In certain embodiments, an alkyl group is an unsubstituted C _1-10 alkyl (such as unsubstituted C _1-6 alkyl, eg, —CH ₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl ( Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n- Bu), unsubstituted tert-butyl (tert-Bu or t -Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In embodiments, an alkyl group is a substituted C _1-10 alkyl (such as a substituted C _1-6 alkyl, eg, -CH ₂ F, -CHF ₂ , -CF ₃ or benzyl (Bn)). or unbranched.

)은 (E)- 또는 (Z)-배치이다.The term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and at least one carbon-carbon double bond (eg, 1, 2, 3, or 4 double bonds). . In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C _1-20 alkenyl”). In some embodiments, an alkenyl group has 1 to 12 carbon atoms (“C _1-12 alkenyl”). In some embodiments, an alkenyl group has 1 to 11 carbon atoms (“C _1-11 alkenyl”). In some embodiments, an alkenyl group has 1 to 10 carbon atoms (“C _1-10 alkenyl”). In some embodiments, an alkenyl group has 1 to 9 carbon atoms (“C _1-9 alkenyl”). In some embodiments, an alkenyl group has 1 to 8 carbon atoms (“C _1-8 alkenyl”). In some embodiments, an alkenyl group has 1 to 7 carbon atoms (“C _1-7 alkenyl”). In some embodiments, an alkenyl group has 1 to 6 carbon atoms (“C _1-6 alkenyl”). In some embodiments, an alkenyl group has 1 to 5 carbon atoms (“C _1-5 alkenyl”). In some embodiments, an alkenyl group has 1 to 4 carbon atoms (“C _1-4 alkenyl”). In some embodiments, an alkenyl group has 1 to 3 carbon atoms (“C _1-3 alkenyl”). In some embodiments, an alkenyl group has 1 to 2 carbon atoms (“C _1-2 alkenyl”). In some embodiments, an alkenyl group has 1 carbon atom (“C ₁ alkenyl”). The one or more carbon-carbon double bonds may be internal (eg in 2-butenyl) or terminal (eg in 1-butenyl). Examples of C _1-4 alkenyl groups include methylidenyl (C ₁ ), ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ), 2-butenyl (C ₄ ), butadienyl (C ₄ ) and the like. Examples of C _1-6 alkenyl groups include pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (C ₆ ), and the like, as well as the aforementioned C _2-4 alkenyl groups. Further examples of alkenyl include heptenyl (C ₇ ), octenyl (C ₈ ), octatrienyl (C ₈ ), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents (“substituted alkenyl”). In certain embodiments, an alkenyl group is an unsubstituted C _1-20 alkenyl. In certain embodiments, an alkenyl group is a substituted C _1-20 alkenyl. In an alkenyl group, a C=C double bond of unspecified stereochemistry (eg -CH=CHCH ₃ or

) is (E)- or (Z)-configuration.

The term “heteroalkenyl” refers to at least one selected from oxygen, nitrogen, or sulfur located within the parent chain (e.g., inserted between adjacent carbon atoms thereof) and/or at one or more terminal position(s) of the parent chain. refers to an alkenyl group that further comprises a heteroatom of (eg, 1, 2, 3, or 4 heteroatoms). In certain embodiments, a heteroalkenyl group refers to a group having 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having 1 to 12 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-12 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having 1 to 11 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-11 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having 1 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-10 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-9 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-8 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-7 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-6 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-5 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-4 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC _1-3 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC _1-2 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted ("unsubstituted heteroalkenyl") or substituted with one or more substituents ("substituted heteroalkenyl"). In certain embodiments, a heteroalkenyl group is an unsubstituted heteroC _1-20 alkenyl. In certain embodiments, a heteroalkenyl group is a substituted heteroC _1-20 alkenyl.

The term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and at least one carbon-carbon triple bond (eg, 1, 2, 3, or 4 triple bonds). ("C _1-20 alkynyl"). In some embodiments, an alkynyl group has 1 to 10 carbon atoms (“C _1-10 alkynyl”). In some embodiments, an alkynyl group has 1 to 9 carbon atoms (“C _1-9 alkynyl”). In some embodiments, an alkynyl group has 1 to 8 carbon atoms (“C _1-8 alkynyl”). In some embodiments, an alkynyl group has 1 to 7 carbon atoms (“C _1-7 alkynyl”). In some embodiments, an alkynyl group has 1 to 6 carbon atoms (“C _1-6 alkynyl”). In some embodiments, an alkynyl group has 1 to 5 carbon atoms (“C _1-5 alkynyl”). In some embodiments, an alkynyl group has 1 to 4 carbon atoms (“C _1-4 alkynyl”). In some embodiments, an alkynyl group has 1 to 3 carbon atoms (“C _1-3 alkynyl”). In some embodiments, an alkynyl group has 1 to 2 carbon atoms (“C _1-2 alkynyl”). In some embodiments, an alkynyl group has 1 carbon atom (“C ₁ alkynyl”). The one or more carbon-carbon triple bonds may be internal (eg in 2-butynyl) or terminal (eg in 1-butynyl). Examples of C _1-4 alkynyl groups include, but are not limited to, methylidinyl (C ₁ ), ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2-butynyl (C ₄ ), and the like. Examples of C _1-6 alkenyl groups include the aforementioned C _2-4 alkynyl groups as well as pentynyl (C ₅ ), hexynyl (C ₆ ), and the like. Further examples of alkynyl include heptynyl (C ₇ ), octynyl (C ₈ ), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (“unsubstituted alkynyl”) or substituted with one or more substituents (“substituted alkynyl”). In certain embodiments, an alkynyl group is an unsubstituted C _1-20 alkynyl. In certain embodiments, an alkynyl group is a substituted C _1-20 alkynyl.

The term "heteroalkynyl" refers to at least one selected from oxygen, nitrogen, or sulfur located within the parent chain (e.g., inserted between adjacent carbon atoms thereof) and/or at one or more terminal position(s) of the parent chain. refers to an alkynyl group further comprising heteroatoms of (eg, 1, 2, 3, or 4 heteroatoms). In certain embodiments, a heteroalkynyl group refers to a group having 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having 1 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-10 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-9 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-8 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-7 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC _1-6 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-5 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-4 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC _1-3 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC _1-2 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC _1-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (“unsubstituted heteroalkynyl”) or substituted with one or more substituents (“substituted heteroalkynyl”). In certain embodiments, a heteroalkynyl group is an unsubstituted heteroC _1-20 alkynyl. In certain embodiments, a heteroalkynyl group is a substituted heteroC _1-20 alkynyl.

"Aralkyl" is a subset of "alkyl" and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on an alkyl moiety.

The term “cycloalkyl” refers to a cyclic alkyl radical having 3 to 10 ring carbon atoms (“C _3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C _3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C _3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C _5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C _5-10 cycloalkyl”). Examples of C _5-6 cycloalkyl groups include cyclopentyl (C ₅ ) and cyclohexyl (C ₅ ). Examples of C _3-6 cycloalkyl groups include the aforementioned C _5-6 cycloalkyl groups as well as cyclopropyl (C ₃ ) and cyclobutyl (C ₄ ). Examples of C _3-8 cycloalkyl groups include the aforementioned C _3-6 cycloalkyl groups as well as cycloheptyl (C ₇ ) and cyclooctyl (C ₈ ). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In certain embodiments, a cycloalkyl group is an unsubstituted C _3-10 cycloalkyl. In certain embodiments, a cycloalkyl group is a substituted C _3-10 cycloalkyl.

), 산소 (예를 들어,

), 또는 황 (예를 들어,

)에 의해 대체된 본원에 정의된 바와 같은 알킬 기를 지칭한다. 헤테로알킬 기는 임의로 본원에 기재된 임의의 치환기로부터 독립적으로 선택되는 1, 2, 3개, 또는 2개 이상의 탄소의 알킬 기의 경우, 4, 5, 또는 6개의 치환기로 치환될 수 있다. 헤테로알킬 기 치환기는 (1) 카르보닐; (2) 할로; (3) C₆-C₁₀ 아릴; 및 (4) C₃-C₁₀ 카르보시클릴을 포함한다. 헤테로알킬렌은 2가 헤테로알킬 기이다.As used herein, the term "heteroalkyl" means that one or more of the constituent carbon atoms is a heteroatom or an optionally substituted heteroatom, such as nitrogen (eg,

), oxygen (eg

), or sulfur (eg

) refers to an alkyl group as defined herein replaced by Heteroalkyl groups may be optionally substituted with 4, 5, or 6 substituents in the case of alkyl groups of 1, 2, 3, or 2 or more carbons independently selected from any of the substituents described herein. Heteroalkyl group substituents are (1) carbonyl; (2) halo; (3) C ₆ -C ₁₀ aryl; and (4) C ₃ -C ₁₀ carbocyclyl. Heteroalkylene is a divalent heteroalkyl group.

As used herein, the term “alkoxy” refers to —OR ^a , where R ^a is, for example, an alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclyl, heterocyclyl, or heteroaryl. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, tert-butoxy, phenoxy, and benzyloxy.

The term “aryl” refers to a monocyclic or polycyclic (eg, bicyclic or tricyclic) 4n+2 aromatic ring system (eg, bicyclic or tricyclic) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system. eg, having 6, 10, or 14 π electrons shared in a cyclic array) (“C _6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C ₆ aryl”; eg, phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C ₁₀ aryl”; eg, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C ₁₄ aryl”; eg, anthracyl). “Aryl” also includes ring systems in which an aryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the radical or point of attachment is on the aryl ring, in which case the number of carbon atoms continues to number the carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (“unsubstituted aryl”) or one or more substituents (eg, -F, -OH or -O(C _1-6 alkyl) ("Substituted aryl"). In certain embodiments, an aryl group is an unsubstituted C _6-14 aryl. In certain embodiments, an aryl group is a substituted C _6-14 aryl.

The term "aryloxy" refers to an -O-aryl substituent.

The term “heteroaryl” refers to a 5 to 14 membered monocyclic or polycyclic (eg, bicyclic, tricyclic) 4n+2 aromatic ring having ring carbon atoms and 1 to 4 ring heteroatoms provided in an aromatic ring system. system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array), wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5 to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, in which case the number of ring members continues to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, wherein the point of attachment is on either the aryl or heteroaryl ring, in which case the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring contains no heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment is to either ring, e.g., containing a heteroatom. It may be on a ring (eg 2-indolyl) or on a ring that does not contain heteroatoms (eg 5-indolyl). In certain embodiments, heteroaryl is a substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen. , nitrogen, or sulfur. In certain embodiments, heteroaryl is a substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. ("5 to 10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. (“5 to 8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. (“5- to 6-membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In certain embodiments, a heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, a heteroaryl group is a substituted 5-14 membered heteroaryl.

The term "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently is selected from nitrogen, oxygen, and sulfur ("3 to 14 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. Heterocyclyl groups are monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., fused, bridged or spiro ring systems such as bicyclic systems (“bicyclic heterocyclyl”) or tricyclic click system ("tricyclic heterocyclyl")), may be saturated, or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also refers to a ring system in which a heterocyclyl ring is fused with one or more carbocyclyl groups, as defined above, wherein the point of attachment is on either the carbocyclyl or heterocyclyl ring, or Includes ring systems in which a heterocyclyl ring as defined is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, in which case the numbering of the ring members is the heterocyclyl ring system Continue to number the ring members in . Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (“unsubstituted heterocyclyl”) or substituted with one or more substituents (“substituted heterocyclyl”). In certain embodiments, a heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, a heterocyclyl group is a substituted 3-14 membered heterocyclyl. In certain embodiments, heterocyclyl is a substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently Oxygen, nitrogen, or sulfur, as the valency permits.

In some embodiments, a heterocyclyl group is a 5 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. (“5- to 10-membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. ("5 to 8 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. (“5- to 6-membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

The term “carbonyl” refers to a group in which the carbon directly attached to the parent molecule is sp ² hybridized and substituted with an oxygen, nitrogen or sulfur atom, such as a ketone (eg, —C(=O)R ^aa ); Carboxylic acids (eg -CO ₂ H), aldehydes (-CHO), esters (eg -CO ₂ R ^aa , -C(=O)SR ^aa , -C(=S)SR ^aa ), amides (eg, -C(=O)N(R ^bb ) ₂ , -C(=O)NR ^bb SO ₂ R ^aa , -C(=S)N(R ^bb ) ₂ ), and imines (eg, -C(=NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa ), -C(=NR ^bb ) N(R ^bb ) ₂ ), wherein R ^aa and R ^bb are as defined herein.

As used herein, the term “amino” refers to —N(R ^N ) ₂ , wherein each R ^N is independently H, OH, NO ₂ , N(R ^N0 ) ₂ , SO ₂ OR ^N0 , SO ₂ R ^N0 , SOR ^N0 , an N-protecting group, alkyl, alkoxy, aryl, cycloalkyl, acyl (eg, acetyl, trifluoroacetyl, or others described herein), each of these listed R ^N groups being optionally substituted can be; or two R ^N are combined to form an alkylene or heteroalkylene, and each R ^N0 is independently H, alkyl, or aryl. An amino group of the present disclosure can be unsubstituted amino (ie, -NH ₂ ) or substituted amino (ie, -N(R ^N ) ₂ ).

As used herein, the term "substituted" means that at least one hydrogen atom is a non-hydrogen atom, such as a halogen atom, such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and to other heteroatoms (but not limited to) in various other groups. "Substituted" also means that one or more hydrogen atoms are heteroatoms such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and higher-order bonds (eg, double- or triple-bonds) to nitrogen in groups such as imines, oxides, hydrazones, and nitriles. For example, in some embodiments “substituted” means that one or more hydrogen atoms are NR _g R _h , NR _g C(=0)R _h , NR _g C(=0)NR _g R _h , NR _g C(=0 )OR _h , NRgSO ₂ R _h , OC(=O)NR _g R _h , OR _g , SR _g , SOR _g , SO ₂ Rg, OSO ₂ R _g , SO ₂ OR _g , =NSO ₂ R _g , and SO ₂ NR _g R _h . "Substituted is also one or more hydrogen atoms C(=O)R _g , C(=O)OR _g , C(=O)NR _g R _h , CH ₂ SO ₂ R _g , CH ₂ SO ₂ NR _g R _h Wherein R _g and R _h are the same or different and are independently hydrogen, alkyl, alkoxy, alkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, Heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl "substituted" further means that one or more hydrogen atoms are aminyl, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylaminyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocycle In addition, each of the foregoing substituents may also be optionally substituted with one or more of the foregoing substituents.

As used herein, the term “salt thereof” or “salts thereof” refers to salts well known in the art. For example, Berge et al., incorporated herein by reference [J. Pharmaceutical Sciences, 1977, 66, 1-19] describe pharmaceutically acceptable salts in detail. Additional information on suitable salts may be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, incorporated herein by reference.

Salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of acid addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or used in the related art. It is a salt of an amino group formed by using another method, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropio nate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulphate, heptanoate, hexanoate, hydroiodide, 2- Hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate , oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate , p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

A "protein", "peptide", or "polypeptide" includes a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein or peptide will be at least 3 amino acids in length. In some embodiments, the peptide is about 3 to about 100 amino acids in length (e.g., about 5 to about 25, about 10 to about 80, about 15 to about 70, or about 20 to about 40 amino acids in length) to be. In some embodiments, the peptide is about 6 to about 40 amino acids in length (e.g., about 6 to about 30, about 10 to about 30, about 15 to about 40, or about 20 to about 30 amino acids in length) to be. In some embodiments, a plurality of peptides may refer to a plurality of peptide molecules wherein each peptide molecule of the plurality comprises a different amino acid sequence than any other peptide molecule of the plurality. In some embodiments, the plurality of peptides is at least one peptide and up to 1,000 peptides (e.g., at least 1 peptide and up to 10, 50, 100, 250, or 500 peptides). In some embodiments, the plurality of peptides comprises 1 to 5, 5 to 10, 1 to 15, 15 to 20, 10 to 100, 50 to 250, 100 to 500, 500 to 1,000, or more different peptides. do. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, but non-natural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain) and/or amino acid analogs as known in the art are alternatives. can be recruited adversarially. In addition, one or more of the amino acids in the protein may be, for example, a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modifications. can be modified by the addition of Proteins can also be single molecules or multi-molecular complexes. A protein or peptide may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination thereof. With regard to the use of substantially any plural and/or singular form herein, those skilled in the art may interpret from plural to singular and/or from singular to plural, as appropriate to the context and/or application. Various singular/plural permutations may be expressly presented herein for clarity.

In general, it will be understood by those skilled in the art that terms used herein, and in particular in the appended claims (eg, the body of the appended claims), are generally intended as "open" terms. (e.g., the term "comprising" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", and the term "comprises" should be interpreted as "including but not limited to" (which should be construed as, but not limited to, etc.). It will be further understood by those skilled in the art that where a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of this phrase does not prevent the introduction of a claim recitation by an indefinite article from containing such an introduced claim recitation, even when the same claim contains the introductory phrase “one or more” or “at least one” and an indefinite article. should not be construed as implying that any particular claim is limited to the embodiment containing only one such recitation (e.g., the indefinite article should be construed to mean "at least one" or "one or more" ); The same is true of the use of the definite article used to introduce claim enumeration. Furthermore, even where a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., "two recitations"). means at least two sequences, or more than two sequences, without other modifiers). Moreover, when a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to mean that those skilled in the art will understand the convention (e.g., "A A system having at least one of A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or a system having A, B, and C together, etc. (including but not limited to).) When a convention similar to "at least one of A, B, or C, etc." is used, such construction is generally meant to mean that those skilled in the art will understand the convention. (e.g., "a system having at least one of A, B, or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or together (including, but not limited to, systems with A, B, and C, etc.) virtually any disjunctive word and/or It will be further understood by those skilled in the art that the phrase should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B." Also, when a feature or aspect of the present disclosure is described in terms of the Markush group, the related art Those skilled in the art will recognize that this disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one of ordinary skill in the relevant art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and It covers combinations of its sub-ranges. Any recited range can be readily recognized as sufficiently delineating and enabling the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily divided into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by one of ordinary skill in the relevant art, all language such as "at most", "at least", "greater than", "less than", etc. include the listed numbers, and as discussed above sub- Refers to a range that can subsequently be divided into ranges. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

Those of ordinary skill in the art will understand that certain compounds described herein can form one or more different isomers (e.g., stereoisomers, geometric isomers, tautomers) and/or isotopes (e.g., different isotopes of one or more valence atoms). , eg hydrogen substituted for deuterium) form. Unless otherwise indicated or clear from context, a depicted structure may be understood to represent any such isomeric or isotopic form, either individually or in combination.

