Methods, systems, kits, and devices comprising peptides

Abstract

Provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of all individual amino acids present in the peptide(s) of interest, an aqueous solution, and uses thereof. The methods comprising peptides of interest may be characterized as methods of synthesizing a peptide. These peptides of interest may be characterized as hydrophobic or partially hydrophobic. These individual amino acids may be characterized as natural amino acids or unnatural amino acids. The aqueous solution may be characterized as pure or substantially pure water. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing a peptide(s) of interest from a template peptide.

Description

[0001] METHODS, SYSTEMS, KITS, AND DEVICES COMPRISING PEPTIDES

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] [1] This application claims the benefit of priority to UK Patent Application 2409424.5, filed June 28, 2024, and UK Patent Application 2502247.6, filed February 14, 2025, and the contents of which are incorporated herein by reference in its entirely.

[0004] BACKGROUND

[0005] [2] A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. Peptides are also increasingly being used in cosmetic applications for application to skin and hair, for example.

[0006] [3] Peptides are currently generally made by recombinant means using bacterial or eukaryotic cell cultures or solid phase peptide synthesis, using FMOC or BOC protected amino acids. Both processes are costly and purification of the peptides can be onerous. Moreover, for solid phase synthesis methods copious amounts of toxic organic solvents are required for the chemistry reactions and removal of the resulting waste material is approximately 10% of the overall cost of peptide production.

[0007] [4] Native peptide ligation of peptides has been undertaken to provide proteins from large peptide fragments. Typically, this requires one half of the peptide to have a reactive thioester chemistry to enable the reaction. Further this technique relies on solid phase synthesis of two portions or halves of the desired peptide before these portions or halves are ligated together.

[0008] SUMMARY

[0009] [5] The present disclosure provides compositions, systems, devices, kits, and methods comprising synthesized and / or amplified peptides and uses thereof. Provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of individual amino acids, an aqueous solution, and uses thereof. The methods comprising peptides of interest may be characterized as methods of synthesizing and / or amplifying a peptide. These peptides of interest may be characterized as hydrophobic or partially hydrophobic. These individual amino acids may be characterized as natural amino acids or unnatural amino acids. The aqueous solution may be characterized as pure or substantially pure water. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing and / or amplifying a peptide(s) of interest from a template peptide.

[0010] [6] The present disclosure relies on studies concerning the synthesis and / or amplification of peptides without the use of complex chemistry, high temperature and / or pressure or DNA templates to facilitate peptide synthesis. Unlike solid phase synthesis of two portions or halves of the desired peptide and then ligation of these portions or halves together, the templated method described herein does not rely on having the majority of the peptide pre-synthesized. The method of synthesizing a peptide from a template peptide described herein mainly requires constituent amino acids.

[0011] [7] Unexpectedly, and as described in the present disclosure, it has been found that a templated synthesis of peptides can occur by providing low amounts of energy to amino acids in solution.

[0012] Certain Embodiments

[0013] [8] The present disclosure provides compositions, systems, devices, kits, and methods comprising synthesized and / or amplified peptides and uses thereof. Provided herein are compositions, systems, kits, devices, and methods comprising a template peptide, a mixture of individual amino acids, an aqueous solution, and optionally a synthesized peptide.

[0014] [9] Provided herein is a method of amplifying a peptide(s) of interest from a biological sample, the method comprising: (a) providing a biological sample comprising a peptide(s) of interest for amplification; (b) providing a mixture of all individual amino acids present in the peptide(s) of interest in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest; (c) contacting the biological sample and the mixture of all individual amino acids in an aqueous solution such that copies of the peptide(s) of interest in the biological sample is produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample; (d) providing an amount of energy to the aqueous solution of the peptide(s) of interest and the mixture of all individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and (e) performing step (d) for a time period sufficient to amplify the peptide(s) of interest with the mixture of all individual amino acids, wherein the amino acid sequence of the amplified peptide is the same as the amino acid sequence of the peptide(s) of interest, wherein the peptide(s) of interest has a length of from about 2 amino acids to about 200 amino acids. In some embodiments, contacting the biological sample and the mixture of all individual amino acids in an aqueous solution occurs in conditions comprising: (a) a reaction temperature of from about 37°C to about 100°C; (b) a pH of from about 3.0-10.0; (c) a pressure of from about 5 mbar to about 1000 mbar; or (d) the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio between 5: 1 and 500: 1. In some embodiments, the reaction temperature is about 40°C, the pH is about 6.0 and the pressure is about 8 mbar. In some embodiments, the amount of energy is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution. In some embodiments, the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of from about 20,000:1 to about 1 :1, or from about 10,000:1 to about 10:1, with respect to a total weight of the mixture of all individual amino acids and the peptide(s) of interest. In some embodiments, the mixture of all individual amino acids is provided at a concentration of from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, the method is performed in the absence of nucleic acids, enzymes, co-enzymes, cellular material or cells. In some embodiments, the mixture of all individual amino acids comprises unnatural amino acids.

[0015]

[0010] Also provided herein is a device comprising: (a) a sample interface configured to receive a biological sample comprising a peptide(s) of interest; (b) one or more of a temperature regulator, a pH regulator, and a pressure regulator; (c) an energy applicator; (d) a chamber fluidically connected to the sample interface, wherein the chamber is configured to receive an aqueous solution that comprises a mixture of all individual amino acids present in the peptide(s) of interest, thereby admixing the peptide(s) of interest with the mixture of all individual amino acids in the aqueous solution; and (e) a processor and a computer readable memory at least transiently storing instructions that, when executed by the processor, causes the device to: (i) maintain one of more of the following conditions in the chamber: (a) a temperature of from about 37°C to about 70°C using the temperature regulator; (b) a pH of from about 3.0 to about 10.0 using the pH regulator; or (c) a pressure of from about 5 mbar to about 1000 mbar using the pressure regulator; and (ii) amplify the peptide(s) of interest using the mixture of all individual amino acids by providing an amount of energy to the chamber using the energy applicator for a time period sufficient to amplify the peptide(s) of interest using the mixture of all individual amino acids, wherein each amplified copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample; (f) optionally wherein the device further comprises a filter for purifying the amplified peptide from the aqueous solution by solid phase extraction. In some embodiments, the amount of energy is provided is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol, and wherein the amount of energy is provided to the aqueous solution in cycles. In some embodiments, the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to the energy applicator at a distance of at least 1 cm from the aqueous solution. In some embodiments, the device further comprises: (a) a component for evaporation and / or condensation regulation; (b) a component for addition and removal of reagents; (c) a component for removal of amplified peptide; (d) a component for monitoring and documenting the contamination profile during peptide amplification and / or after peptide amplification is terminated; (e) a component for sampling the reaction mixture to assess the progress of peptide amplification; (f) a detector for measuring the peptide(s) of interest, aqueous solution, individual amino acids, amplified peptide, or combinations thereof; or (g) any combination thereof. In some embodiments, the instructions further comprise at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof.

[0016]

[0011] Also provided herein is a system for amplifying a peptide(s) of interest from a biological sample, the system comprising: a mixture of all individual amino acids present in the peptide(s) of interest in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest, wherein the mixture of all individual amino acids is configured to be contacted with the biological sample in an aqueous solution such that copies of the peptide(s) of interest in the biological sample are produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample; an energy generating component configured to provide an amount of energy to the aqueous solution of the peptide(s) of interest and the mixture of all individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and wherein the system is configured to amplify the peptide(s) of interest upon provision of the amount of energy. In some embodiments, contacting the biological sample and the mixture of all individual amino acids in an aqueous solution occurs in conditions comprising: a reaction temperature of from about 30°C to about 100°C; a pH of from about 3.0-10.0; or a pressure of from about 5 mbar to about 1000 mbar. In some embodiments, the reaction temperature is about 40°C, the pH is about 6.0 and the pressure is about 8 mbar. In some embodiments, the peptide(s) of interest comprises a length of from about 2 amino acids to about 200 amino acids. In some embodiments, the amount of energy is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of from about 20,000: 1 to about 1 : 1, or from about 10,000: 1 to about 10: 1, with respect to a total weight of the mixture of all individual amino acids and the peptide(s) of interest. In some embodiments, the mixture of all individual amino acids is provided at a concentration of from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, the system is configured to perform peptide synthesis in the absence of nucleic acids, enzymes, co-enzymes, cellular material or cells. In some embodiments, the mixture of all individual amino acids comprises unnatural amino acids.