Peptide surface immobilization

In certain single molecule assay methods, the molecule to be analyzed is immobilized onto a surface so that the molecule can be monitored without interference from other reactive components in solution. In some embodiments, surface immobilization of the molecule allows the molecule to be localized to a desired area of the surface for real-time monitoring of reactions involving the molecule.

Thus, in some aspects, the present application provides a method of immobilizing a peptide to a surface by attaching any one of the compounds described herein to the surface of a solid support. In some embodiments, the method comprises contacting the surface of a solid support with a compound of Formula (V), (X), (XV) , or a salt thereof. In some embodiments, the surface is functionalized with complementary functional moieties configured for attachment (eg, covalent or non-covalent attachment) to the functionalized terminal end of the peptide. In some embodiments, a solid support comprises a plurality of sample wells formed at a surface of the solid support. In some embodiments, a method comprises immobilizing a single peptide to the surface of each of a plurality of sample wells. In some embodiments, localizing a single peptide per sample well is a single molecule detection method, e.g., It is advantageous for single molecule peptide sequencing.

In some embodiments, surface as used herein refers to the surface of a substrate or solid support. In some embodiments, a solid support refers to a material, layer, or other structure having a surface, such as a receptive surface, capable of supporting a deposited material, such as a functionalized peptide described herein. In some embodiments, the receiving surface of the substrate may optionally have one or more features, including nanoscale or microscale intrusion features, such as an array of sample wells. In some embodiments, an array is a planar arrangement of elements, such as sensors or sample wells. Arrays can be 1 or 2 dimensional. A one-dimensional array is an array having one row or column of elements in a first dimension and multiple rows or columns in a second dimension. The number of rows or columns in the first and second dimensions may or may not be the same. In some embodiments, an array can include, for example, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , or 10 ⁷ sample wells.

An exemplary scheme of peptide surface immobilization is shown in FIG. 9 . As indicated, panels (I) to (II) depict the process of immobilizing a peptide 900 comprising a functionalized terminal end 902 . In panel (I), a solid support containing sample wells is shown. In some embodiments, a sample well is formed by a bottom surface comprising non-metallic layer 910 and a sidewall surface comprising metallic layer 912 . In some embodiments, the non-metallic layer 910 includes a transparent layer (eg, glass, silica). In some embodiments, metallic layer 912 includes a metal oxide surface (eg, titanium dioxide). In some embodiments, metallic layer 912 includes a passivating coating 914 (eg, a phosphorus-containing layer such as an organophosphonate layer). As shown, the bottom surface comprising non-metallic layer 910 includes complementary functional moieties 904 . Methods of selective surface modification and functionalization are described in further detail in US Patent Publication No. 2018/0326412 and US Provisional Application No. 62/914,356, the contents of each of which are incorporated herein by reference.

In some embodiments, a peptide ( 900 ) comprising a functionalized terminal end ( 902 ) is contacted with a complementary functional moiety ( 904 ) of a solid support to form a covalent or non-covalent linking group. In some embodiments, functionalized terminal end ( 902 ) and complementary functional moiety ( 904 ) comprise partner click chemical handles that form a covalent linking group between, for example, peptide ( 900 ) and a solid support. Suitable click chemistry handles are described elsewhere herein. In some embodiments, functionalized terminal end ( 902 ) and complementary functional moiety ( 904 ) form a non-covalent binding partner, for example forming a non-covalent linking group between peptide ( 900 ) and a solid support. include An example of a non-covalent binding partner is a complementary oligonucleotide strand (e.g., complementary nucleic acid strands, including DNA, RNA, and variants thereof), protein-protein binding partners (e.g., Barnaze and Barsta), and protein-ligand binding partners (e.g., biotin and streptavidin).

In panel (II), peptide 900 is shown immobilized to the bottom surface via a linking group formed by contacting a functionalized terminal end 902 and a complementary functional moiety 904 . In this example, peptide ( 900 ) is attached via a non-covalent linking group shown in the enlarged region of panel (III). As shown, in some embodiments, the non-covalent linking group comprises an avidin protein ( 920 ). Avidin proteins are generally biotin-binding proteins that have a biotin binding site on each of the four subunits of the avidin protein. Avidin proteins include, for example, avidin, streptavidin, trapavidin, tamavidin, bradavidin, xenavidin, and homologs and variants thereof. In some embodiments, the avidin protein ( 920 ) is streptavidin. The multiplicity of the avidin protein ( 920 ) can allow for a variety of linkage configurations, since each of the four binding sites can independently bind a biotin molecule (shown as white circles).

As shown in panel (III), in some embodiments, the non-covalent linkage is to an avidin protein ( 920 ) bound to a first bis-biotin moiety ( 922 ) and a second bis-biotin moiety ( 924 ) is formed by In some embodiments, the functionalized terminal end ( 902 ) comprises a first bis-biotin moiety ( 922 ) and the complementary functional moiety ( 904 ) comprises a second bis-biotin moiety ( 924 ) do. In some embodiments, functionalized terminal end ( 902 ) comprises an avidin protein ( 920 ) before being contacted with complementary functional moiety ( 904 ). In some embodiments, complementary functional moiety ( 904 ) comprises an avidin protein ( 920 ) prior to being contacted with functionalized terminal end ( 902 ).

In some embodiments, the functionalized terminal end ( 902 ) comprises a first bis-biotin moiety ( 922 ) and a water soluble moiety, wherein the water soluble moiety comprises a first bis-biotin moiety ( 922 ) and a peptide ( 900 ) of amino acids (eg, terminal amino acids). Water-soluble moieties are described in detail elsewhere herein.

Protein sequencing process

Aspects of the present disclosure also involve methods of protein sequencing and identification, methods of protein sequencing and identification, methods of amino acid identification, and compositions, systems, and apparatus for performing such methods. Such protein sequencing and confirmation is, in some embodiments, performed on the same instrument that performs sample preparation and/or genome sequencing, described in more detail herein. In some aspects, methods of determining the sequence of a target protein are described. In some embodiments, a target protein is enriched prior to determining the sequence of the target protein (e.g., enriched using an electrophoretic method, eg, affinity SCODA). In some aspects, a sample (e.g., a plurality of proteins (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 50, or more) are described. In some embodiments, a sample is prepared as described herein prior to determining the sequence of a target protein or plurality of proteins present in the sample (eg, lysed, purified, fragmented, and/or enriched for target protein). In some embodiments, the target protein is an enriched target protein (eg, enriched using an electrophoretic method, eg, affinity SCODA).

In some embodiments, the present disclosure provides methods for sequencing and/or identifying individual proteins in a sample comprising a plurality of proteins by identifying one or more types of amino acids in the proteins from a mixture. In some embodiments, one or more amino acids (e.g., terminal amino acids) of a protein are labeled (e.g., directly or indirectly, e.g., using a binding agent), and the relative position of the labeled amino acids in the protein. is determined In some embodiments, the relative position of amino acids in a protein is determined using a series of amino acid labeling and cleavage steps. In some embodiments, the relative position of a labeled amino acid in a protein is measured by translocating the labeled protein through a pore (e.g., a protein channel) and through the pore to determine the relative position of the labeled amino acid in the protein molecule. It can be determined without removing the amino acid from the protein by detecting the signal (eg, Foerster resonance energy transfer (FRET) signal) from the labeled amino acid(s) during translocation.

In some embodiments, terminal amino acids (e.g., the identity of the N-terminal or C-terminal amino acid) is determined before the terminal amino acid is removed and the identity of the next amino acid at the terminal end is evaluated; This process can be repeated until multiple consecutive amino acids in the protein have been evaluated. In some embodiments, assessing the identity of an amino acid comprises determining the type of amino acid present. In some embodiments, determining the type of amino acid is determining the actual amino acid identity (e.g., which of the 20 naturally-occurring amino acids, e.g., using specific binding agents for individual terminal amino acids to determine). However, in some embodiments, assessing the identity of terminal amino acid types may include determining a subset of potential amino acids that may be present at the ends of a protein. In some embodiments, this may be accomplished by determining that an amino acid is not one or more specific amino acids (ie, and thus may be any other amino acid). In some embodiments, this determines which of a specified subset of amino acids can be at the end of a protein (e.g., based on size, charge, hydrophobicity, binding properties) (e.g., of two or more terminal amino acids). (using a binding agent that binds to a specified subset).

In some embodiments, a protein can be digested into a plurality of smaller proteins, and sequence information can be obtained from one or more of these smaller proteins (e.g., by sequentially evaluating the terminal amino acids of the protein and those amino acids). using a method that entails exposing the next amino acid at the terminus by removing

In some embodiments, a protein is sequenced from its amino (N) terminus. In some embodiments, a protein is sequenced from its carboxy (C) terminus. In some embodiments, a first end (eg, N or C terminus) of a protein is immobilized and the other end (eg C or N terminus) is sequenced as described herein.

Sequencing a protein as used herein refers to determining sequence information about a protein. In some embodiments, this may involve determining the identity of each sequential amino acid for part (or all) of a protein. In some embodiments, this may involve determining the identity of a fragment (eg, a fragment of a target protein or a fragment of a sample comprising a plurality of proteins). In some embodiments, this includes assessing the identity of a subset of amino acids in a protein (eg, and determining the relative position of one or more types of amino acids without determining the identity of each amino acid in the protein). may entail In some embodiments amino acid content information can be obtained from a protein without directly determining the relative positions of different types of amino acids in the protein. Amino acid content alone can be used to infer the identity of proteins present (eg, by comparing amino acid content to a database of protein information and determining which protein(s) have the same amino acid content).

In some embodiments, sequence information for a target protein or a plurality of protein fragments obtained from a sample comprising a plurality of proteins (eg, via enzymatic and/or chemical cleavage) is the target protein or plurality present in the sample. can be analyzed to reconstruct or infer the sequence of the protein of Thus, in some embodiments, one or more types of amino acids are identified by detecting luminescence of one or more labeled affinity reagents that selectively bind to one or more types of amino acids. In some embodiments, one or more types of amino acids are identified by detecting the luminescence of a labeled protein.

In some embodiments, the present disclosure provides a composition, apparatus for sequencing a protein by identifying over time a series of amino acids present at the termini of the protein (eg, by iterative detection and cleavage of amino acids at the termini). , and methods are provided. In still other embodiments, the present disclosure provides compositions, devices, and methods for sequencing proteins by determining the labeled amino content of the protein and comparing it to a reference sequence database.

In some embodiments, the present disclosure provides compositions, devices, and methods for sequencing a protein by sequencing a plurality of fragments of the protein. In some embodiments, sequencing a protein comprises combining sequence information for a plurality of protein fragments to identify and/or determine a sequence for the protein. In some embodiments, combining sequence information may be performed by computer hardware and software. The methods described herein can allow a set of related proteins, such as an organism's entire proteome, to be sequenced. In some embodiments, a plurality of single molecule sequencing reactions are performed in parallel (eg, on a single chip or cartridge) according to aspects of the present disclosure. For example, in some embodiments, a plurality of single molecule sequencing reactions are each performed in separate sample wells on a single chip or cartridge.

In some embodiments, the methods provided herein can be used for sequencing and identification of individual proteins in a sample comprising a plurality of proteins. In some embodiments, the present disclosure provides methods for uniquely identifying individual proteins in a sample comprising a plurality of proteins. In some embodiments, individual proteins are detected in a mixed sample by determining partial amino acid sequences of the proteins. In some embodiments, a partial amino acid sequence of a protein is within a contiguous stretch of approximately 5 to 50, 10 to 50, 25 to 50, 25 to 100, or 50 to 100 amino acids. While not wishing to be bound by any particular theory, it is expected that most human proteins can be identified using incomplete sequence information by reference to proteomic databases. For example, simple modeling of the human proteome has shown that approximately 98% of proteins can be uniquely identified by detecting only 4 types of amino acids within a stretch of 6 to 40 amino acids (see, e.g., Swaminathan , et al. PLoS Comput Biol. 2015, 11(2):e1004080] and [Yao, et al. Phys. Biol. 2015, 12(5):055003). Thus, a sample comprising multiple proteins can be fragmented (eg, chemically digested, enzymatically digested) into short protein fragments of approximately 6 to 40 amino acids, and sequencing of this protein-based library It will reveal the identity and abundance of each of the proteins present in the original sample. Compositions and methods for identifying proteins by selective amino acid labeling and determining partial sequence information are described in detail in US Patent Application Serial No. 15/510,962, filed September 15, 2015, entitled "Single Molecule Peptide Sequencing" , which is incorporated herein by reference in its entirety.

Sequencing according to the present disclosure, in some aspects, involves proteins (e.g., on the surface of a solid support, e.g., a chip or cartridge, e.g., in a sequencing device or module as described herein) of a substrate. , target protein). In some embodiments, a protein may be immobilized on a surface of a sample well on a substrate (eg, on a bottom surface of a sample well). In some embodiments, the N-terminal amino acid of the protein is immobilized (eg, attached to a surface). In some embodiments, the C-terminal amino acid of the protein is immobilized (e.g., attached to the surface). In some embodiments, one or more non-terminal amino acids are immobilized (eg, attached to a surface). The immobilized amino acid(s) may be attached using any suitable covalent or non-covalent linkage, for example as described in this disclosure. In some embodiments, the plurality of proteins are present in a plurality of sample wells (e.g., with one protein attached to a surface, eg, a bottom surface, of each sample well), e.g., in an array of sample wells on a substrate. attached

In some embodiments, the identity of a terminal amino acid (eg, an N-terminal or C-terminal amino acid) is determined, then the terminal amino acid is removed, and the identity of the next amino acid at the terminal end is determined. This process can be repeated until a plurality of contiguous amino acids in the protein have been determined. In some embodiments, determining the identity of an amino acid comprises determining the type of amino acid present. In some embodiments, determining the type of amino acid is the actual amino acid identity, for example by determining which of the 20 naturally-occurring amino acids is a terminal amino acid (eg, using a binding agent specific for the individual terminal amino acid). includes determining In some embodiments, the type of amino acid is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, selected from tyrosine and valine. In some embodiments, determining the identity of a terminal amino acid type may include determining a subset of potential amino acids that may be present at the end of a protein. In some embodiments, this may be accomplished by determining that an amino acid is not one or more specific amino acids (and thus may be any other amino acid). In some embodiments, this determines which of a specified subset of amino acids can be at the end of a protein (e.g., based on size, charge, hydrophobicity, post-translational modifications, binding properties) (e.g., two (using a binding agent that binds to a specified subset of the above terminal amino acids).

In some embodiments, assessing the identity of terminal amino acid types comprises determining which amino acids contain post-translational modifications. Non-limiting examples of post-translational modifications include acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, four dylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, sumoylation, and ubiquitination.

In some embodiments, a protein or proteins can be digested into a plurality of smaller proteins, and sequence information can be obtained from one or more of these smaller proteins (e.g., by sequentially evaluating the terminal amino acids of the protein, using a method that involves removing that amino acid to expose the terminus of the next amino acid).

In some embodiments, sequencing of a protein molecule comprises at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 , at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more) amino acids. In some embodiments, at least two amino acids are contiguous amino acids. In some embodiments, at least two amino acids are non-adjacent amino acids.

In some embodiments, sequencing of a protein molecule comprises less than 100% (e.g., less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, 70%) of all amino acids in the protein molecule. less than, 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). For example, in some embodiments, sequencing of a protein molecule includes identification of less than 100% of one type of amino acid in a protein molecule (eg, identification of a portion of all amino acids of one type in a protein molecule). . In some embodiments, sequencing of a protein molecule comprises identifying less than 100% of each type of amino acid in the protein molecule.

In some embodiments, sequencing of a protein molecule comprises at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 or more types of identification.

A non-limiting example of protein sequencing by repetitive terminal amino acid detection and truncation is shown in FIG. 14A . In some embodiments, protein sequencing comprises providing a protein ( 1000 ) immobilized via a linking group ( 1002 ) to a surface ( 1004 ) of a solid support (e.g., attached to a bottom or sidewall surface of a sample well) do. In some embodiments, linking group ( 1002 ) is formed by a covalent or non-covalent linkage between the functionalized terminal end of protein ( 1000 ) and a complementary functional moiety on surface ( 1004 ). For example, in some embodiments, linking group ( 1002 ) is a biotin moiety of protein ( 1000 ) (eg, functionalized according to the present disclosure) and the avidin protein of the surface ( 1004 ). In some embodiments, linking group ( 1002 ) comprises a nucleic acid.

In some embodiments, protein ( 1000 ) is immobilized to surface ( 1004 ) via a functionalization moiety at one terminal end such that the other terminal end is free for detection and cleavage of terminal amino acids in a sequencing reaction. Thus, in some embodiments, reagents used in certain protein sequencing reactions preferentially interact with terminal amino acids at the non-immobilized (eg, free) end of the protein ( 1000 ). In this way, the protein ( 1000 ) remains immobilized over repeated cycles of detection and cleavage. To this end, in some embodiments, linker 1002 can be designed according to a desired set of conditions used for detection and cleavage, for example to limit detachment of protein 1000 from surface 1004 . have. Suitable linker compositions and techniques for functionalizing proteins (eg, which can be used to immobilize proteins to surfaces) are described in detail elsewhere herein.

In some embodiments, as shown in FIG. 14A , protein sequencing can proceed by (1) contacting the protein ( 1000 ) with one or more amino acid recognition molecules that associate with one or more types of terminal amino acids. As shown, in some embodiments, a labeled amino acid recognition molecule ( 1006 ) interacts with a protein ( 1000 ) by associating with a terminal amino acid.