[0017] INCORPORATION BY REFERENCE

[0018]

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020]

[0013] FIGURES 1A-1C show an exemplary workflow of peptides amplification from a biological sample in a bench top device. FIG. 1A shows is a representative schematic of steps (a)-(e) according to the methods disclosed herein. FIG. IB shows an exemplary peptide amplification device with consumable peptide amplification kit. FIGURE 1C shows exemplary consumables for peptide amplification, including plate and individual vail capability-specific plastics, an amplification kit according to the present disclosure for targeted amplification, universal amplification kit, and kit with different labels for multiplexing samples.

[0021]

[0014] FIGURES 2A-2B show ratio comparisons of light proteins (native proteins) and heavy proteins (amplified proteins) from biological sample with insulin treatment. FIG. 2A is a bar plot showing the ratio of both light and heavy proteins after insulin treatment to both light and heavy proteins without insulin treatment after peptide amplification for proteins involved in insulin pathway proteins. Y-axis represents intensity of peptides. X-axis represents proteins identified. FIG. 2B is a bar plot with three variables that show the ratio of amplified proteins after insulin treatment versus amplified proteins without insulin treatment, ratio of native proteins with insulin treatment versus amplified proteins without insulin treatment, and ratio of proteins (including both light and heavy peptides) with insulin treatment versus proteins without insulin treatment after peptide amplification for proteins involved in insulin pathway proteins. Y-axis represents relative abundance. X-axis shows protein identified.

[0022]

[0015] FIGURE 3 shows the intensity of peptide signal at different ratios of light peptide (nonamplified peptide) versus the heavy peptide (amplified peptide) from biological samples treated with rapamycin or without treatment of rapamycin. Y-axis represents the intensity of peptide signal from UV-Vis spectra. X-axis represents the ratios.

[0023]

[0016] FIGURES 4A and 4B are Venn diagrams demonstrating the results for both peptides and proteins after the amplification of a biological sample according to the methods disclosed herein. FIG. 4A is a Venn diagram showing the number of light peptides identified (left circle), and the number of heavy peptides identified (right), and the number of peptides that overlap (intersection of the two sets). FIG. 4B is a Venn diagram showing the number of light proteins identified (left circle), and the number of heavy proteins identified (right), and the number of proteins that overlap (intersection of the two sets).

[0024]

[0017] FIGURE 5 shows the peptide sequence of a representative peptide identified according to the methods disclosed herein, which was identified using with heavy-labelled peptide fragments and light peptide fragments. Underline represents only light peptides detected, bold represents only heavy-labelled peptides detected, and bold and underline represents peptides that both heavy and light chains were detected.

[0025]

[0018] FIGURE 6 is a volcano plot a quantification of protein expression comparing protein expression (as measured by both light and heavy-labelled peptides) in rapamycin treated cells as compared to untreated (control). Y-axis represents the negative logarithm of a probability value (-Log p-value). X-axis represents fold-change, proteins with higher intensity in the control sample (left) and in the sample treatment with rapamycin (rapamycin).

[0026]

[0019] FIGURES 7 A - 7D are plots showing proteins with higher intensity in the control biological sample after amplification (FIG. 7A), and the functional enrichment networks identified from the proteins upregulated in control (FIG. 7B) and proteins with higher intensity in the biological sample treated with rapamycin after amplification (FIG. 7C) ), and the functional enrichment networks identified from the proteins upregulated in rapamycin treated cells (FIG. 7D).

[0027]

[0020] FIGURE 8 is a bar plot showing the quantities of peptides prior to amplification and post amplification. Y-axis represents the amount of protein (ug). X-axis shows the treatment group (control, C; rapamycin, R), and amount of starting material used in the methods disclosed herein (1 pg, 2 pg and 10 pg) for four biological replicates each (BR1-BR4).

[0028] DETAILED DESCRIPTION

[0029]

[0021] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0030]

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031]

[0023] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0032] Definitions

[0033]

[0024] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0034]

[0025] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0035]

[0026] The terms, “or” and “and / or,” as used herein, include any and all combinations of one or more of the associated listed items.

[0036]

[0027] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0028] The terms, “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0037]

[0029] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0038]

[0030] The term “actuator,” as used herein in reference to a microfluidic device, refers to a component that causes a machine or other device to operate. An actuator may be a component of a machine that is responsible for moving and controlling a mechanism or system, such as, for example, controlling the opening or closing of a valve.

[0039]

[0031] The terms, “amplification,” “amplifying,” and grammatical equivalents thereof, as used herein, refer to a process by which a peptide copied to generate a plurality of peptides containing the same sequence as the original peptide or a distinguishable portion thereof.

[0040]

[0032] The terms, “bind,” “binding,” “contacting,” “interact” and “interacting,” as used herein, refer to a non-covalent interaction between macromolecules (e.g., between two peptides, between a peptide and a nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific.

[0041]

[0033] The term “cancer,” as used herein, can refer to a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication. The term cancer may be used interchangeably with the terms “carcino-,” “onco-,” and “tumor.”

[0042]

[0034] The term, “chamber,” and “channel,” when used interchangeably herein with reference to a device, such as a microfluidic device, refers to a compartment, which is at least partially enclosed, in the device, such as a separate section, area, or passageway, in which a composition, system, sample, fluid, gas, or loose material may be contained in isolation and / or in which an activity, such as a reaction, can occur. A chamber or channel is generally connected or communicating with another component of the device. A chamber or channel may contain or have the ability to contain matter, such as reagents. Contained materials, such as a composition, system, sample, fluid, gas, or loose material, may be obstructed or allowed movement through a structural component of the device in a controlled manner. Contained materials may be allowed movement from one structural component of the device to another. Alternatively, or in addition, a chamber or channel can also direct or vent air or gases. By way of non-limiting example, a chamber or channels may comprise one or more hydrogels, a well, a flow strip, a heating element, or combinations thereof. Also, by way of non-limiting example, one or more chambers or channels may be in fluid communication, optical communication, or thermal communication. As another non-limiting example, the chambers or channels may be arranged in a sequence, in parallel, or both.

[0043]

[0035] The term, “detection event,” as used herein in reference to a microfluidic device, generally refers to a moment in which compositions within the detection region of a microfluidic device bind to a synthesized peptide or detect peptide bond formation, in accordance to the assay(s) being performed. A detection event may produce a detectable product or a detectable signal.

[0044]

[0036] The term, “detectable product,” as used herein, refers to a unit produced after peptide bond formation that is capable of being discovered, identified, perceived or noticed. A detectable product can comprise a detectable label and / or moiety that emits a detectable signal. A detectable product may include other components that are not capable of being readily discovered, identified, perceived or noticed at the same time as the detectable signal. For example, a detectable product may comprise remnants of a reporter.

[0045]

[0037] The term, “detectable signal,” as used herein, refers to an act, event, physical quantity or impulse that can be detected, discovered, identified, perceived or noticed using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.

[0046]

[0038] The term, “detection region,” as used herein in reference to a microfluidic device, generally refers to a structural component which may comprise detection reagents that are immobilized, dried, or otherwise deposited thereto. A detection region may comprise one or more dried and / or immobilized amplification reagents. Accordingly, in some instances, a detection region may comprise a plurality of microwells, detection chambers or channels, in fluid communication with amplification region(s). By way of a non-limiting example, a detection region may comprise parallel detection chambers, each coupled to a single amplification region. One of ordinary skill in the art will recognize that the relative numbers of and relationships between amplification region(s) and detection region(s) may be varied depending on the assay(s) being performed. Also, by way of a non-limiting example, compositions within the detection region of a microfluidic device may be agitated (e.g., via a spring-loaded valve piston).

[0047]

[0039] The terms “heater”, “heating unit”, “heating element”, “heat source”, and the like, as used herein in reference to a device, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device.

[0048]

[0040] The term, “zzz vitro,'' as used herein, refers to describing something outside an organism. An in vitro system, composition or method may take place in a container for holding laboratory reagents such that it is separated from the biological source from which a material in the container is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term “z ? vivo" is used to describe an event that takes place within an organism. The term “ex vivo" is used to describe an event that takes place in a cell that has been obtained from an organism. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.