In some embodiments, the method further comprises identifying amino acids (terminal amino acids) of the protein ( 1000 ) by detecting labeled amino acid recognition molecules ( 1006 ). In some embodiments, detecting comprises detecting luminescence from a labeled amino acid recognition molecule ( 1006 ). In some embodiments, the luminescence is uniquely associated with the labeled amino acid recognition molecule ( 1006 ), and the luminescence is associated with the type of amino acid to which the labeled amino acid recognition molecule ( 1006 ) selectively binds. Thus, in some embodiments, the type of amino acid is identified by determining one or more luminescent properties of a labeled amino acid recognition molecule ( 1006 ).

In some embodiments, protein sequencing proceeds by (2) removing terminal amino acids by contacting protein ( 1000 ) with exopeptidase ( 1008 ), which binds to and cleave terminal amino acids of protein (1000). Upon removal of terminal amino acids by exopeptidase ( 1008 ), protein sequencing proceeds by (3) subjecting the protein ( 1000 ) (with n-1 amino acids) to additional cycles of terminal amino acid recognition and cleavage. In some embodiments, steps (1) to (3) occur in the same reaction mixture, eg, as in a dynamic peptide sequencing reaction. In some embodiments, steps (1) to (3) may be performed using other methods known in the art, such as peptide sequencing by Edman digestion.

Edman degradation involves repeated cycles of modifying and truncating the terminal amino acids of a protein, where each successively truncated amino acid is identified to determine the protein's amino acid sequence. Referring to FIG. 14A , peptide sequencing by conventional Edman digestion can be performed by (1) contacting a protein ( 1000 ) with one or more amino acid recognition molecules that selectively bind to one or more types of terminal amino acids. In some embodiments, step (1) further comprises removing any one or more labeled amino acid recognition molecules that do not selectively bind to protein ( 1000 ). In some embodiments, step (2) is the terminal amino acid (eg, PITC) of the protein ( 1000 ) by contacting the terminal amino acid with an isothiocyanate (eg, PITC) to form an isothiocyanate-modified terminal amino acid. , free terminal amino acids). In some embodiments, isothiocyanate-modified terminal amino acids are more susceptible to removal by cleavage reagents (eg, chemical or enzymatic cleavage reagents) than unmodified terminal amino acids.

In some embodiments, Edman degradation is performed by (2) removing terminal amino acids by contacting the protein ( 1000 ) with an exopeptidase ( 1008 ) that specifically binds to and cleaves isothiocyanate-modified terminal amino acids. It goes on. In some embodiments, exopeptidase ( 1008 ) comprises a modified cysteine protease. In some embodiments, the exopeptidase ( 1008 ) is a modified cysteine protease, such as from Trypanosoma cruzi. cysteine proteases (see, e.g., Borgo, et al. (2015) Protein Science 24:571-579]). In yet another embodiment, step (2) comprises removing terminal amino acids by subjecting the protein ( 1000 ) to chemical (eg, acidic, basic) conditions sufficient to cleave the isothiocyanate-modified terminal amino acids. include In some embodiments, Edman degradation proceeds by washing the protein ( 1000 ) after (3) terminal amino acid cleavage. In some embodiments, washing comprises removing exopeptidase ( 1008 ). In some embodiments, washing comprises restoring the protein ( 1000 ) to neutral pH conditions (eg, after chemical cleavage by acidic or basic conditions). In some embodiments, sequencing by Edman decomposition comprises repeating steps (1) to (3) for a plurality of cycles.

In some embodiments, peptide sequencing may be performed in a dynamic peptide sequencing reaction. In some embodiments, referring again to FIG. 10A , the reagents required to perform steps (1) and (2) are combined within a single reaction mixture. For example, in some embodiments, steps (1) and (2) can be performed without exchanging one reaction mixture for another and without washing steps as in conventional Edman digests. Thus, in this embodiment, a single reaction mixture comprises a labeled amino acid recognition molecule ( 1006 ) and an exopeptidase ( 1008 ). In some embodiments, the exopeptidase ( 1008 ) is present in the mixture at a concentration less than that of the labeled amino acid recognition molecule ( 1006 ). In some embodiments, an exopeptidase ( 1008 ) binds a protein ( 1000 ) with a binding affinity that is less than that of a labeled amino acid recognition molecule ( 1006 ).

In some embodiments, dynamic protein sequencing is performed in real time by evaluating binding interactions of terminal amino acids with labeled amino acid recognition molecules and cleavage reagents (eg, exopeptidases). 14B shows an example of a method of sequencing in which discrete combining events generate signal pulses of signal output. The inset panel of FIG. 14B (left) illustrates the general scheme of real-time sequencing by this approach. As shown, the labeled amino acid recognition molecule associates with (e.g., binds to) and dissociates from a terminal amino acid (represented herein as phenylalanine), resulting in a series of signal outputs that can be used to identify terminal amino acids. generates a pulse of In some embodiments, a series of pulses provides a pulsing pattern (eg, a characteristic pattern) by which the identity of the corresponding terminal amino acid can be diagnosed.

As further shown in the inset panel of FIG . 14B (left), in some embodiments, the sequencing reaction mixture further comprises an exopeptidase. In some embodiments, the exopeptidase is present in the mixture at a concentration less than that of the labeled amino acid recognition molecule. In some embodiments, an exopeptidase exhibits broad specificity such that it cleave most or all types of terminal amino acids. Thus, a dynamic sequencing approach may involve monitoring recognition molecule binding at the end of a protein over the course of a degradation reaction catalyzed by exopeptidase cleavage activity.

14B further shows the progression of signal output intensity over time (right panel). In some embodiments, terminal amino acid cleavage by the exopeptidase(s) occurs at a lower frequency than binding pulses of labeled amino acid recognition molecules. In this way, the amino acids of a protein can be counted and/or identified in a real-time sequencing process. In some embodiments, one type of amino acid recognition molecule may associate with more than one type of amino acid, wherein the different characteristic patterns correspond to the association of one type of labeled amino acid recognition molecule with different types of terminal amino acids. . For example, in some embodiments, different characteristic patterns (as exemplified by each of phenylalanine (F, Phe), tryptophan (W, Trp), and tyrosine (Y, Tyr)) are labeled over the course of degradation. amino acid recognition molecules (e.g., ClpS protein) and the association of different types of terminal amino acids. In some embodiments, multiple labeled amino acid recognition molecules can be used, each capable of associating a different subset of amino acids.

In some embodiments, dynamic peptide sequencing is performed by observing different association events, e.g., between an amino acid recognition molecule and an amino acid at the terminal end of a peptide, wherein each association event lasts for a duration of time. to generate a change in the magnitude of a signal, eg, a luminescent signal. In some embodiments, observing different association events, eg, association events between an amino acid recognition molecule and an amino acid at the terminal end of a peptide, can be performed during the peptide degradation process. In some embodiments, a transition from one characteristic signal pattern to another is indicative of amino acid cleavage (eg, amino acid cleavage resulting from peptidic degradation). In some embodiments, amino acid truncation refers to the removal of at least one amino acid from the end of a protein (eg, removal of at least one terminal amino acid from a protein). In some embodiments, amino acid cleavage is determined by inference based on the duration of time between characteristic signal patterns. In some embodiments, amino acid cleavage is determined by detecting a change in signal produced by the association of a labeled cleavage reagent with an amino acid at the terminus of a protein. As amino acids are sequentially cleaved from the ends of the protein during degradation, a series of magnitude changes, or series of signal pulses, are detected.

In some embodiments, signal pulse information can be used to identify amino acids based on characteristic patterns in a series of signal pulses. In some embodiments, the characteristic pattern comprises a plurality of signal pulses, each signal pulse comprising a pulse duration. In some embodiments, a plurality of signal pulses may be characterized by a summary statistic (eg, mean, median, time resolved constant) of a distribution of pulse durations in a characteristic pattern. In some embodiments, the average pulse duration of the characteristic pattern is from about 1 millisecond to about 10 seconds (e.g., from about 1 ms to about 1 s, from about 1 ms to about 100 ms, from about 1 ms to about 10 ms, about 10 ms to about 10 s, about 100 ms to about 10 s, about 1 s to about 10 s, about 10 ms to about 100 ms, or about 100 ms to about 500 ms). In some embodiments, different characteristic patterns corresponding to different types of amino acids in a single protein can be distinguished from each other based on statistically significant differences in summary statistics. For example, in some embodiments, one characteristic pattern is 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) from another characteristic pattern. It is recognized that, in some embodiments, smaller differences in average pulse duration between different characteristic patterns may require a greater number of pulse durations within each characteristic pattern to distinguish one from another with statistical reliability. It should be.

Sequencing device or module

Sequencing of nucleic acids or proteins according to the present disclosure can, in some aspects, be performed using systems that perform single molecule analysis. A system can include a sequencing device or module and an instrument configured to interface with the sequencing device or module. A sequencing device or module may include an array of pixels, where an individual pixel includes a sample well and at least one photodetector. A sample well of the sequencing device or module may be formed on or through a surface of the sequencing device or module and configured to receive a sample placed on the surface of the sequencing device or module. In some embodiments, a sample well is a cartridge that can be inserted into a device (e.g., Disposable or single-use cartridges). Collectively, the sample wells can be considered an array of sample wells. The plurality of sample wells may have a suitable size and shape such that at least a portion of the sample wells accommodates a sample comprising a single target molecule or a plurality of molecules (eg, target nucleic acids or target proteins). In some embodiments, the number of molecules in a sample well is such that some sample wells are 1 molecule (e.g., target nucleic acid or target protein), while others may be distributed among the sample wells of a sequencing device or module to contain zero, two, or multiple molecules.

In some embodiments, a sequencing device or module is positioned to receive a sample comprising a target molecule or plurality of molecules (eg, a target nucleic acid or target protein) from a sample preparation device or module. In some embodiments, a sequencing device or module is directly connected to a sample preparation device or module (e.g., physically attached) or indirectly connected.

Excitation light is provided to the sequencing device or module from one or more light sources external to the sequencing device or module. An optical component of the sequencing device or module may receive excitation light from a light source, direct the light toward an array of sample wells of the sequencing device or module, and illuminate an illuminated area within the sample wells. In some embodiments, a sample well may have a configuration that allows a sample comprising the target molecule or plurality of molecules to be held proximate to the surface of the sample well, which allows for transmission of excitation light to the sample well and the target molecule or plurality of molecules. Detection of emitted light from a sample containing the molecule can be facilitated. A target molecule or a sample comprising a plurality of molecules positioned within the illumination area may emit emission light in response to being illuminated by 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 through illumination with an excitation light. Emitted light emitted by the target molecule or sample comprising the plurality of molecules may then be detected by one or more photodetectors in pixels corresponding to sample wells having the sample comprising the target molecule or plurality of molecules being analyzed. Multiple sample wells may be analyzed in parallel when performed across an array of sample wells, which may range in number from approximately 10,000 pixels to 1,000,000 pixels according to some embodiments.

A sequencing device or module may include an optical system for receiving excitation light and directing the excitation light among the array of sample wells. The optical system may include one or more grating couplers configured to couple the excitation light to the sequencing device or module and to direct the excitation light to other optical components. The optical system may include an optical component that directs the excitation light from the grating coupler towards the sample well array. Such optical components may include optical splitters, optical combiners, and waveguides. In some embodiments, one or more optical splitters may couple the excitation light from the grating coupler and pass the excitation light to at least one of the waveguides. According to some embodiments, the optical splitter can have an arrangement that allows delivery of the excitation light to be substantially uniform across all waveguides such that each of the waveguides receives substantially similar amounts of excitation light. Such embodiments may improve the performance of a sequencing device or module by improving the uniformity of the excitation light received by a sample well of the sequencing device or module. Examples of suitable components for coupling excitation light to sample wells and/or directing emission light to a photodetector, eg, included in a sequencing device or module, are those entitled "Probing, Detecting, and Analyzing Molecules". U.S. Patent Application Serial No. 14/821,688, filed Aug. 7, 2015, "Integrated Device for Searching, Detecting, and Analyzing Molecules," and Nov. 17, 2014, entitled "Integrated Device with External Light Source for Searching, Detecting, and Analyzing Molecules." U.S. Patent Application No. 14/543,865, filed on 14/7, both of which are incorporated herein by reference in their entirety. Examples of suitable grating couplers and waveguides that can be implemented in a sequencing device or module are described in U.S. Patent Application Serial No. 15/844,403, filed on December 15, 2017, entitled "Optical Couplers and Waveguide Systems," which Its entirety is hereby incorporated by reference.

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

A component located from a sequencing device or module can be used to position and align an excitation source to the sequencing device or module. Such components may include lenses, mirrors, prisms, windows, diaphragms, attenuators, and/or optical components including optical fibers. Additional mechanical components allowing control of one or more alignment components may be included in the device. These 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 Serial No. 15/161,088, entitled "Pulsed Lasers and Systems," filed May 20, 2016, incorporated herein by reference in its entirety. included as Another example of a beam-steering module is described in U.S. Patent Application Serial No. 15/842,720, filed on December 14, 2017, entitled "Compact Beam Shaping and Steering Assembly," which is incorporated herein by reference in its entirety. included as Additional examples of suitable excitation sources are described in U.S. Patent Application Serial No. 14/821,688, filed August 7, 2015, entitled "Integrated Apparatus for Searching, Detecting, and Analyzing Molecules," which is incorporated herein by reference in its entirety. incorporated herein by reference.

Photodetector(s) positioned with individual pixels of a sequencing device or module may be configured and positioned to detect emission light from corresponding sample wells of the pixels. Examples of suitable photodetectors are described in US patent application Ser. No. 14/821,656, filed Aug. 7, 2015, entitled "Integrated Apparatus for Temporal Binning of Accepted Photons," which is incorporated herein by reference in its entirety. included as In some embodiments, a sample well and its respective photodetector(s) may be aligned along a common axis. In this way, the photodetector(s) can overlap sample wells within a pixel.

Characteristics of the detected emitted light may provide an indication for identifying a marker associated with the emitted light. These characteristics may be of any suitable type, including the time of arrival 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. can include In some embodiments, the photodetector may have a configuration that allows detection of one or more timing characteristics (eg, luminescence lifetime) associated with the emitted light of the sample. The photodetector is capable of detecting a distribution of photon arrival times after a pulse of excitation light propagates through a sequencing device or module, the distribution of arrival times determining a timing characteristic of the sample's emission light (eg, as a proxy for luminescence lifetime). Can provide instructions. In some embodiments, one or more photodetectors provide an indication of the probability of emitted light (eg, luminescence intensity) emitted by the marker. In some embodiments, a plurality of photodetectors may be sized and arranged to capture a spatial distribution of emitted light. An output signal from one or more photodetectors can then be used to distinguish a marker from among a plurality of markers, where the plurality of markers can be used to identify a sample within a sample. In some embodiments, a sample can be excited by multiple excitation energies, and emitted light emitted by the sample in response to multiple excitation energies and/or timing characteristics of the emitted light can distinguish a marker from a plurality of markers. .

In operation, parallel analysis of samples within the sample wells is performed by exciting some or all of the samples within the wells using an excitation light and detecting a signal from the sample emission with a photodetector. Light emitted from the sample can be detected by a corresponding photodetector and converted into at least one electrical signal. Electrical signals may be transmitted along conductive lines in circuitry of the sequencing device or module, which may be coupled to equipment connected to the sequencing device or module. The electrical signal may then be processed and/or analyzed. Processing and/or analysis of the electrical signals may occur on a suitable computing device located on or external to the device.

The instrument may include a user interface for controlling operation of the instrument and/or sequencing device or module. The user interface may be configured to allow a user to enter information into the device, such as commands and/or settings used to control functions of the device. In some embodiments, a user interface may include buttons, switches, dials, and/or a microphone for voice commands. The user interface may allow the user to receive feedback on the performance of the device and/or sequencing device or module, such as pertinent alignments and/or information obtained by a readout signal from a photodetector on the sequencing device or module. In some embodiments, the user interface may provide feedback using a speaker that provides acoustic feedback. In some embodiments, the user interface may include indicator lights and/or display screens to provide visual feedback to the user.

In some embodiments, an instrument or device described herein may include a computer interface configured to interface with 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, a computing device may be a server (eg, a cloud-based server) accessible over a wireless network through a suitable computer interface. A computer interface may facilitate communication of information between a device and a computing device. Input information for controlling and/or configuring the device may be provided to the computing device and transmitted to the device via a computer interface. Output information generated by the device may be received by the computing device through a computer interface. The output information may include feedback regarding the performance of the instrument, the performance of the sequencing device or module, and/or data generated from the read signal of the photodetector.

In some embodiments, an instrument may include a processing device configured to analyze data received from one or more photodetectors of a sequencing device or module and/or transmit control signals to the excitation source(s). In some embodiments, a processing device includes a general purpose processor, and/or a special-adapted processor (e.g., a central processing unit (CPU), such as one or more microprocessor or microcontroller cores, a field-programmable gate array (FPGA)) , 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 device'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 only by the sequencing device or processing device of the module.

According to some embodiments, an instrument configured to analyze a sample comprising a target molecule or a plurality of molecules based on luminescent emission characteristics is a difference in luminescence lifetime and/or intensity between different luminescent molecules, and/or the same in different environments. Differences between lifetimes and/or intensities of luminescent molecules can be detected. The inventors have recognized and appreciated that differences in luminescence emission lifetimes can be used to discriminate between the presence or absence of different luminescent molecules and/or between different environments or conditions in which the luminescent molecules are treated. In some cases, identifying light emitting molecules based on lifetime (eg, rather than emission wavelength) can simplify aspects of the system. By way of example, wavelength-identifying optics (such as wavelength filters, dedicated detectors for each wavelength, dedicated pulsed light sources at different wavelengths, and/or diffractive optics) can identify light emitting molecules based on their lifetime in a number of cases. may be reduced or eliminated. In some cases, a single pulsed light source operating at a single characteristic wavelength can be used to excite different luminescent molecules that emit 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 identify different luminescent molecules emitting in the same wavelength region may be less complex to operate and maintain, and may be more compact. and can be manufactured at a lower cost.

While an assay system based on luminescence lifetime analysis may have certain benefits, the amount of information and/or accuracy of detection obtained by the assay system may be increased by allowing for 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, luminescence intensity can additionally or alternatively be used to differentiate between different luminescent labels. For example, some luminescent labels may emit at significantly different intensities or have significant differences in their probabilities of excitation (eg, differences of at least about 35%), even though their decay rates may be similar. By referencing the binned signals for the measured excitation light, it may be possible to distinguish different luminescent labels based on intensity levels.

According to some embodiments, different luminescent lifetimes can be distinguished with a photodetector configured to time-bin the luminescent emission events after excitation of the luminescent label. Time binning can occur during a single charge-accumulation cycle for the photodetector. A charge-accumulation cycle is the interval between read events in which light-generated carriers accumulate in bins of a time-binned photodetector. An example of a time-binning photodetector is described in US patent application Ser. No. 14/821,656, filed Aug. 7, 2015, entitled "Integrated Apparatus for Temporal Binning of Received Photons," which is incorporated herein in its entirety. is incorporated by reference in In some embodiments, the time-binned photodetector can generate charge carriers in the photon absorption/carrier generation region and directly transfer the charge carriers to charge carrier storage bins in the charge carrier storage region. In such an embodiment, the time-binned photodetector may not include a vesicle glide/trap region. Such time-binning photodetectors may be referred to as "directly binning pixels". An example of a time-binning photodetector with direct binning pixels is described in U.S. Patent Application Serial No. 15/852,571, filed on December 22, 2017, entitled "Integrated Photodetector with Direct Binning Pixels", which Its entirety is hereby incorporated by reference.

In some embodiments, different numbers of fluorophores of the same type are used to target molecules (e.g., a target nucleic acid or target protein) or a plurality of molecules present in a sample (e.g., multiple nucleic acids or multiple proteins). 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. Because of the different number of fluorophores, there may be different excitation and fluorophore emission probabilities associated with different molecules. For example, there may be more emission events for the second labeled molecule during the signal accumulation interval such that the apparent intensity of the bin is significantly higher for the first labeled molecule.

The inventors have recognized and appreciated that distinguishing nucleic acids or proteins based on fluorophore decay rate and/or fluorophore intensity may allow for simplification of optical excitation and detection systems. For example, optical excitation can be performed with a single-wavelength source (eg, a source that produces one characteristic wavelength rather than multiple sources or sources operating at multiple different characteristic wavelengths). Additionally, wavelength discriminating optics and filters may not be required in the detection system. Alternatively, a single photodetector can be used for each sample well to detect emission from a different fluorophore. The phrase "characteristic wavelength" or "wavelength" is used to refer to a central or predominant wavelength with a limited bandwidth of radiation. For example, the limited bandwidth of radiation may include a center or peak wavelength within a 20 nm bandwidth output by a pulsed light source. In some cases, “characteristic wavelength” or “wavelength” may be used to refer to a peak wavelength within the total bandwidth of radiation output by a source.

Combined sample preparation and sequencing device

In some embodiments, a device herein comprising a sample preparation module further comprises a sequencing module. In some embodiments, a device comprising a sample preparation module and a sequencing module carries a sequencing chip or cartridge embedded into a sample preparation cartridge such that the two cartridges comprise a single, non-removable consumable. In some embodiments, the sequencing chip or cartridge requires consumable supporting electronics (eg, wirebond, PCB substrate with electrical contact). The consumable support electronics may be in direct physical contact with the sequencing chip or cartridge. In some embodiments, a sequencing chip or cartridge requires an interface for peristaltic pump, temperature control, and/or electrophoretic contact. These interfaces can allow precise geometric registration for many electrical contacts and laser alignment. In some embodiments, different sections of a chip or cartridge may include different temperatures, physical forces, electrical interfaces of varying voltages and currents, vibrations, and/or competing alignment requirements. In some embodiments, disparate instrument sub-systems associated with a sample preparation or sequencing module must be in close proximity to share resources. In some embodiments, a device comprising a sample preparation module and a sequencing module is hands-free (ie, can be used without the use of hands).

In some embodiments, a device comprising a sample preparation module and a sequencing module is an average generated using a control method (eg, a sage bluepipin method, a manual method (eg, a manual bead-based size selection method)) Produce target nucleic acids with an average read-length for downstream sequencing applications that are longer than the read-length (e.g., enriched or purified). In some embodiments, the sample preparation device is configured to have a voltage of at least 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2400, 2500, 2600; Produce target nucleic acids with average read-lengths for sequencing that include lengths of 2700, 2800, 2900, or 3000 nucleotides. In some embodiments, the sample preparation device has a range of 700 to 3000, 1000 to 3000, 1000 to 2500, 1000 to 2400, 1000 to 2300, 1000 to 2200, 1000 to 2100, 1000 to 2000, 1000 to 1900, 1000 to 1800 to 1700, 1000 to 1600, 1000 to 1500, 1000 to 1400, 1000 to 1300, 1000 to 1200, 1500 to 3000, 1500 to 2500, 1500 to 2000, or 2000 to 3000 nucleotides in length. A target nucleic acid having read-length is produced.

In some embodiments, an apparatus comprising a sample preparation module and a sequencing module shortens the time between initiation of sample preparation and detection of a target molecule contained within the sample than a control or traditional method (eg, the sage bluepipin method followed by sequencing). allowed time In some embodiments, an apparatus comprising a sample preparation module and a sequencing module is used in less time (eg, 2x, 3x, 4x) than a control or traditional method (eg, the sage bluepipin method followed by sequencing). , 5-fold, or 10-fold less time) can be used to detect the target molecule.

In some embodiments, a device comprising a sample preparation module and a sequencing module is capable of detecting a target molecule with a lower input of a sample than a control or traditional method (eg, the sage bluepipin method followed by sequencing). In some embodiments, a device of the present disclosure is capable of carrying as little as 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, or 1 μg of sample (e.g., biological sample) is requested. In some embodiments, a device of the present disclosure may be used in 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 130 μL, 150 μL, Samples as small as 175 μL, 200 μL, 225 μL, or 250 μL (eg, biological samples, such as blood) are required.

device or module

In some embodiments, a device or module (e.g., a sample preparation device; a sequencing device; a combined sample preparation and sequencing device) can in some cases small volume(s) of fluid with a well-defined fluid flow resolution, and well It is configured to accurately transport at a defined flow rate. In some embodiments, the device or module is configured to transport a fluid at a flow rate of at least 0.1 μL/s, at least 0.5 μL/s, at least 1 μL/s, at least 2 μL/s, at least 5 μL/s, or more do. In some embodiments, a device or module herein provides a flow rate of 100 μL/s or less, 75 μL/s or less, 50 μL/s or less, 30 μL/s or less, 20 μL/s or less, 15 μL/s or less, or less It is configured to transport a fluid at a flow rate of Combinations of these ranges are possible. For example, in some embodiments, a device or module herein is configured to transport a fluid at a flow rate of greater than or equal to 0.1 μL/s and less than or equal to 100 μL/s, or greater than or equal to 5 μL/s and less than or equal to 15 μL/s. For example, in certain embodiments, the systems, devices, and modules herein have fluid flow resolution on the order of tens of microliters or hundreds of microliters. Additional discussion of fluid flow resolution is described elsewhere herein. In certain embodiments, systems, devices, and modules are configured to transport small volumes of fluid through at least a portion of a cartridge.

Some aspects relate to the placement of pumps and instruments that include rollers (eg, in combination with a crank-and-rocker mechanism). Another aspect is a cartridge comprising a channel (eg, a microchannel) having a cross-sectional shape (eg, a substantially triangular shape), a valve, a deep section, and/or a surface layer (eg, a flat elastomeric membrane). It is about. Certain aspects relate to the separation of certain components of a peristaltic pump (eg, rollers) from other components of the pump (eg, pumping lanes). In some cases, certain elements of the appliance (eg, edges of rollers) are selected in such a way that any of a variety of benefits are achieved (eg, through engagement and disengagement) elements of the cartridge (eg, surface layers and specific shape of the channel). In some non-limiting embodiments, certain inventive features and arrangements of instruments, cartridges, and pumps described herein contribute to improved automation of the fluid pumping process (e.g., a number of different due to the use of separate cartridges and convertible rollers containing fluid channels). In some cases, features described herein are useful for handling a relatively high number of different fluids (e.g., for multiplexing with a large number of samples) with a relatively high number of batches using a relatively small number of hardware components. capability (eg, due to the use of separate cartridges having a number of different channels, each of which can be accessible to a roller). As an example, in some cases, the features described herein allow more than one device to be paired with a cartridge to pump more than one lane simultaneously or to use two pumps in one lane for other functionality. . In some cases, the feature contributes to a reduction in required fluid volume and/or a less stringent resistance to roller/channel interaction (e.g., due to the inventive cross-sectional shape of the edges of the channels and/or rollers; and/or due to the use of inventive valves and/or dip sections in the channels). In some cases, the features described herein result in a reduction in the required cleaning of hardware components (eg, due to the separation of the instrument and cartridge of a peristaltic pump). In some embodiments, aspects of the instruments, cartridges, and pumps described herein are useful for preparing samples. For example, some of these aspects may be incorporated into a sample preparation module upstream of a detection module (eg, for analysis/sequencing/confirmation of biologically-derived samples).

In another aspect, a peristaltic pump is provided. In some embodiments, a peristaltic pump comprises a roller and a cartridge, wherein the cartridge comprises a base layer having a surface comprising channels, at least a portion of at least some of the channels having (1) a single apex at the base of the channels; On the surface of the base layer, it has a substantially triangular-shaped cross section with two different vertices, and (2) a surface layer comprising an elastomer configured to substantially seal the surface openings of the channels. Embodiments of peristaltic pumps are further described elsewhere herein.

In some embodiments, a system (eg, pump, device) described herein undergoes a pump cycle. In some embodiments, a pump cycle corresponds to one revolution of the system's crank. In some embodiments, each pump cycle is capable of transporting at least 1 μL, at least 2 μL, at least 4 μL, at least 10 μL, at most 8 μL, and/or at most 6 μL of fluid. Combinations of the above-mentioned ranges are also possible (eg, 1 μL to 10 μL). Other ranges of volume of fluid are also possible.

In some embodiments, the systems described herein have a specific stroke length. In certain embodiments, where each pump cycle can transport on the order of 1 μL to 10 μL of fluid, and/or when the channel dimensions are preferably on the order of 1 mm wide and on the order of 1 mm deep ( eg, as may be machined or molded to reduce channel volume and maintain reasonable tolerances), the stroke length is 10 mm or more, 12 mm or more, 14 mm or more, 20 mm or less, 18 mm or less, and/or or 16 mm or less. Combinations of the above-mentioned ranges are also possible (eg, 10 mm to 20 mm). Other ranges are also possible. As used herein, “stroke length” refers to the distance a roller slides while being engaged with a substrate. In certain embodiments, the substrate comprises a cartridge.

In another aspect, a cartridge is provided. In some embodiments, a cartridge includes a base layer having a surface comprising channels, at least a portion of at least some of the channels having (1) a single vertex at the base of the channels and two other vertices at the surface of the base layer. It has a substantially triangular-shaped cross section, and (2) has a surface layer comprising an elastomer configured to substantially seal the surface openings of the channels. Embodiments of the cartridge are further described elsewhere herein.

In some embodiments, the cartridge includes a base layer. In some embodiments, the base layer has a surface comprising one or more channels. For example, FIG. 8 is a schematic diagram of a cross-sectional view of cartridge 100 along the width of channel 102, in accordance with some embodiments. The illustrated cartridge 100 includes a base layer 104 having a surface 111 that includes channels 102 . In certain embodiments, at least some of the channels are microchannels. For example, in some embodiments, at least a portion of channels 102 are microchannels. In certain embodiments, all channels are microchannels. For example, referring again to FIG. 8 , in certain embodiments, all channels 102 are microchannels. As used herein, the term "channel" will be known to those skilled in the art and may refer to a structure configured to contain and/or transport a fluid. Channels are generally walls; a base (eg, a base coupled to and/or formed from a wall); and surface openings that can be open, covered, and/or sealed in one or more portions of the channels.

As used herein, the term "microchannel" refers to a channel comprising at least one dimension of a size of 1000 micrometers or less. For example, a microchannel may include at least one dimension (eg, width, height) of a size of 1000 micrometers or less (eg, 100 micrometers or less, 10 micrometers or less, 5 micrometers or less). can In some embodiments, a microchannel comprises at least one dimension of 1 micrometer or greater (eg, 2 micrometers or greater, 10 micrometers or greater). Combinations of the above-mentioned ranges are also possible (eg, greater than or equal to 1 micrometer and less than or equal to 1000 micrometers, greater than or equal to 10 micrometers and less than or equal to 100 micrometers). Other ranges are also possible. In some embodiments, the microchannels have a hydrodynamic diameter of 1000 micrometers or less. As used herein, the term "hydrodynamic diameter" (DH) will be known to those skilled in the art and can be determined as DH = 4A/P, where A is the cross-section of the flow of fluid through a channel; P is the perimeter of the cross section (circumference of the cross section of the channel contacted by the fluid).

In some embodiments, at least a portion of at least some channel(s) has a substantially triangular-shaped cross section. In some embodiments, at least a portion of at least some channel(s) has a substantially triangular-shaped cross-section with a single vertex at the base of the channel and two other vertices at the surface of the base layer. Referring again to FIG. 24 , in some embodiments, at least a portion of at least some of the channels 102 have a substantially triangular-shaped cross-section with a single vertex at the base of the channel and two other vertices at the surface of the base layer. have

As used herein, the term "triangle" is used to refer to a shape in which a triangle can be circumscribed or circumscribed such that it approximates or is equivalent to a real shape, but is not purely limited to a triangle. For example, a triangular cross section may include non-zero curvature in one or more portions.

The triangular cross section may include a wedge shape. As used herein, the term "wedge shape" will be 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 shape has an axis of symmetry from the thick end to the thin end. For example, a wedge shape can have a thick end (e.g., the surface opening of a channel), taper to a thin end (e.g., the base of a channel), and have an axis of symmetry from the thick end to the thin end. have.

Additionally, in certain embodiments, a substantially triangular cross section (ie, “v-groove(s)”) may have a variety of aspect ratios. As used herein, the term "aspect ratio" for a v-groove refers to the height-to-width ratio. For example, in some embodiments, the v-groove(s) can 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-mentioned ranges are also possible (eg, 0.1 to 2, 0.2 to 1). Other ranges are also possible.

In some embodiments, at least a portion of at least some channel(s) has a cross section comprising a substantially triangular portion and a second portion opening into the substantially triangular portion and extending substantially below the triangular portion relative to the surface of the channel. In some embodiments, the second portion has a diameter (eg, an average diameter) that is significantly smaller than the average diameter of the substantially triangular segments. Referring again to FIG. 24 , in some embodiments, at least a portion of at least some of the channels 102 includes a substantially triangular portion 101 and a second portion 103 open into the substantially triangular portion 101 , and , with a cross section extending substantially below the triangular portion 101 relative to the surface 105 of the channel, wherein the second portion 103 is significantly smaller than the mean diameter 109 of the substantially triangular portion 101. It has a diameter of 107. In some such cases, the second portion of the channel having a diameter significantly smaller than that of the average diameter of the substantially triangular portion of the channel is a substantially triangular portion accessible to the rollers of the appliance and a deformed portion of the surface layer, but the rollers and a deformed part of the surface layer and a second part inaccessible to the surface layer. For example, referring again to FIG. 24 , according to certain embodiments, substantially triangular portions 101 of channels 102 are accessible to rollers (not shown) and deformed portions of surface layer 106 while , the second portion 103 is inaccessible to the roller and to the deformed portion of the surface layer 106 . In some such cases, a seal with the surface layer 106 cannot be achieved in the portion of the channel 102 with the second portion 103, since the surface layer 106 is such that it substantially encloses the triangular portion 101. This is because, although filling, the fluid can still move freely in the second part 103 even if the second part 103 is otherwise deformed by the roller. In some embodiments, a portion along the length of the channel can have both a substantially triangular portion and a second portion ("deep section"), while a different portion along the length of the channel has only a substantially triangular portion. In some such embodiments, when the instrument (eg, roller) engages a portion having both a substantially triangular portion and a second portion (the deep section), no pumping action is initiated, indicating that the seal with the surface layer because it is not achieved. However, as the instrument engages along the length of the channel, if the instrument deforms the surface layer in a portion of the channel having only a substantially triangular section, the pumping action begins, which in that portion results in a second portion (deep section). ) allows a seal (and consequently differential pressure) to be created. Thus, in some cases, the presence and absence of a dip section along the length of a channel of a cartridge may allow control of a portion of the channel to undergo pumping action upon engagement with an instrument.

Inclusion of such a "deep section" as a second portion of at least a portion of a channel of a cartridge can contribute to any of a variety of potential benefits. For example, this dip section (eg, second portion 103 ) in some cases contributes to a reduction in pump volume in a peristaltic pumping process. In some such cases, the pump volume can be reduced by a factor of 2 or more for higher volume resolution. In some cases, these dip sections can also provide a well-defined starting point for pump volumes that are not determined by where the roller lands on the channel. For example, the interface between a portion of a channel having both a substantially triangular portion and a second portion (the dip section) and a portion of a channel having only a substantially triangular portion, in some cases, serves as a well-defined starting point for the pump volume. can be used, since only the fluid occupying the volume of the latter channel section can be pumped. In some cases, where the roller lands on the channel may have some error associated with it depending on any of a variety of factors, such as cartridge registration. Inclusion of a dip section may, in some cases, reduce or eliminate the fluctuations in pump volume associated with these errors.

As used herein, the average diameter of a 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.

SCODA

SCODA may involve providing a time-varying driving field component that applies a force to a particle in some medium in combination with a time-varying mobility-altering field component that affects the particle's mobility in the medium. The mobility-altering field component is correlated with the driving field component to provide the time-averaged net motion of the particle. SCODA can be applied to cause selected particles to move towards the focus area.