[0049]

[0041] The terms, “length” and “linked” as used herein, refer to a nucleic acid (polynucleotide) or polypeptide, may be expressed as “kilobases” (kb) or “base pairs (bp),”. Thus, a length of 1 kb refers to a length of 1000 linked nucleotides, and a length of 500 bp refers to a length of 500 linked nucleotides. Similarly, a protein having a length of 500 linked amino acids may also be simply described as having a length of 500 amino acids.

[0050]

[0042] The terms, “non-naturally occurring” and “engineered,” as used herein, refer to indicate involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a nonlimiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0051]

[0043] The terms, “peptide,” “polypeptide” and “protein,” as used herein, refer to a polymeric form of amino acids. A polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more engineered modifications, or both. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some instances, when a heterologous peptide, such as a fusion partner protein, protein tag or NLS, is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein may be removed or absent.

[0052]

[0044] The term “reagent mix”, “reagent master mix”, “reagents”, and the like, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the formulation is intended for.

[0053]

[0045] The term, “sample,” as used herein, refers to something comprising a peptide(s) of interest. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of nonlimiting example, samples may be modified or manipulated with purification techniques, digestion techniques, heat, nucleic acid amplification, salts and buffers.

[0054]

[0046] The terms “sample interface” and “sample input,” as used herein in reference to a microfluidic device, generally refer to a structural component capable of receiving a composition comprising a peptide(s) of interest as disclosed herein (e.g., a sample). The composition comprising a peptide(s) of interest may be a sample as defined above, which may be collected with a sample collector (e.g., swab, tube, etc.) before being received in a sample interface. By way of a non-limiting example, the sample may be directly collected at the sample interface (e.g., without the use of a separate sample collector). In some instances, a sample interface may be in fluid communication with a plurality of chambers, channels, or reservoirs of a microfluidic device. In some instances, the sample interface is fluidically connected to the plurality of chambers via lysis, preparation, amplification, or detection regions. The term, “subject,” as used herein, refers to an animal. The subject may be a mammal. The subject may be a human. The subject may be diagnosed or at risk for a disease.

[0055]

[0047] The term, “syndrome,” as used herein, refers to a group of symptoms which, taken together, characterize a condition.

[0056]

[0048] The terms, “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.

[0057]

[0049] The term, “template peptide,” as used herein, refers to a peptide that is (designed or intended to be) used as a reference in the synthesis of a peptide, such that the synthesized peptide has the same amino acid sequence as the template peptide.

[0058]

[0050] The terms, “treatment” and “treating,” as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0059]

[0051] The term, “valve,” as used herein, refers to a mechanism or device for directing, regulating, controlling, or obstructing the passage of fluid, gas, or loose materials through an opening or passageway. A valve may regulate the movement of fluid through an opening in one direction only. A valve may operate automatically, pneumatically, hydraulically, mechanically, electrically, chemically or combinations thereof.

[0060]

[0052] The term, “variant,” as used herein, refers to a form or version of a peptide that differs from the wild-type peptide. A variant may have a different function or activity relative to the wild-type peptide.

[0061] Introduction

[0062]

[0053] Disclosed herein are compositions, systems, devices, kits, and methods comprising at least one of: a) A template peptide; b) A mixture of amino acids; and c) An aqueous solution.

[0063]

[0054] Also provided herein are compositions, systems, kits, devices, and methods comprising peptides of interest, mixture of individual amino acids, an aqueous solution, and uses thereof. Various compositions, systems, kits, devices, or methods of the present disclosure may involve synthesizing and / or amplifying a peptide from a template peptide. The methods comprising peptides may be characterized as methods of synthesizing and / or amplifying a peptide. In some embodiments, a template peptide comprises the same amino acid sequence as a peptide of interest. In some embodiments, a synthesized peptide is referred to as a peptide or peptide product. In some embodiments, the template peptide or peptide are characterized as hydrophobic or partially hydrophobic. In some embodiments, the individual amino acids are characterized as natural amino acids or unnatural amino acids. In some embodiments, the aqueous solution is characterized as pure or substantially pure water.