In one embodiment of the SCODA-based purification described herein as electrophoretic SCODA, a time-varying electric field also provides a periodic driving force and drags (or equivalently mobility) can also be changed. For example, DNA molecules have a mobility that depends on the magnitude of the applied electric field as they move through a sieving matrix such as agarose or polyacrylamide. By applying an appropriate periodic electric field pattern to the separating matrix (eg agarose or polyacrylamide gel), a convergent velocity field can be created for every molecule in the gel whose mobility depends on the electric field. Field-dependent mobility is the result of interactions between repetitive DNA molecules and the sieving matrix, and is a common feature of charged molecules with high conformational entropy and high charge-to-mass ratios that migrate through the sieving matrix. Since nucleic acids tend to be the only molecules present in most biological samples with both high conformational entropy and high charge-to-mass ratio, electrophoretic SCODA-based purification has been shown to be highly selective for nucleic acids.

The ability to detect specific biomolecules in a sample has wide applications in the fields of diagnosis and treatment of diseases. Research continues to uncover a number of biomarkers associated with various disorders. Exemplary biomarkers include gene mutations, presence or absence of specific proteins, elevated or decreased expression of specific proteins, elevated or decreased levels of specific RNA, presence of modified biomolecules, and the like. Biomarkers and methods for detecting biomarkers are potentially useful for the diagnosis, prognosis, and monitoring of treatment of a variety of disorders, including cancer, disease, infection, organ failure, and the like.

Differential modification of biomolecules in vivo is an important feature of many biological processes including development and disease progression. One example of differential modification is DNA methylation. DNA methylation involves the addition of methyl groups to nucleic acids. For example, a methyl group can be added at the 5' position on the pyrimidine ring in cytosine. Methylation of cytosines in CpG islands is commonly used in eukaryotes for long-term regulation of gene expression. Abnormal methylation patterns have been implicated in many human diseases including cancer. DNA can also be methylated at nitrogen 6 of the adenine purine ring.

Chemical modification of a molecule, for example by methylation, acetylation or other chemical alteration, can alter the binding affinity of a target molecule and an agent that binds to the target molecule. For example, methylation of cytosine residues increases the binding energy of hybridization compared to unmethylated two strands. The effect is small. A previous study reports an increase in the melting temperature of both strands of approximately 0.7°C per site of methylation in a 16 nucleotide sequence when comparing two strands with both strands unmethylated to two strands with both strands methylated. .

Affinity SCODA

SCODAphoresis is a method of injecting a biomolecule into a gel and preferentially concentrating the nucleic acid or other biomolecule of interest in the center of the gel. SCODA can be applied to, for example, DNA, RNA and other molecules. After concentration, purified molecules can be removed for further analysis. SCODA phoresis-affinity In one specific embodiment of SCODA, a binding site specific to a biomolecule of interest can be immobilized in a gel. In doing so, it is possible to create a non-linear motive response to an electric field for a biomolecule that binds to a specific binding site. One specific application of affinity SCODA is sequence-specific SCODA. Here oligonucleotides can be immobilized in a gel to allow enrichment of DNA molecules complementary to only bound oligonucleotides. All other non-complementary DNA molecules may focus weakly or not at all, and thus may be washed from the gel by application of a small DC bias.

SCODA-based transport first applies a time-varying forcing (i.e. driving) field to induce periodic motion of a particle, and then a time-varying perturbation that periodically changes the particle's drag (or equivalently mobility) over this forcing field. It is a general technique for moving particles through a medium by overlapping fields (i.e., mobility-altering fields). The application of the mobility-altering field is coordinated with the application of the forcing field such that the particle moves more during one part of the forcing cycle than in other parts of the forcing cycle.

By varying the particle's drag (i.e., mobility) at the same frequency as the externally applied force, net drift can be induced with zero time-averaged forcing. Appropriate selection of driving forces and drag coefficients that vary in time and space can create convergent velocity fields in one or two dimensions. The time-varying drag coefficient and driving force can be determined even when the differences between a target molecule and one or more non-target molecules, such as molecules that are differentially modified at one or more positions, or nucleic acids that differ in sequence at one or more bases, are very small. It can be used in practical systems that only specifically enrich (ie preferentially focus) certain molecules.

Affinity matrices can be created by immobilizing agents (ie, probes) that have binding affinity for a target molecule in a medium. Using such a matrix, operating conditions can be selected in which the target molecule transiently binds to the affinity matrix, which has the effect of reducing the overall mobility of the target molecule as it migrates through the affinity matrix. The strength of these transient interactions varies over time, which has the effect of altering the mobility of the target molecule of interest. Therefore, SCODA drift may be generated. This technique is referred to as affinity SCODA and is generally applicable to any target molecule that has affinity for the matrix.

Affinity SCODA can selectively enrich for nucleic acids based on sequence content with single nucleotide resolution. In addition, affinity SCODA can result in different values of k for molecules that have the same DNA sequence but with subtly different chemical modifications, such as methylation. Thus, affinity SCODA can be used to enrich for (i.e., preferentially focus on) molecules that differ subtly in binding energy for a given probe, specifically methylated, unmethylated, hypermethylated, or hypomethylated. can be used to enrich for sequences.

Exemplary media that can be used to perform affinity SCODA include any medium in which molecules of interest can migrate and affinity agents can be immobilized to provide an affinity matrix. In some embodiments, polymeric gels are used, including polyacrylamide gels, agarose gels, and the like. In some embodiments, a microfabricated/microfluidic matrix is used.

Exemplary operating conditions that can be varied to provide the mobility altering field include temperature, pH, salinity, concentration of denaturant, concentration of catalyst, application of an electric field to physically tear the two strands apart, and the like.

Exemplary affinity agents that can be immobilized on a matrix to provide an affinity matrix are nucleic acids having a sequence complementary to a nucleic acid sequence of interest, proteins having different binding affinities for differentially modified molecules, modified or antibodies specific for unmodified molecules, nucleic acid aptamers specific for modified or unmodified molecules, other molecules or chemical agents that bind preferentially to modified or unmodified molecules, and the like.

The affinity agent may be immobilized within the medium in any suitable manner. For example, where the affinity agent is an oligonucleotide, the oligonucleotide can be covalently linked to the medium, the acrydite modified oligonucleotide can be directly incorporated into a polyacrylamide gel, or the oligonucleotide can be physically incorporated into the medium. can be covalently bound to an accompanying bead or other construct; and the like.

When the affinity agent is a protein or antibody, in some embodiments the protein is physically entrained in the medium (eg the protein can be cast directly into an agarose or polyacrylamide gel), covalently coupled to the medium, or a second parent covalently coupled to a bead entrained in the medium (e.g., through the use of cyanogen bromide to couple the protein to an agarose gel), or directly coupled to the medium or to a bead entrained in the medium; capable of being bound to chemoatizing agents (eg hexahistidine tag coupled to NTA-agarose), and the like.

If the affinity agent is a protein, the conditions under which the affinity matrix is prepared and the conditions under which the sample is loaded must be controlled so as not to denature the protein (e.g., the temperature must be kept below a level likely to denature the protein; The concentration of any denaturant in the buffer or in the sample used to prepare the medium or perform SCODA focusing should be kept below a level that would likely denature the protein).

Where the affinity agent is a small molecule that interacts with the molecule of interest, the affinity agent may be covalently coupled to the medium in any suitable manner.

One exemplary embodiment of affinity SCODA is sequence-specific SCODA. In sequence-specific SCODA, the target molecule is or comprises a nucleic acid molecule having a specific sequence, and the affinity matrix contains immobilized oligonucleotide probes complementary to the target nucleic acid molecule. In some embodiments, sequence specific SCODA is used both to isolate a specific nucleic acid sequence from a sample and to isolate and/or detect whether that specific nucleic acid sequence is differentially modified within the sample. In some such embodiments, affinity SCODA is performed under conditions such that both the nucleic acid sequence and the differentially modified nucleic acid sequence are enriched by application of the SCODA field. Contaminant molecules, including nucleic acids with undesired sequences, can be washed from the affinity matrix during SCODA focusing. A wash bias can then be applied in conjunction with a SCODA focusing field to isolate differentially modified nucleic acid molecules, as described below, by preferentially focusing molecules with a higher binding energy to the immobilized oligonucleotide probe.

Example

Embodiments of the present invention are further described with reference to the following examples, which are intended to be illustrative in nature and not limiting.

Example 1 - Use of sample preparation device

Samples of DNA extracted from human blood were prepared using the automated sample preparation device of the present disclosure.

The sample preparation device includes a fluidics module (including a peristaltic pumping system), a temperature control module (to provide temperature and mechanical accuracy), and a user to select any process-specific parameters (e.g., the desired size of the nucleic acid). range, desired degree of homology for target molecule capture, etc.) on the device, and a lid that the user can open to insert a sample preparation cartridge of the present disclosure. The device was powered with a 1000-volt electrode supply. The sample preparation cartridge contained 13 discrete microfluidic channels (or pumping lanes) and was constructed such that it could perform end-to-end sample preparation. The microfluidic channel is designed to make reagents and cartridges possible in an automated sequence: (1) pipette introduction of lysis+combined sample lysis using lysis buffer and subsequent extraction of target DNA; (2) DNA purification; (3) DNA tagging using the transposase Tn5 followed by DNA repair; (4) selection of DNA fragments in a specific size range using nucleic acid capture probes and SCODA; and (5) DNA cleaning.

100 μL of whole human blood was mixed with lysis buffer, proteinase K was incubated at 55° C. for 10 minutes, then mixed with isopropanol; The lysate mixture was then added to the sample port in the sample preparation cartridge, the loaded cartridge was inserted into the sample preparation device, and DNA was extracted. An automated device as described above produced 1.2 μg extracted DNA; 1 μg of that extracted DNA was further processed using the sequential steps described above to generate 530 ng of DNA library at a concentration of 6.5 nM. This purified DNA library produced by the sample preparation device was then subjected to sequencing using a glass sequencing chip.

As a control experiment, 100 μL of whole human blood (from the same sample as above) was manually processed to generate a DNA library for sequencing using traditional DNA extraction and purification techniques.

The present inventors found that sequencing data obtained using a DNA library prepared using an automated sample preparation device was superior in quality (eg, sequencing data obtained using DNA manually prepared using traditional DNA extraction and purification techniques). , as assessed by mean read length) were similar. As shown in Table 3, the automated device produced more total reads than the manual process (72 total reads using the automated process compared to 27 total reads using the manual process) and greater read length ( 1989.0 ± 760.1 base pair read lengths were generated using the automated process compared to 1132.1 ± 324.5 base pair read lengths using the manual process, with significant differences observed between processes in terms of accuracy and GC content of reads generated. There was no difference.

Table 3. Sequencing results from DNA libraries generated from whole human blood.

Example 2 - Use of a Sample Preparation Device to Enrich DNA for Sequencing

E. coli cultured using the automated sample preparation device of the present disclosure. Samples of DNA extracted from E. coli cells were prepared.

The sample preparation device includes a fluidics module (including a peristaltic pumping system), a temperature control module (to provide temperature and mechanical accuracy), and a user to select any process-specific parameters (e.g., the desired size of the nucleic acid). range, desired degree of homology for target molecule capture, etc.) on the device, and a lid that the user can open to insert a sample preparation cartridge of the present disclosure. The device was powered with a 1000-volt electrode supply. The sample preparation cartridge contained 13 discrete microfluidic channels (or pumping lanes) and was constructed such that it could perform end-to-end sample preparation. The microfluidic channel is designed to make reagents and cartridges possible in an automated sequence: (1) pipette introduction of combined sample + lysis buffer and subsequent extraction of target DNA; (2) DNA purification; (3) DNA tagging using the transposase Tn5 followed by DNA repair; (4) selection of DNA fragments in a specific size range using SCODA; and (5) DNA cleaning.

700 million E. coli from overnight cultures mixed with lysis buffer and proteinase K. Samples of E. coli cells were incubated at 55° C. for 10 minutes, then mixed with isopropanol; The lysate mixture was added to the sample port in the sample preparation cartridge, the loaded cartridge was inserted into the sample preparation device, and DNA was extracted. Automated processing continued to provide DNA into the DNA library ready for sequencing, with short breaks for the user to add the DNA repair enzyme and DNA repair buffer mix to the cartridge immediately prior to the DNA repair step. An automated device delivered the DNA repair enzyme and DNA repair buffer mix to the reaction positions in the cartridge. An automated device as described above produced 0.96 μg extracted DNA; A subsequent automated step generated a DNA library of 279 ng at a concentration of 2.89 nM.

As a control experiment, 700 million teeth. A sample of E. coli cells (from the same sample as above) was manually processed to generate DNA using conventional DNA extraction and purification techniques. This manually prepared DNA was subjected to the same automated library preparation process on an automated device to generate 199 ng of DNA library at a concentration of 2.65 nM.

The purified DNA library produced by the sample preparation device was enriched using Aline beads and then subjected to sequencing on a Pacific Biosciences® RSII DNA sequencer.

The present inventors found that sequencing data obtained using purified DNA and prepared in library format using an automated sample preparation device were obtained using traditional DNA extraction and purification techniques, followed by DNA manually prepared by automated DNA library preparation. It was found that it generated sequencing reads that were slightly shorter in length compared to the data, but similar in quality (as assessed by Rsq score) (FIG. 25).

As shown in Table 4, the fully automated library generated reads with the same read quality (Rsq 0.82) as those generated by manual DNA extraction, and approximately equivalent read length (851 base average read length vs. 922) for the manual.

Table 4. Extracted and purified E. coli through an automated sample preparation device run on the same automated device. Sequencing results from DNA libraries generated from E. coli cells versus manually extracted and purified DNA

Example 3 - Use of a Sample Preparation Device to Enrich DNA for Sequencing

Using the automated sample preparation device of the present disclosure, E. DNA fragments in a specific size range were selected using SCODA for DNA libraries manually prepared from E. coli cultured cells.

4 micrograms of manually purified louse. coli DNA was treated with Tn5a tagging and then split into 4 separate samples of 1 μg each. The selection of DNA fragments of a specific size was performed in four different ways: (1) Sage Bluepipin with a program to collect fragments between 3 kb and 10 kb in size, (2) collection of fragments larger than 4 kb to 10 kb in size. (3) manual align bead size selection with 0.45x bead addition, or (4) the SCODA technique as in an automated sample preparation device (described in Example 8.0). did

After size selection, each sample was separately prepared into a DNA library and sequenced on a Pacific Biosciences® RSII DNA sequencer.

We found that sequencing data obtained using DNA library size selection using an automated sample preparation device were superior or equivalent to replicating DNA libraries selected for size by standard manual bead-based processes or automated Sage bluepipin size selection methods. was found (FIG. 26).

As shown in Table 5 (below), the automated device produced read lengths that were longer than the manual size selection process and equivalent to the bluepippin method, in terms of accuracy and GC content of the reads generated. There was no significant difference in

Table 5. Sequencing metrics from DNA libraries generated from automated size selection compared to those derived from sample sizes selected by commercial and manual methods.

Example 4 - Preparation of biological samples for sequencing

sample dissolution

A cultured cell or tissue sample containing one or more target molecules (eg, proteins) is lysed using any method known to those skilled in the art. The biological sample is suspended in lysis buffer (eg RIPA buffer, GCl (guanidine-HCl) buffer, GlyNP40 buffer) and mechanically homogenized to break the cell wall (eg in a lysis cartridge). Once the cells are disrupted, the target molecule is then precipitated and the supernatant is discarded. Precipitation can be achieved using centrifugation with a washing step (eg, addition of either a mix of chloroform/methanol or trichloroacetic acid). See FIG. 3 .

enrichment

The lysed sample is then optionally enriched (eg, using an affinity matrix) to capture target molecules, and the remaining non-target molecules discarded (eg, in an enrichment cartridge). Enrichment can include a depletion strategy (eg, using an affinity matrix) used to reduce sample complexity by sequestering non-target molecules. See FIG. 4 .

fragmentation

The lysed sample (if not enriched) or the enriched sample can then be fragmented (eg digested) (eg in a fragmentation cartridge). This step in the sample process converts the target molecule into smaller fragments or subunits. This step can be performed using non-enzymatic and/or enzymatic processes. Non-enzymatic methods include (but are not limited to) acid hydrolysis, cleavage via cyanogen bromide, hydroxylamine, and 2-nitro-5-thiocyanobenzoic acid, and electrochemical oxidation. Enzymatic methods include (but are not limited to) the use of nucleases or proteases. See FIG. 6 .

sensualization

Prior to sequencing, a fragmented sample can be functionalized (eg, in a functionalization cartridge) at one of its terminal moieties (eg, the N-terminus or C-terminus of a protein fragment). For example, a digested peptide can be labeled with some moiety that can immobilize the peptide onto a sequencing substrate. Functionalization can be achieved through a variety of chemical or enzymatic methods. See Figures 6 and 7.

Example 5 - Preparation of protein samples

This example describes the preparation of a protein sample using the device of the present disclosure, wherein the incubation, functionalization, quenching, immobilization complex formation, and purification steps were performed on a single cartridge. Proteins were prepared by pull-down from spiked plasma, where enriched proteins were purified using either antibodies or DNA aptamers on solid supports. The protein was then equilibrated with the desired buffer by gel filtration or by pH adjustment. Enriched protein samples (50-200 μM in 100 μL) containing equal mixtures of 2, 3, or 4 proteins were then incubated in 100 mM HEPES or sodium phosphate (pH 6-9) with 10-20% acetonitrile. prepared, a solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl, 200 mM in water, 1 μL) serving as reducing agent, and a freshly dissolved iodoacetamide solution serving as amino acid side chain capping agent. (9 mg in 97.3 μL water for 500 mM, 2 μL), and trypsin (1 μg/μL, 0.5-1 μL) to act as a protein digestion agent. Next, the peptide samples were incubated at 37° C. for 6-10 hours in the digestion section, where the proteins were denatured and digested. This resulted in the formation of a digested peptide sample.

Next, the digested peptide sample was automatically transported through a series of reservoirs, where it was mixed with a functionalization agent, a first (catalytic) reagent, and a second (pH-adjusting) reagent. Initially, the digested peptide samples were automatically added to potassium carbonate (1 M, 5 μL) to adjust the pH to a value between 10 and 11. The digested peptide samples were then automatically exposed to an azide delivery agent, imidazole-1-sulfonyl azide solution ("ISA" 200 mM in 200 mM KOH, 1.2 μL). Next, the digested peptide sample was automatically mixed with copper sulfate (catalytic reagent) solution. Finally, the digested peptide samples were automatically transferred to the functionalized portion of the modular cartridge, which was incubated for 1 hour at room temperature. This resulted in the formation of an unquenched mixture comprising one or more derivatized peptides.

After functionalization of the peptide in the functionalization area, 50 μL of the unquenched sample was automatically transported to a portion of a modular cartridge, where it was mixed with a plurality of polystyrene beads (solid substrate) and 10 actively mixed quenching step, followed by a static mixing step for a total of 23 minutes. Finally, the resulting quenched mixture was passed through an on-cartridge column to filter it from a plurality of polystyrene beads.

Next, the pH of the quenched peptide sample was adjusted to 7-8 through the addition of 6 μL of 1 M acetic acid. The quenched mixture was then automatically mixed with the immobilization complex DBCO-Q24-SV (50 μM, 6 μL) and then incubated at 37° C. on the device for 4 hours. The peptide samples were then automatically transported onto the column in a modular cartridge consisting of Zeba desalting column resin with a cutoff of 40 kDa equilibrated first with 10 mM TRIS, 10 mM potassium acetate buffer, pH 7.5. Finally, purified peptide samples resulting from this workflow were frozen and stored at temperatures below -20 °C.

Later, the purified peptide samples were sequenced and the observed peptides were identified based on their correspondence to protein sequences. 27a to 27d present the results in the form of bar charts. 27A corresponds to a mixture of two proteins—GIP and ADM. 27B corresponds to a mixture of three proteins—GLP1, insulin, and ADM. 27C corresponds to a mixture of four proteins—GLP1, ADM, insulin, and GIP. Figure 27d corresponds to a mixture of four peptides - GLP1, ADM, insulin, and GIP. A small number of off-target assignments (801) are indicated, but in general the sequenced peptides were correctly assigned to proteins prepared from the peptide samples. Moreover, the libraries generated in this example had similar or higher total reads than libraries prepared manually with replicates of the same protein mix. This example demonstrates that purified peptide samples can be prepared in an automated manner on modular cartridges of the type disclosed herein.

Example 6 - Use of devices of the present disclosure

This example describes an exemplary device wherein the incubation, functionalization, quenching, immobilization complex formation, and purification steps can be performed using a device of the present disclosure comprising multiple modular cartridges. Although the modular cartridges of this embodiment were not connected, peptide samples were prepared by following the protocol of Example 5. Protein samples were loaded and then incubated (eg at 37° C. for 5 hours), where the proteins were denatured and digested. The cartridge further included a pump lane to facilitate pumping of the fluid within the cartridge, as well as a reagent/sample mixture source.

After incubation, the peptide sample became a digested peptide sample. The digested peptide sample was then automatically delivered to a second cartridge, where it was automatically transported through a series of reservoirs, where it was combined with a functionalization agent, a first (catalytic) reagent, and a second (pH-adjusting) reagent. mixed. The digested peptide sample was transported through the sample inlet to a second cartridge. The digested peptide sample mixed sequentially with a functionalization agent, a first (catalytic) reagent, and a second (pH-adjusting) reagent was automatically transported. Finally, the digested peptide samples are incubated for a period of time (eg 1 hour at room temperature). This resulted in the formation of an unquenched mixture. The second cartridge further included a pump lane.

A portion of the unquenched sample was automatically transported to a third cartridge containing a sample inlet, a filter for beads, a small volume acidic reagent reservoir, and a mixing channel. Here, the unquenched mixture was quenched at room temperature. Finally, the resulting quenched mixture was passed through an on-cartridge column to remove a plurality of polystyrene beads and the pH was adjusted to 7-8 by addition of acetic acid from an acidic reagent reservoir.

The quenched mixture was then mixed with the DBCO-Q24-SV immobilized complex in the mixture source of the first modular cartridge and then incubated at 37°C.

Finally, the peptide sample was automatically transported to a fourth cartridge that controlled the flow of the quenched peptide sample through a commercial Zeba desalting column resin. Additional equilibration buffer was dispensed through the column to ensure that the peptides were transported through the column. A purified peptide sample was collected from a specific fraction of the fluid passing through the column, while the remaining fluid was sent to a waste reservoir. This example demonstrates that, in some embodiments, purified peptide samples can be produced automatically using a device comprising multiple cartridges.

Additional Embodiments

Additional embodiments of the present disclosure are encompassed by the numbered paragraphs below:

1. A device for enriching a target molecule from a biological sample, the device comprising an automated sample preparation module comprising a cartridge housing configured to receive a removable cartridge.

2. The device of paragraph 1, wherein the removable cartridge is a single-use cartridge or a multi-use cartridge.

3. The device of paragraphs 1 or 2, wherein the removable cartridge is configured to receive a biological sample.

4. The device of paragraph 3, wherein the removable cartridge further contains a biological sample.

5. The device of any of paragraphs 1-4, wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in the sample preparation process.

6. The device of any of paragraphs 1-5, wherein the cartridge comprises one or more affinity matrices, each affinity matrix comprising an immobilized capture probe having binding affinity for a target molecule.

7. The device of any of paragraphs 1-6, wherein the biological sample is a blood, saliva, sputum, feces, urine or buccal swab sample.

8. The device of any of paragraphs 1-7, wherein the target molecule is a target nucleic acid.

9. The device of paragraph 8, wherein the target nucleic acid is an RNA or DNA molecule.

10. The device of any of paragraphs 3-9, wherein the immobilized capture probe is an oligonucleotide capture probe, and wherein the oligonucleotide capture probe comprises a sequence that is at least partially complementary to the target nucleic acid.

11. The apparatus of paragraph 10, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to the target nucleic acid.

12. The device of any of paragraphs 8-11, wherein the device produces target nucleic acids having an average read-length for downstream sequencing applications that is greater than the average read-length generated using the control method.

13. The device of any of paragraphs 1-7, wherein the target molecule is a target protein.

14. The device of any of paragraphs 1-7 or 13, wherein the immobilized capture probe is a protein capture probe that binds to a target protein.

15. The device of paragraph 13, wherein the protein capture probe is an aptamer or an antibody.

16. The protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, 10 ^-5 to 10 ^-4 M , 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M binding affinity to the target protein.

17. The device of any of paragraphs 1-16, wherein the device further comprises a sequencing module.

18. The apparatus of paragraph 17, wherein the automated sample preparation module is directly or indirectly connected to the sequencing module.

19. The device of paragraphs 17 or 18, wherein the device is configured to transfer the target molecule from the automated sample preparation module to the sequencing module.

20. The apparatus of any of paragraphs 17-19, wherein the sequencing module performs nucleic acid sequencing.

21. The apparatus of paragraph 20, wherein the nucleic acid sequencing comprises single-molecule real-time sequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, and/or Sanger sequencing.

22. The apparatus of any of paragraphs 17-19, wherein the sequencing module performs protein sequencing.

23. The apparatus of paragraph 22, wherein the protein sequencing comprises Edman digestion or mass spectrometry.

24. The apparatus of any of paragraphs 17-19, wherein the sequencing module performs single-molecule protein sequencing.

25. A method for purifying a target molecule from a biological sample;

(i) lysing the biological sample;

(ii) fragmenting the lysed sample of (i); and

(iii) enriching the sample with an affinity matrix comprising immobilized capture probes having binding affinity for the target molecule, thereby purifying the target molecule;

How to include.

26. The method of paragraph 25, wherein the target molecule is a molecule and the target nucleic acid.

27. The method of paragraph 26 wherein the target nucleic acid is an RNA or DNA molecule.

28. The method of any of paragraphs 25-27, wherein the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to the target nucleic acid.

29. The method of paragraph 28, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to the target nucleic acid.

30. The method of paragraph 28 wherein the target molecule is a target protein.

31. The method of paragraph 25 or 30 wherein the immobilized capture probe is a protein capture probe that binds to a target protein.

32. The method of paragraph 31 wherein the protein capture probe is an aptamer or an antibody.

33. The protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, 10 ^-5 to 10 ^-4 M , the method of paragraph 31 or 32, which binds to the target protein with a binding affinity of 10 ^-4 to 10 ^-3 M, or 10 ^-3 to 10 ^-2 M.

34. The method of any one of paragraphs 25-33, wherein step (i) comprises an electrolytic method, an enzymatic method, a detergent-based method, and/or mechanical homogenization.

35. The method of any one of paragraphs 25-34, wherein step (i) comprises a plurality of dissolution methods wherein step (i) is performed sequentially.

36. The method of any of paragraphs 25 to 35, wherein the sample is purified after dissolution and before step (ii) or (iii).

37. The method of any of paragraphs 25-36, wherein step (ii) comprises a mechanical, chemical and/or enzymatic fragmentation method.

38. The method of any of paragraphs 25-37, wherein the sample is purified after fragmentation and before step (iii).

39. The method of any of paragraphs 25-38, wherein step (iii) comprises enrichment using an electrophoretic method.

40. The method of paragraph 39 wherein the electrophoretic method is affinity SCODA, FIGE, or PFGE.

41. The method of any of paragraphs 25-40, further comprising (iv) detecting the target molecule.

42. The method of paragraph 41, wherein step (iv) comprises detection using absorbance, fluorescence, mass spectrometry, and/or sequencing methods.

43. The method of any of paragraphs 25-42 wherein the biological sample is a blood, saliva, sputum, feces, urine or buccal sample.

44. The method of any of paragraphs 25-43, wherein the biological sample is from a human, non-human primate, rodent, dog, cat, or equine.

45. The method of any of paragraphs 25-44, wherein the biological sample comprises a bacterial cell or population of bacterial cells.

46. A device for enriching a target molecule from a biological sample, comprising an automated sample preparation module, wherein the automated sample preparation module performs the following steps:

(i) receiving the biological sample;

(ii) lysing the biological sample;

(iii) fragmenting the sample of (ii); and

(iv) enriching the sample with an affinity matrix comprising immobilized capture probes having binding affinity for the target molecule.

47. The apparatus of paragraph 46, wherein the target molecule is a molecule and the target nucleic acid.

48. The device of paragraph 46 wherein the target nucleic acid is an RNA or DNA molecule.

49. The device of any of paragraphs 46-48, wherein the immobilized capture probe is an oligonucleotide capture probe, and wherein the oligonucleotide capture probe comprises a sequence that is at least partially complementary to the target nucleic acid.

50. The apparatus of paragraph 49, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to the target nucleic acid.

51. The apparatus of paragraph 46 wherein the target molecule is a target protein.

52. The apparatus of paragraphs 46 or 51, wherein the immobilized capture probe is a protein capture probe that binds to a target protein.

53. The apparatus of paragraph 52 wherein the protein capture probe is an aptamer or an antibody.

54. The protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, 10 ^-5 to 10 ^-4 M , 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M binding affinity to the target protein.

55. The device of any of paragraphs 46-54, wherein the device further comprises a sequencing module.

56. The apparatus of paragraph 55, wherein the automated sample preparation module is directly or indirectly connected to the sequencing module.

57. The apparatus of paragraphs 55 or 56, wherein the apparatus is configured to transfer the target molecule from the automated sample preparation module to the sequencing module.

58. The apparatus of any of paragraphs 55-57, wherein the sequencing module performs nucleic acid sequencing.

59. The apparatus of paragraph 58, wherein the nucleic acid sequencing comprises single-molecule real-time sequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, and/or Sanger sequencing.

60. The apparatus of any of paragraphs 55-57, wherein the sequencing module performs protein sequencing.

61. The apparatus of paragraph 60, wherein the protein sequencing comprises Edman digestion or mass spectrometry.

62. The apparatus of any of paragraphs 55-57, wherein the sequencing module performs single-molecule protein sequencing.

63. The device of any of paragraphs 55-59, wherein the device produces a target nucleic acid having an average sequencing read-length greater than the average sequencing read-length generated using the control method.

Additional aspects of the invention

Aspects of the exemplary embodiments and examples described above can be combined in various combinations and subcombinations to produce additional embodiments of the invention. To the extent that aspects of the illustrative embodiments and examples described above are not mutually exclusive, all such combinations and subcombinations are intended to be within the scope of the present invention. It will be apparent to those skilled in the art that embodiments of the present invention include many aspects. Therefore, the scope of the claims is not to be limited by the preferred embodiments presented in the description and examples, but is to be given the broadest interpretation consistent with the description as a whole.

SEQUENCE LISTING <110> Quantum Si Incorporated <120> DEVICES AND METHODS FOR SEQUENCING <130> R0708.70095WO00 <140> PCT/US2021/028471 <141> 2021-04-21 <150> US 63/014,071 <151> 2020-04-22 <150> US 63/014,087 <151> 2020-04-22 <150> US 63/014,093 <151> 2020-04-22 <150> US 63/014,106 <151> 2020-04-22 <150> US 63/041,206 <151> 2020-06-19 <150> US 63/139,339 <151> 2021-01-20 <150> US 63/139,343 <151> 2021-01-20 <150> US 63/139,346 <151> 2021-01-20 <150> US 63/139,348 <151> 2021-01-20 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 1 Ile Asn Thr Ile Tyr Leu Gly Gly Pro Phe Ser Pro Asn Val Leu Asn 1 5 10 15 Trp Arg <210> 2 <211> 22 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 2 Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala Phe Gly Gln 1 5 10 15 Thr Asp Met Ser Arg Lys 20 <210> 3 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 3 Gly Tyr Ser Ile Phe Ser Tyr Ala Thr Lys 1 5 10 <210> 4 <211> 11 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 4 Lys Leu Ile Ser Glu Glu Asp Leu Lys Ala Arg 1 5 10 <210> 5 <211> 223 <212> PRT <213> artificial sequence <220> <223> synthetic <220> <221> MOD_RES <222> (31)..(31) <223> Xaa may be Lys or Glu <400> 5 Met Glu Lys Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala 1 5 10 15 Phe Gly Gln Thr Asp Met Ser Arg Lys Ala Phe Val Phe Pro Xaa Ser 20 25 30 Asp Thr Ser Tyr Val Ser Leu Lys Ala Pro Leu Thr Lys Pro Leu Lys 35 40 45 Ala Phe Thr Val Cys Leu His Phe Tyr Thr Glu Leu Ser Ser Thr Arg 50 55 60 Gly Tyr Ser Ile Phe Ser Tyr Ala Thr Lys Arg Gln Asp Asn Glu Ile 65 70 75 80 Leu Ile Phe Trp Ser Lys Asp Ile Gly Tyr Ser Phe Thr Val Gly Gly 85 90 95 Ser Glu Ile Leu Phe Glu Val Pro Glu Val Thr Val Ala Pro Val His 100 105 110 Ile Cys Thr Ser Trp Glu Ser Ala Ser Gly Ile Val Glu Phe Trp Val 115 120 125 Asp Gly Lys Pro Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr Thr Val 130 135 140 Gly Ala Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln Asp Ser Phe Gly 145 150 155 160 Gly Asn Phe Glu Gly Ser Gln Ser Leu Val Gly Asp Ile Gly Asn Val 165 170 175 Asn Met Trp Asp Phe Val Leu Ser Pro Asp Glu Ile Asn Thr Ile Tyr 180 185 190 Leu Gly Gly Pro Phe Ser Pro Asn Val Leu Asn Trp Arg Ala Leu Lys 195 200 205 Tyr Glu Val Gln Gly Glu Val Phe Thr Lys Pro Gln Leu Trp Pro 210 215 220 <210> 6 <211> 21 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 6 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> 7 <211> 30 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 7 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 <210> 8 <211> 4 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 8 Asn Tyr Cys Asn One <210> 9 <211> 17 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 9 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu <210> 10 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 10 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu 1 5 10 <210> 11 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 11 Arg Gly Phe Phe Tyr Thr Pro Lys Thr 1 5 <210> 12 <211> 4 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 12 Gly Ile Val Glu One <210> 13 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 13 Ala Leu Tyr Leu Val Cys Gly Glu 1 5 <210> 14 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 14 Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu 1 5 10

Claims (288)