[0064]

[0055] In general, compositions, methods and systems described herein are not found in nature or not naturally occurring. In some embodiments, compositions, systems, devices, kits, and methods described herein comprise at least one non-naturally occurring component. In some embodiments, compositions, systems, devices, kits, and methods comprise at least two components that do not naturally occur together. In some embodiments, compositions, systems, devices, kits, and methods described herein comprise a peptide that is similar to a naturally occurring peptide. In some embodiments, the peptide lacks a portion of the naturally occurring peptide. In some embodiments, the peptide comprises a mutation relative to the naturally-occurring peptide, wherein the mutation is not found in nature. In some embodiments, the peptide also comprises at least one additional amino acid relative to the naturally-occurring peptide. Conversely, and for clarity, a peptide that is “natural,” “naturally-occurring,” or “found in nature” includes peptides from cells or organisms that have not been genetically modified by a human or machine.

[0065] Peptide

[0066]

[0056] Provided herein are compositions, systems and methods comprising a peptide or peptides of interest. A “peptide(s) of interest” as described herein can also be referred to as a template peptide in the present disclosure. In some embodiments, peptide(s) of interest comprises one peptide of interest. In some embodiments, peptide(s) of interest comprises two or more peptides of interest.

[0067]

[0057] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample.

[0068]

[0058] In some embodiments, the peptide comprises a length of at least 2 amino acids to at least 200 amino acids. In some embodiments, the peptide comprises at least 2 amino acids to at least 200 amino acids. In some embodiments, the peptide comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids.

[0069]

[0059] In some embodiments, the peptide comprises a length of about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises about 2 amino acids to about 200 amino acids. In some embodiments, the peptide comprises about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 110 amino acids, about 120 amino acids, about 130 amino acids, about 140 amino acids, about 150 amino acids, about 160 amino acids, about 170 amino acids, about 180 amino acids, about 190 amino acids, about 200 amino acids, about 210 amino acids, about 220 amino acids.

[0070]

[0060] In some embodiments, the template peptide comprises a length of at least 2 amino acids to at least 200 amino acids. In some embodiments, the template peptide comprises at least 2 amino acids to at least 200 amino acids. In some embodiments, the template peptide comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids.

[0071]

[0061] In some embodiments, the template peptide comprises a length of about 2 amino acids to about 200 amino acids. In some embodiments, the template peptide comprises about 2 amino acids to about 200 amino acids. In some embodiments, the template peptide comprises about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 110 amino acids, about 120 amino acids, about 130 amino acids, about 140 amino acids, about 150 amino acids, about 160 amino acids, about 170 amino acids, about 180 amino acids, about 190 amino acids, about 200 amino acids, about 210 amino acids, about 220 amino acids.

[0072]

[0062] In some embodiments, the peptide comprises amino acids that are about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 65% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 70% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 75% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 80% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 85% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 90% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 95% hydrophobic. In some embodiments, the peptide comprises amino acids that are about 100% hydrophobic.

[0073]

[0063] In some embodiments, the template peptide comprises amino acids that are about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 65% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 70% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 75% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 80% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 85% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 90% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 95% hydrophobic. In some embodiments, the template peptide comprises amino acids that are about 100% hydrophobic.

[0074]

[0064] In some embodiments, the peptide comprises a secondary structure. In some embodiments, the peptide comprises a helical secondary structure. In some embodiments, the peptide comprises a beta sheet secondary structure. In some embodiments the peptide comprises a random coil structure. In some embodiments, the peptide comprises a secondary structure that is a combination of a helical, random coil and beta sheet. In some embodiments, the peptide comprises a structural conformation determined by one or more disulfide bonds between amino acids in the peptide. In some embodiments, the peptide comprises a structural conformation that requires folding and / or non-linear assembly. In some embodiments, the peptide is any one peptide described in TABLE 1. In some embodiments, the peptide is insulin. In some embodiments, the peptide is a peptide with a therapeutic target described in TABLE 2. In some embodiments, the peptide is a peptide with a therapeutic target associated with a disease or syndrome described in TABLE 3.

[0075]

[0065] In some embodiments, the peptide is a peptide variant. In some embodiments, the peptide variant is a form or version of a peptide that differs from a naturally occurring peptide or a wild-type peptide. In some embodiments, the peptide variant comprises a different function or activity relative to the naturally occurring peptide or the wild-type peptide. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying the peptide variant, the naturally occurring peptide or the wild-type peptide. In some embodiments, methods for synthesizing and / or amplifying the peptide described herein comprise synthesizing and / or amplifying a peptide comprising the naturally occurring peptide sequence or the wild-type peptide sequence. In some embodiments, methods for synthesizing and / or amplifying the peptide described herein comprise synthesizing and / or amplifying a peptide comprising a peptide variant sequence.

[0076] Aqueous Solution

[0077]

[0066] Provided herein are compositions, systems and methods comprising an aqueous solution. An aqueous solution as described herein can also be referred to as a composition. In some embodiments, the aqueous solution described herein comprises system components or method components.

[0078]

[0067] In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises pure water. For example, the aqueous solution comprises pure or substantially pure water, the template peptide and the individual amino acids capable of forming copies of the template peptide. In some embodiments, the aqueous solution comprises the template peptide and a mixture of individual amino acids.

[0079]

[0068] In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the pure or substantially pure water is MilliQ® water, which is a form of ultrapure water of Type 1 (as defined by ISO 3696 (1987)). In some embodiments, the pure or substantially pure water is provided by capacitive deionization, reverse osmosis, carbon filtering, microfiltration, ultrafiltration, ultraviolet oxidation and the like. In some embodiments, the pure or substantially pure water comprises a low level of solids, low organics and low conductivity. In some embodiments, the pure or substantially pure water comprises less than 5 pg / ml solids, less than 1 pg / ml solids, or even less than 0.1 pg / ml solids. In some embodiments, the pure or substantially pure water comprises organics content of less than 100pg / l, or less than 50pg / l total organic carbon. Additionally, or alternatively, pure or substantially pure water may have a conductivity of less than IpS-cm-l, less than 0.1 pS cm- 1, or even less than 0.01 pS.cm-1 at 25°C.

[0080]

[0069] In some embodiments, the aqueous solution is free or substantially free of biological contaminants. In some embodiments, the aqueous solution is free or substantially free of nucleic acids, enzymes, co-enzymes (such as adenosine triphosphate), cells, cellular material and / or organic solvents. For example, the aqueous solution may be free or substantially free from bacteria, viruses, eukaryotic cells and / or components thereof, such as organelles.

[0081]

[0070] In some embodiments, the aqueous solution is filtered. In some embodiments, the aqueous solution is sterilized. In some embodiments, the aqueous solution is irradiated. In some embodiments, the aqueous solution is irradiated with gamma irradiation. In some embodiments, the aqueous solution is sterile.

[0082]

[0071] In some embodiments, the aqueous solution comprises a buffer. In some embodiments, the buffer comprises phosphate buffers, Tris buffers, HEPES buffers, or combinations thereof. In some embodiments, the buffer comprises a saline solution. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution comprises the water, the buffer, the saline solution, the acid, the base, or combinations thereof. In some embodiments, the aqueous solution further comprises the template peptide and the individual amino acids capable of forming copies of the template peptide.

[0083]

[0072] In some embodiments, the aqueous solution comprises a source of phosphate. In some embodiments, the aqueous solution comprises phosphate in an amount of at least 50pM, such as lOOpM, or 1 mM. In some embodiments, the aqueous solution comprises water comprising disodium hydrogen phosphate or phosphate buffered saline solution (PBS). In some embodiments, the PBS comprises disodium hydrogen phosphate, sodium chloride. In some embodiments, the PBS comprises potassium chloride and potassium dihydrogen phosphate. In some embodiments, the PBS comprises:

[0084]

[0073] In some embodiments, the aqueous solution comprises a source of phosphate, a peptide, and individual amino acids. In some embodiments, the aqueous solution comprises a source of phosphate, a template peptide, and a mixture of individual amino acids.

[0074] In some embodiments, the aqueous solution comprises individual amino acids. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a peptide. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a template peptide. In some embodiments, the aqueous solution comprising individual amino acids is contacted with a peptide(s) of interest from a biological sample.

[0085]

[0075] In some embodiments, the aqueous solution comprises a mixture of individual amino acids. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a peptide. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a template peptide. In some embodiments, the aqueous solution comprising a mixture of individual amino acids is contacted with a peptide(s) of interest from a biological sample.