A device for preparing a biological sample for sequencing, wherein the device comprises:
(i) a lysis cartridge comprising one or more microfluidic channels and configured to take a biological sample comprising one or more target molecules and produce a lysed sample;
(ii) an enrichment cartridge comprising one or more microfluidic channels and configured to enrich for at least one of the one or more target molecules to produce an enriched sample;
(iii) a fragmentation cartridge comprising one or more microfluidic channels and configured to digest or fragment at least one of the one or more target molecules to produce a fragmented sample; and
(iv) a functionalization cartridge comprising one or more microfluidic channels and configured to functionalize a terminal moiety of at least one of the one or more target molecules to form a functionalized sample.
and an automation module configured to receive at least two of the cartridges selected from The method of claim 1 , wherein the device comprises more than three of the cartridges selected from (i), (ii), (iii), or (iv), optionally wherein the device comprises (i), (ii), (iii), and each cartridge selected from (iv). The method of claim 1, wherein the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, plant sample, blood sample, saliva sample, sputum sample, stool sample, urine sample, buccal swab sample, amnion sample, semen sample. , a synovial fluid sample, a spinal cord sample, or a pleural fluid sample. 4. The device according to any one of claims 1 to 3, wherein the one or more target molecules are nucleic acids. 4. The device according to any one of claims 1 to 3, wherein the at least one target molecule is a protein. 6. The device of any preceding claim, wherein the one or more microfluidic channels are configured to contain and/or transport fluid(s) and/or reagent(s). 7. The device of any one of claims 1 to 6, wherein the lysis cartridge contains reagents that dissolve the sample but do not degrade or fragment the one or more target molecules. 8. The device of any one of claims 1 to 7, wherein the lysis cartridge comprises a reagent that facilitates at least partial isolation or purification of one or more target molecules from non-target molecules in the sample. 9. The device of claim 7 or 8, wherein the reagent comprises a detergent, acid, and/or base. 10. The device of any one of claims 7-9, wherein the reagent comprises a lysis buffer. 11. The device of claim 10, wherein the lysis buffer is selected from the group consisting of RIPA buffer, GCl (guanidine-HCl) buffer, and GlyNP40 buffer. 12. The device of any one of claims 1-11, wherein the one or more microfluidic channels in the lysis cartridge promotes shearing of cells and/or tissue (eg, shear flow of cells and/or tissue). . 12. The device of any one of claims 1-11, wherein the lysis cartridge comprises a needle passageway that promotes mechanical shearing of cells and/or tissue. 14. The device of claim 13, wherein the needle passageway has an inside diameter of 0.1 to 1 mm. 15. The device of any one of claims 1-14, wherein one or more microfluidic channels in the dissolution cartridge comprises an array of posts. 16. The device of any one of claims 1-15, wherein the dissolution cartridge is configured to be heated at an elevated temperature (eg, 20 to 60 °C). 17. The device of any one of claims 1-16, wherein the device is configured to heat the dissolution cartridge at an elevated temperature (eg, 20 to 60 °C). 18. The device of any one of claims 1-17, wherein the device is configured to microwave or sonicate the dissolution cartridge. 18. The device of any one of claims 1-17, wherein the module is further configured to receive an enrichment cartridge. 20. The device of claim 19, wherein the enrichment cartridge is positioned to receive the lysed sample from the lysing cartridge. 21. The device of claim 19 or 20, wherein the lysis cartridge and the enrichment cartridge are connected by one or more microfluidic channels. 22. The device of any preceding claim, wherein the enrichment cartridge comprises one or more affinity matrices. 23. The device of claim 22, wherein the one or more affinity matrices are in the microfluidic channels of the enrichment cartridge. 24. The device of claim 23, wherein the one or more target molecules is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one of the one or more target molecules. . 25. The device of claim 24, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to the target molecule. 26. The device according to any one of claims 22 to 25, wherein the device produces nucleic acids having an average read-length greater than the average read-length generated using the control method. 23. The device of claim 22, wherein the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds to at least one of the one or more target molecules. 28. The device of claim 27, wherein the protein capture probe is an aptamer or an antibody. The method of claim 27 or 28, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, The device binds to the target protein with a binding affinity of 10 ⁻⁵ to 10 ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. 23. The device of claim 22, wherein the at least one target molecule is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one non-target molecule. . 31. The device of claim 30, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to a non-target molecule. 32. The device of claim 30 or 31, wherein the oligonucleotide capture probe is not complementary to one or more target molecules. 23. The device of claim 22, wherein the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds to at least one non-target molecule. 34. The device of claim 33, wherein the protein capture probe is an aptamer or an antibody. The method of claim 33 or 34, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, and binds to a non-target protein with a binding affinity of 10 ⁻⁵ to 10 ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. 36. The device of any one of claims 33-35, wherein the protein capture probe does not bind one or more target molecules. 37. The device of any one of claims 30-36, wherein the enrichment cartridge is configured to deplete the sample of non-target molecules. 38. The device of any one of claims 1-37, wherein the module is further configured to receive a fragmentation cartridge. 39. The device of claim 38, wherein the fragmentation cartridge is positioned to receive the lysed sample from the lysis cartridge. 40. The device of claim 38 or 39, wherein the lysis cartridge and the fragmentation cartridge are connected by one or more microfluidic channels. 39. The device of claim 38, wherein the fragmentation cartridge is positioned to receive the enriched sample from the enrichment cartridge. 42. The device of claim 41, wherein the enrichment cartridge and fragmentation cartridge are connected by one or more microfluidic channels. 39. The device of claim 38, wherein the lysed sample can be removed from the device (eg to enable passive enrichment). 44. The device of any one of claims 38-43, wherein the device is configured such that the lysed sample is enriched prior to fragmentation. 45. The device of any one of claims 1-17 and 38-44, wherein the fragmentation cartridge comprises a non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules. . 46. The device of claim 45, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules comprises a detergent, acid, and/or base. 47. The method of claim 45 or 46, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules is cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide, hydrochloric acid, BNPS -skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], and/or 2-nitro-5-thiocyanobenzoic acid. 45. The device of any one of claims 1-17 and 38-44, wherein the fragmentation cartridge comprises one or more enzymatic reagents that digest or fragment at least one of the one or more target molecules. 49. The device of claim 48, wherein the one or more enzymatic reagents comprise one or more proteases. 50. The device of claim 49, wherein the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC and ArgC. 49. The device of claim 48, wherein the one or more enzymatic reagents comprise one or more endonucleases or exonucleases. 52. The device of any one of claims 1-17 and 38-51, wherein the fragmentation cartridge can be heated at an elevated temperature (eg, 20-60 °C). 53. The device of any one of claims 1-17 and 38-52, wherein the device is configured to heat the fragmentation cartridge at an elevated temperature (eg, 20 to 60 °C). 54. The device of any one of claims 1-17 and 38-53, wherein the device is configured to microwave or sonicate the fragmentation cartridge. 55. The device of any one of claims 1-54, wherein the module is further configured to receive a functionalization cartridge. 56. The device of claim 55, wherein the dissolution cartridge and functionalization cartridge are connected by one or more microfluidic channels. 56. The device of claim 55, wherein the enrichment cartridge and functionalization cartridge are connected by one or more microfluidic channels. 56. The device of claim 55, wherein the fragmentation cartridge and functionalization cartridge are connected by one or more microfluidic channels. 59. The device of claim 58, wherein the functionalization cartridge is positioned to receive the fragmented sample from the fragmentation cartridge. 57. The device of claim 55 or 56, wherein the dissolved sample is enriched prior to functionalization. 61. The device of any one of claims 55-60, wherein the dissolved sample is fragmented prior to functionalization. 62. The method of any one of claims 55-61, wherein the functionalization cartridge comprises a reagent that covalently transforms moiety M ⁰ of one or more target molecules, or of one or more fragments thereof, into modified moiety M ¹ . A device comprising one chamber. 63. The device of claim 62, wherein the reagent is non-enzymatic. 64. The device of claim 62 or 63, wherein the covalent modification is regiospecific. 65. The device of any one of claims 62-64, wherein a portion of the one or more target molecules, or of one or more fragments thereof, is a C-terminal carboxylate group or a C-terminal amino group. 66. The device of any one of claims 62-65, wherein the reagent comprises a buffer, salt, organic compound, acid, and/or base. 67. The device of any one of claims 62-66, wherein a portion of, or of one or more fragments of, the one or more target molecules is a C-terminal amino group and the covalent modification is diazo delivery. 68. The apparatus of claim 67, wherein moiety M ⁰ is -NH ₂ and moiety M ¹ is -N ₃ . 67. The device of claim 66, wherein the reagent comprises imidazole-1-sulfonyl azide and a copper salt (eg, copper sulfate), and a buffer having a pH of about 10 to 11. 70. The device of any one of claims 55-69, wherein the first chamber is connected to the second chamber through one or more microfluidic channels, and/or optionally a purification chamber. 71. The device of claim 70, wherein the second chamber contains a reagent that covalently modifies moiety M ¹ to produce a functionalized peptide. 72. The device of claim 71, wherein the covalent modification is an electrocyclization click reaction. 73. The method of claim 71 or 72, wherein the reagent comprises a DBCO-labeled DNA-streptavidin conjugate and a buffer, and optionally the DBCO-labeled DNA-streptavidin conjugate is immobilized on the surface of the second chamber. Device. 74. The device of claim 73, wherein the functionalized peptide is functionalized with a DBCO-labeled DNA-streptavidin conjugate. 73. The method according to any one of claims 70 to 72, comprising a purification chamber positioned between a first chamber and a second chamber comprising a resin that facilitates purification or enrichment of the modified target molecule, or fragment thereof. Device. 76. The device of claim 75, wherein the resin is a Sephadex resin, optionally a G-10 Sephadex resin. 77. The device of any one of claims 55-76, wherein the functionalized cartridge can be heated at an elevated temperature (eg, 20-60 °C). 78. The device of any one of claims 55-77, wherein the device is configured to heat the functionalized cartridge at an elevated temperature (eg, 20-60 °C). 79. The device of any one of claims 55-78, wherein the functionalized cartridge is amenable to microwave or sonication. 80. The device of any one of claims 55-79, wherein the device is configured to microwave or sonicate the functionalized cartridge. 81. The device of any one of claims 1-80, wherein the device further comprises a peristaltic pump configured to transport one or more fluids into, into, or out of any of the cartridges held by the device. 82. The device of any preceding claim, wherein the device further comprises a peristaltic pump configured to transport one or more fluids into or through any of the microfluidic channels of a cartridge received by the device. . 83. The device of any one of claims 1-82, wherein the device is configured to transport fluid with a fluid flow resolution of 1000 microliters or less, 100 microliters or less, 50 microliters or less, or 10 microliters or less. . 84. The device of any one of claims 1-83, wherein one of the cartridges comprises a base layer having a surface comprising channels. 85. The device of claim 84, wherein the channel comprises one or more microfluidic channels. 86. The device of claim 84 or 85, wherein at least a portion of at least some of the channels have a substantially triangular-shaped cross-section with a single vertex at the base of the channels and two other vertices at the surface of the base layer. 87. The device of any preceding claim, wherein at least a portion of at least a portion of the channels of any one of the cartridges has a surface layer comprising an elastomer configured to substantially seal surface openings of the channels. 88. The device of claim 87, wherein the elastomer comprises silicone. 89. The device of any one of claims 1-88, wherein at least one portion of at least a portion of the channel has walls and a base comprising a substantially rigid material compatible with the biological material. 90. The device of any one of claims 1-89, wherein any one of the cartridges includes one or more fluid reservoirs. 91. The device of any one of claims 1-90, wherein at least a portion of the channel is connected to a reservoir in a temperature zone. 92. The device of any one of claims 1-91, wherein at least a portion of the channel is connected to an electrophoretic gel. 93. The device of any one of claims 1-92, wherein the device is configured to simultaneously receive two or more cartridges. 94. The device of claim 93, wherein the device is configured to establish fluid communication between two or more cartridges received by the device at the same time. 95. The device of any preceding claim, wherein the device is configured to sequentially receive two or more cartridges. 96. The device of any preceding claim, wherein the device further comprises a sequencing module. 97. The device of claim 96, wherein the device is configured to deliver the one or more target molecules to the sequencing module. 98. The device of claim 96 or 97, wherein the sequencing module performs nucleic acid sequencing. 99. The device of claim 98, wherein nucleic acid sequencing comprises single-molecule real-time sequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, and/or Sanger sequencing. 99. The device of claim 96 or 98, wherein the sequencing module performs protein sequencing. 101. The device of claim 100, wherein protein sequencing comprises Edman digestion or mass spectrometry. 99. The device of claim 96 or 98, wherein the sequencing module performs single-molecule protein sequencing. (i) lysing a biological sample comprising one or more target molecules;
(ii) enriching at least one of the one or more target molecules and/or at least one non-target molecule;
(iii) fragmenting one or more target molecules; and
(iv) functionalizing the terminal moiety of one or more target molecules
An apparatus for preparing one or more target molecules, configured to perform two or more of the steps selected from 104. The device of claim 103, wherein one or more of the steps selected from (i), (ii), (iii), and (iv) are performed in a cartridge. 104. The apparatus of claim 103, wherein more than one step is performed on the same cartridge. 106. The device of claim 104 or 105, wherein the cartridge is a single-use cartridge or a multi-use cartridge. 107. The device of any one of claims 104-106, wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in any of the automated steps. 107. The device of any one of claims 104-106, wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport one or more target molecules between any of the automated steps. 109. The device of any one of claims 104-108, wherein the cartridge contains resin for purification of one or more target molecules between any of the automated steps. 110. The device of claim 109, wherein the resin is a Sephadex resin, optionally a G-10 Sephadex resin. 111. The device of any one of claims 103-110, wherein the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, or plant sample. 112. The method of any one of claims 103-111, wherein the biological sample is a blood sample, a saliva sample, a sputum sample, a stool sample, a urine sample, a buccal swab sample, an amnion sample, a semen sample, a synovial fluid sample, a spinal cord sample, or a pleural fluid sample. sample-in device. 113. The device of any one of claims 103-112, wherein the one or more target molecules are nucleic acids. 113. The device of any one of claims 103-112, wherein the one or more target molecules are proteins. 115. The device of any one of claims 104-114, wherein step (i) is performed in the dissolution cartridge or dissolution section of the cartridge. 116. The device of claim 115, wherein the lysis cartridge or lysis section of the cartridge contains reagents that lyse the sample but do not degrade or fragment the one or more target molecules. 117. The device of claim 115 or 116, wherein the lysis cartridge or lysis section of the cartridge contains a reagent that promotes at least partial isolation or purification of one or more target molecules from non-target molecules in the sample. 118. The device of claim 116 or 117, wherein the reagent comprises a detergent, acid, and/or base. 119. The device of any one of claims 116-118, wherein the reagent comprises a lysis buffer. 120. The device of claim 119, wherein the lysis buffer is selected from the group consisting of RIPA buffer, GCl (guanidine-HCl) buffer, and GlyNP40 buffer. 121. The method of any one of claims 115-120, wherein the lysis cartridge or one or more microfluidic channels in the lysis section of the cartridge conduct shear of cells and/or tissue (eg, shear flow of cells and/or tissue). A device that promotes. 122. The device of any one of claims 115-121, wherein the lysis cartridge or lysis section of the cartridge comprises a needle passageway that promotes mechanical shearing of cells and/or tissue. 123. The device of claim 122, wherein the needle passageway has an inside diameter of 0.1 to 1 mm. 124. The device of any one of claims 115-123, wherein the dissolution cartridge or one or more microfluidic channels in the dissolution section of the cartridge comprises an array of posts. 125. The device of any one of claims 115-124, wherein the dissolution cartridge or dissolution section of the cartridge is configured to be heated at an elevated temperature (eg, 20 to 60 °C). 126. The device of any one of claims 115-125, wherein the device is configured to heat the dissolution cartridge or dissolution section of the cartridge at an elevated temperature (eg, 20 to 60 °C). 127. The device of any one of claims 115-126, wherein the device is configured to subject the dissolution cartridge or dissolution section of the cartridge to microwave or sonication. 128. The device of any one of claims 104-127, wherein step (ii) is performed in an enrichment cartridge or an enrichment section of a cartridge. 129. The device of claim 128, wherein the enrichment cartridge is positioned to receive the lysed sample from the lysing cartridge, or the enrichment section of the cartridge is positioned to receive the lysed sample from the lysing section of the cartridge. 130. The device of claim 128 or 129, wherein the dissolution cartridge and the enrichment cartridge or the dissolution section of the cartridge and the enrichment section of the cartridge are connected by one or more microfluidic channels. 131. The device of any one of claims 128-130, wherein the enrichment cartridge or enrichment section of the cartridge comprises one or more affinity matrices. 132. The device of claim 131, wherein the one or more affinity matrices are in a microfluidic channel of an enrichment cartridge or an enrichment section of a cartridge. 132. The device of claim 131, wherein the one or more target molecules is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one of the one or more target molecules. . 134. The device of claim 133, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90%, 95%, or 100% complementary to the target molecule. 135. The device of any one of claims 131-134, wherein the device produces nucleic acids with an average read-length greater than the average read-length generated using the control method. 132. The device of claim 131, wherein the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds to at least one of the one or more target molecules. 137. The device of claim 136, wherein the protein capture probe is an aptamer or an antibody. The method of claim 136 or 137, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, The device binds to the target protein with a binding affinity of 10 ⁻⁵ to 10 ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. 132. The device of claim 131, wherein the at least one target molecule is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one non-target molecule. . 140. The device of claim 139, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to a non-target molecule. 141. The device of claim 139 or 140, wherein the oligonucleotide capture probe is not complementary to one or more target molecules. 132. The device of claim 131, wherein the at least one target molecule is a protein and the immobilized capture probe is a protein capture probe that binds at least one non-target molecule. 143. The device of claim 142, wherein the protein capture probe is an aptamer or an antibody. The method of claim 142 or 143, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, and binds to a non-target protein with a binding affinity of 10 ⁻⁵ to 10 ⁻⁴ M, 10 ⁻⁴ to 10 ⁻³ M, or 10 ⁻³ to 10 ⁻² M. 145. The device of any one of claims 142-144, wherein the protein capture probe does not bind one or more target molecules. 146. The device of any one of claims 139-145, wherein the enrichment cartridge or enrichment section of the cartridge is configured to deplete the sample of non-target molecules. 147. The apparatus of any one of claims 115-146, wherein step (iii) is performed on a fragmentation cartridge or a fragmentation section of a cartridge. 148. The device of claim 147, wherein the fragmentation cartridge is positioned to receive the lysed sample from the lysis cartridge, or the fragmentation section of the cartridge is positioned to receive the lysed sample from the lysis section of the cartridge. 149. The device of claim 147 or 148, wherein the dissolution cartridge and the fragmentation cartridge or the dissolution section of the cartridge and the fragmentation section of the cartridge are connected by one or more microfluidic channels. 148. The device of claim 147, wherein the fragmentation cartridge is positioned to receive the enriched sample from the enrichment cartridge, or the fragmentation section of the cartridge is positioned to receive the enriched sample from the enrichment section of the cartridge. 151. The device of claim 150, wherein the enrichment cartridge and the fragmentation cartridge or the enrichment section of the cartridge and the fragmentation section of the cartridge are connected by one or more microfluidic channels. 148. The device of claim 147, wherein the lysed sample can be removed from the device (eg to enable passive enrichment). 153. The device of any one of claims 147-152, wherein the device is configured such that the lysed sample is enriched prior to fragmentation. 154. The device of any one of claims 115-153, wherein the fragmentation cartridge or fragmentation section of the cartridge comprises a non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules. 155. The device of claim 154, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules comprises a detergent, acid, and/or base. 156. The method of claim 154 or 155, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules is cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide, hydrochloric acid, BNPS -skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], and/or 2-nitro-5-thiocyanobenzoic acid. 154. The device of any one of claims 115-153, wherein the fragmentation cartridge or fragmentation section of the cartridge comprises one or more enzymatic reagents that digest or fragment at least one of the one or more target molecules. 158. The device of claim 157, wherein the one or more enzymatic reagents comprise one or more proteases. 159. The device of claim 158, wherein the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC and ArgC. 158. The device of claim 157, wherein the one or more enzymatic reagents comprise one or more endonucleases or exonucleases. 161. The device of any one of claims 115-160, wherein the fragmentation cartridge or fragmentation section of the cartridge can be heated at an elevated temperature (eg, 20 to 60 °C). 162. The device of any one of claims 115-161, wherein the device is configured to heat the fragmentation cartridge or fragmentation section of the cartridge at an elevated temperature (eg, 20 to 60 °C). 163. The device of any one of claims 115-162, wherein the device is configured to microwave or sonicate the fragmentation cartridge or fragmentation section of the cartridge. 164. The device of any one of claims 115-163, wherein step (iv) is performed in a functionalization cartridge or a functionalization section of a cartridge. 165. The device of claim 164, wherein the dissolution cartridge and the functionalized cartridge or the dissolving section of the cartridge and the functionalized section of the cartridge are connected by one or more microfluidic channels. 165. The device of claim 164, wherein the enrichment cartridge and the functionalization cartridge or the enrichment section of the cartridge and the functionalization section of the cartridge are connected by one or more microfluidic channels. 165. The device of claim 164, wherein the fragmentation cartridge and the functionalization cartridge or the fragmentation section of the cartridge and the functionalization section of the cartridge are connected by one or more microfluidic channels. 168. The device of claim 167, wherein the functionalization cartridge is positioned to receive the fragmented sample from the fragmentation cartridge. 166. The device of claim 164 or 165, wherein the dissolved sample is enriched prior to functionalization. 170. The device of any one of claims 164-169, wherein the dissolved sample is fragmented prior to functionalization. 171. The method of any one of claims 164-170, wherein the functionalized cartridge or functionalized section of the cartridge covalently modifies moiety M ⁰ of one or more target molecules, or of one or more fragments thereof, with modified moiety M ¹ . A device comprising a first chamber containing a reagent to be tested. 172. The device of claim 171, wherein the reagent is non-enzymatic. 173. The device of claim 171 or 172, wherein the covalent modification is regiospecific. 174. The device of any one of claims 171-173, wherein a portion of the one or more target molecules, or of one or more fragments thereof, is a C-terminal carboxylate group or a C-terminal amino group. 175. The device of any one of claims 171-174, wherein the reagent comprises a buffer, salt, organic compound, acid, and/or base. 176. The device of any one of claims 171-175, wherein a portion of, or of one or more fragments thereof, of the one or more target molecules is a C-terminal amino group and the covalent modification is diazo delivery. 177. The apparatus of claim 176, wherein moiety M ⁰ is -NH ₂ and moiety M ¹ is -N ₃ . 176. The device of claim 175, wherein the reagent comprises imidazole-1-sulfonyl azide and a copper salt (eg, copper sulfate), and a buffer having a pH of about 10 to 11. 179. The device of any one of claims 164-178, wherein the first chamber is connected to the second chamber through one or more microfluidic channels, and/or optionally a purification chamber. 180. The device of claim 179, wherein the second chamber contains reagents that covalently modify moiety M ¹ to produce a functionalized peptide. 181. The device of claim 180, wherein the covalent modification is an electrocyclization click reaction. 182. The method of claim 180 or 181, wherein the reagent comprises a DBCO-labeled DNA-streptavidin conjugate and a buffer, and optionally the DBCO-labeled DNA-streptavidin conjugate is immobilized to the surface of the second chamber. Device. 183. The device of claim 182, wherein the functionalized peptide is functionalized with a DBCO-labeled DNA-streptavidin conjugate. 182. The method of any one of claims 179-181 comprising a purification chamber positioned between a first chamber and a second chamber comprising a resin that facilitates purification or enrichment of the modified target molecule, or fragment thereof. Device. 185. The device of claim 184, wherein the resin is a Sephadex resin, optionally a G-10 Sephadex resin. 186. The device of any one of claims 164-185, wherein the functionalized cartridge or functionalized section of the cartridge can be heated at an elevated temperature (eg, 20 to 60 °C). 187. The device of any one of claims 164-186, wherein the device is configured to heat the functionalized cartridge or functionalized section of the cartridge at an elevated temperature (eg, 20 to 60 °C). 188. The device of any one of claims 164-187, wherein the functionalized cartridge or functionalized section of the cartridge can be subjected to microwave or sonication. 189. The device of any one of claims 164-188, wherein the device is configured to microwave or sonicate the functionalized cartridge or functionalized section of the cartridge. 190. The device of any one of claims 103-189, wherein the device further comprises a peristaltic pump configured to transport one or more fluids into, into, or out of any of the cartridges held by the device. 191. The device of any one of claims 103-190, wherein the device further comprises a peristaltic pump configured to transport one or more fluids into or through any of the microfluidic channels of a cartridge received by the device. . 192. The device of any one of claims 103-191, wherein the device is configured to transport fluid with a fluid flow resolution of 1000 microliters or less, 100 microliters or less, 50 microliters or less, or 10 microliters or less. . 193. The device of any one of claims 103-192, wherein one of the cartridges comprises a base layer having a surface comprising channels. 194. The device of claim 193, wherein the channel comprises one or more microfluidic channels. 195. The device of claim 193 or 194, wherein at least a portion of at least some of the channels have a substantially triangular-shaped cross-section with a single vertex at the base of the channel and two other vertices at the surface of the base layer. 196. The device of any one of claims 103-195, wherein at least a portion of at least a portion of the channels of any one of the cartridges has a surface layer comprising an elastomer configured to substantially seal surface openings of the channels. 197. The device of claim 196, wherein the elastomer comprises silicone. 198. The device of any one of claims 103-197, wherein at least one portion of at least a portion of the channel has walls and a base comprising a substantially rigid material compatible with the biological material. 199. The device of any one of claims 103-198, wherein any one of the cartridges includes one or more fluid reservoirs. 200. The device of any one of claims 103-199, wherein at least a portion of the channel is connected to a reservoir in a temperature zone. 201. The device of any one of claims 103-200, wherein at least a portion of the channel is connected to an electrophoretic gel. 202. The device of any one of claims 103-201, wherein the device is configured to simultaneously receive two or more cartridges. 203. The device of claim 202, wherein the device is configured to establish fluid communication between two or more cartridges received by the device at the same time. 204. The device of any one of claims 103-203, wherein the device is configured to sequentially receive two or more cartridges. 205. The device of any one of claims 103-204, wherein the device further comprises a sequencing module. 206. The device of claim 205, wherein the device is configured to deliver one or more target molecules to the sequencing module. 207. The apparatus of claim 205 or 206, wherein the sequencing module performs nucleic acid sequencing. 208. The device of claim 207, wherein nucleic acid sequencing comprises single-molecule real-time sequencing, sequencing-by-synthesis, sequencing-by-ligation, nanopore sequencing, and/or Sanger sequencing. 208. The device of claim 205 or 207, wherein the sequencing module performs protein sequencing. 210. The device of claim 209, wherein protein sequencing comprises Edman digestion or mass spectrometry. 208. The device of claim 205 or 207, wherein the sequencing module performs single-molecule protein sequencing. (i) lysing a biological sample comprising one or more target molecules;
(ii) enriching at least one of the one or more target molecules and/or at least non-target molecules;
(iii) fragmenting one or more target molecules; and
(iv) functionalizing the terminal moiety of the one or more fragmented target molecules.
A method for producing one or more target molecules, comprising two or more of the steps selected from;
steps here (e.g. wherein at least one of steps (i), (ii), (iii), and/or (iv)) is performed in an automated sample preparation device. 213. The method of claim 212, wherein the biological sample is a single cell, mammalian cell tissue, animal sample, fungal sample, or plant sample. 213. The method of claim 212, wherein the biological sample is a blood sample, saliva sample, sputum sample, stool sample, urine sample, buccal swab sample, amnion sample, semen sample, synovial fluid sample, spinal cord sample, or pleural fluid sample. 215. The method of any one of claims 212-214, wherein the one or more target molecules are nucleic acids. 215. The method of any one of claims 212-214, wherein the one or more target molecules are proteins. 213. The method of claim 212, wherein the two steps are performed in an automated sample preparation device. 213. The method of claim 212, wherein the three steps are performed in an automated sample preparation device. 213. The method of claim 212, wherein the four steps are performed in an automated sample preparation device. 220. The method of any one of claims 212-219, wherein step (i) is performed using a dissolution cartridge. 221. The method of claim 220, wherein the dissolution cartridge comprises one or more microfluidic channels configured to contain and/or transport fluid(s) and/or reagent(s). 222. The method of claim 220 or 221, wherein the lysis cartridge contains reagents that lyse the sample but do not degrade or fragment the one or more target molecules. 223. The method of any one of claims 220-222, wherein the lysis cartridge comprises reagents that facilitate at least partial isolation or purification of one or more target molecules from non-target molecules in the sample. 224. The method of claim 222 or 223, wherein the reagent comprises a detergent, acid, and/or base. 225. The method of any one of claims 222-224, wherein the reagent comprises a lysis buffer. 226. The method of claim 225, wherein the lysis buffer is selected from the group consisting of RIPA buffer, GCl (guanidine-HCl) buffer, and GlyNP40 buffer. 227. The method of any one of claims 220-226, wherein the one or more microfluidic channels in the lysis cartridge promotes shearing of cells and/or tissue (eg, shear flow of cells and/or tissue). . 228. The method of any one of claims 220-227, wherein the lysis cartridge comprises a needle passageway that promotes mechanical shearing of cells and/or tissue. 229. The method of claim 228, wherein the needle passage has an inside diameter of 0.1 to 1 mm. 230. The method of any one of claims 220-229, wherein one or more microfluidic channels in the lysis cartridge comprises an array of posts. 231. The method of any one of claims 220-230, wherein the dissolution cartridge is configured to be heated at an elevated temperature (eg, 20 to 60 °C). 232. The method of any one of claims 220-231, wherein the device is configured to heat the dissolution cartridge at an elevated temperature (eg, 20 to 60 °C). 233. The method of any one of claims 220-232, wherein the device is configured to microwave or sonicate the dissolution cartridge. 234. The method of any one of claims 212-233, wherein step (ii) is performed in an automated sample preparation device. 235. The method of claim 234, wherein step (ii) is performed using an enrichment cartridge. 236. The method of claim 235, wherein the enrichment cartridge comprises one or more affinity matrices. 237. The method of claim 236, wherein the one or more affinity matrices are in a microfluidic channel of an enrichment cartridge. 237. The method of claim 236, wherein the one or more target molecules is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one of the one or more target molecules. . 239. The method of claim 238, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to the target molecule. 237. The method of claim 236, wherein the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds to at least one of the one or more target molecules. 241. The method of claim 240, wherein the protein capture probe is an aptamer or an antibody. The method of claim 240 or 241, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, The method of binding to the target protein with a binding affinity of 10 ^-5 to 10 ^-4 M, 10 ^-4 to 10 ^-3 M, or 10 ^-3 to 10 ^-2 M. 237. The method of claim 236, wherein the at least one target molecule is a nucleic acid, the immobilized capture probe is an oligonucleotide capture probe, and the oligonucleotide capture probe comprises a sequence that is at least partially complementary to at least one non-target molecule. . 244. The method of claim 243, wherein the oligonucleotide capture probe comprises a sequence that is at least 80%, 90% 95%, or 100% complementary to a non-target molecule. 245. The method of claim 243 or 244, wherein the oligonucleotide capture probe is not complementary to one or more target molecules. 237. The method of claim 236, wherein the one or more target molecules are proteins and the immobilized capture probe is a protein capture probe that binds at least one non-target molecule. 247. The method of claim 246, wherein the protein capture probe is an aptamer or an antibody. 247. The method of claim 246 or 247, wherein the protein capture probe is 10 ^-9 to 10 ^-8 M, 10 ^-8 to 10 ^-7 M, 10 ^-7 to 10 ^-6 M, 10 ^-6 to 10 ^-5 M, and binds to a non-target protein with a binding affinity of 10 ^-5 to 10 ^-4 M, 10 ^-4 to 10 ^-3 M, or 10 ^-3 to 10 ^-2 M. 249. The method of any one of claims 246-248, wherein the protein capture probe does not bind one or more target molecules. 250. The method of any one of claims 243-249, wherein the enrichment cartridge is configured to deplete the sample of non-target molecules. 251. The method of any one of claims 212-250, wherein step (iii) is performed in an automated sample preparation device. 252. The method of claim 251, wherein step (iii) is performed using a fragmentation cartridge. 253. The method of any one of claims 212-233, 251 or 252, wherein the fragmentation cartridge comprises a non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules. . 254. The method of claim 253, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules comprises a detergent, acid, and/or base. 254. The method of claim 253 or 254, wherein the non-enzymatic reagent that digests or fragments the sample and/or one or more target molecules is cyanogen bromide, hydroxylamine, iodosobenzoic acid, dimethyl sulfoxide, hydrochloric acid, BNPS -skatole [2-(2-nitrophenylsulfenyl)-3-methylindole], and/or 2-nitro-5-thiocyanobenzoic acid. 256. The method of any one of claims 252-255, wherein the fragmentation cartridge comprises one or more enzymatic reagents that digest or fragment at least one of the one or more target molecules. 257. The method of claim 256, wherein the one or more enzymatic reagents comprise one or more proteases. 258. The method of claim 257, wherein the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, LysC, LysN, AspN, GluC and ArgC. 258. The method of claim 257, wherein the one or more enzymatic reagents comprise one or more endonucleases or exonucleases. 260. The method of any one of claims 252-259, wherein the fragmentation cartridge can be heated at an elevated temperature (eg, 20 to 60 °C). 261. The method of any one of claims 252-260, wherein the method is configured to heat the fragmentation cartridge at an elevated temperature (eg, 20 to 60 °C). 262. The method of any one of claims 252-261, wherein the method is configured to subject the fragmentation cartridge to microwave or sonication. 263. The method of any one of claims 212-262, wherein step (iv) is performed in an automated sample preparation device. 264. The method of claim 263, wherein step (iv) is performed using a functionalized cartridge. 265. The method of claim 264, wherein the functionalization cartridge comprises a first chamber comprising a reagent that covalently transforms moiety M ⁰ of one or more target molecules, or of one or more fragments thereof, to modified moiety M ¹ . . 266. The method of claim 265, wherein the reagent is non-enzymatic. 267. The method of claim 265 or 266, wherein the covalent modifications are regiospecific. 268. The method of any one of claims 265-267, wherein a portion of one or more target molecules, or one or more fragments thereof, is a C-terminal carboxylate group or a C-terminal amino group. 269. The method of any one of claims 265-268, wherein the reagent comprises a buffer, salt, organic compound, acid, and/or base. 270. The method of any one of claims 265-269, wherein a portion of one or more target molecules, or one or more fragments thereof, is a C-terminal amino group and the covalent modification is diazo delivery. 271. The method of claim 270, wherein moiety M ⁰ is -NH ₂ and moiety M ¹ is -N ₃ . 270. The method of claim 269, wherein the reagent comprises imidazole-1-sulfonyl azide and a copper salt (eg, copper sulfate), and a buffer having a pH of about 10 to 11.
[claim 271]
273. The method of any one of claims 264-272, wherein the first chamber is connected to the second chamber through one or more microfluidic channels, and/or optionally a purification chamber.
[claim 272]
272. The method of claim 271, wherein the second chamber contains reagents that covalently modify moiety M ¹ to produce a functionalized peptide. 273. The method of claim 272, wherein the covalent modification is an electrocyclization click reaction. 274. The method of claim 272 or 273, wherein the reagent comprises a DBCO-labeled DNA-streptavidin conjugate and a buffer, and optionally the DBCO-labeled DNA-streptavidin conjugate is immobilized to the surface of the second chamber. Way. 275. The method of claim 274, wherein the functionalized peptide is functionalized with a DBCO-labeled DNA-streptavidin conjugate. 274. The method of any one of claims 271-273, comprising a purification chamber positioned between a first chamber and a second chamber comprising a resin that facilitates purification or enrichment of the modified target molecule, or fragment thereof. Way. 277. The method of claim 276, wherein the resin is a Sephadex resin, optionally a G-10 Sephadex resin. 278. The method of any one of claims 264-277, wherein the functionalization cartridge can be heated at an elevated temperature (eg, 20-60 °C). 279. The method of any one of claims 264-278, wherein the method is configured to heat the functionalized cartridge at an elevated temperature (eg, 20 to 60 °C). 280. The method of any one of claims 264-279, wherein the functionalized cartridge is amenable to microwave or sonication. 281. The method of any one of claims 264-280, wherein the method is configured to subject the functionalized cartridge to microwave or sonication. 220. The method of any one of claims 212-219, wherein two or more of steps (i), (ii), and (iii) are performed in a single cartridge. (i) lysing a biological sample comprising one or more target molecules;
(ii) enriching at least one of the one or more target molecules and/or at least one non-target molecule;
(iii) fragmenting one or more target molecules; and
(iv) functionalizing the terminal moiety of one or more target molecules
A cartridge for preparing one or more target molecules, configured to perform two or more of the steps selected from 284. The cartridge of claim 283, wherein the cartridge is a single-use cartridge or a multi-use cartridge. 285. The cartridge of claim 283 or 284, wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport a fluid used in any of the automated steps. 285. The cartridge of claim 283 or 284, wherein the cartridge comprises one or more microfluidic channels configured to contain and/or transport one or more target molecules between any of the automated steps. 287. The cartridge of any one of claims 283-286, wherein the cartridge comprises a resin for purification of one or more target molecules between any of the automated steps. 288. The cartridge of claim 287, wherein the resin is a Sephadex resin, optionally a G-10 Sephadex resin.

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