[0086]

[0076] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide in an aqueous solution. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide in an aqueous solution comprising a mixture of individual amino acids. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the methods described herein comprise an aqueous solution described herein.

[0087] Individual Amino Acids

[0088]

[0077] Provided herein are compositions, systems and methods comprising individual amino acids or a mixture of individual amino acids. In some embodiments, the individual amino acids or the mixture of individual amino acids comprise the amino acids present in a peptide or peptide(s) of interest. As used herein the term “mixture of all individual amino acids” refers to all amino acids present in the peptide(s) of interest or the template peptide. In some embodiments, the individual amino acids or the mixture of individual amino acids comprise the amino acids present in a template peptide. In some embodiments, the individual amino acids or the mixture of individual amino acids are present in an aqueous solution as described herein, which can also be referred to as a composition.

[0089]

[0078] In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the mixture of individual amino acids comprises amino acids that comprise chemical modifications. In some embodiments, the mixture of individual amino acids comprises amino acids with post-translational modifications. In some embodiments, the mixture of amino acids comprises amino acids with naturally occurring moieties. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof.

[0090]

[0079] In some embodiments, the mixture of individual amino acids comprises an amount that is at least equal to the stoichiometric amount of each amino acid in a template peptide sequence. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the template peptide. In some embodiments, the mixture of individual amino acids comprises a concentration in the aqueous solution from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the mixture of individual amino acids in the aqueous solution are contacted with the template peptide. In some embodiments, the mixture of individual amino acids and the template peptide are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the template peptide.

[0091]

[0080] In some embodiments, the total weight of the amino acids and the weight of the template peptide may be provided in a w / w (weight by weight) ratio of anywhere between 20,000 to 1 and 5 to 1. For example, a w / w ratio between 15,000 to 1; 10,000 to 1; 5 000 to 1; 1 000 to 1; 500 to 1, 100 to 1, 50 to 1, 10 to 1, and 5 to 1 w / w; By way of further example, the solution may comprise about 21.5 to 1 w / w of the amino acids to the template peptide.

[0092]

[0081] In some embodiments, the total weight of the amino acids in a solution result in a concentration between about 0.001 g / mL and 10 g / mL, or between about 0.005 g / mL and 5 g / mL, or between about 0.01 g / mL and 1 g / mL. In some embodiments, the solution comprises a concentration of amino acids about 0.02 g / mL. In some embodiments, the concentration of the solution depends upon the composition of the template peptide. In some embodiments, the template peptide comprises a relatively higher proportion of amino acids having higher water solubility, such that a more concentrated solution is possible for peptide synthesis.

[0093] Method of Peptide Synthesis

[0082] Provided herein are methods comprising a peptide or peptide(s) of interest. Surprisingly, the methods for peptide synthesis described herein can be performed in an aqueous solution as described herein and without the need for chemical solvents or toxic solvents. Thus, the methods for peptide synthesis described herein results in the desired yield of synthesized peptide without the waste of chemical solvents or toxic solvents common in methods of peptide synthesis known in the art (Solid Phase Peptide Synthesis - SPPS).

[0094]

[0083] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the methods described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample (e.g., a blood sample, a tissue sample, a cell sample). In some embodiments, the biological sample can include but is not limited to, blood, serum, urine, saliva, mucus, cerebrospinal fluid, or any bodily fluid. In some embodiments, a biological sample comprise a biopsy sample, a tumor tissue, a skin sample, a hair follicle, or any cells removed from a subject. In some embodiments, the biological sample is isolated from any suitable biological tissue or biopsy including, but not limited to, adipose tissue, cardiac tissue, liver tissue, lung tissue, tissue of the gastrointestinal tract (e.g., esophagus, intestine, intestinal polyps, colon, etc.), kidney tissue, bladder tissue, tumor tissue (cancerous or non-cancerous, benign), thyroid tissue, muscle tissue, prostate tissue, skin, or blood. In some embodiments, peptide synthesis as described herein comprises a template peptide, mixture of individual amino acids, an aqueous solution, or combinations thereof. In some embodiments, the peptide provided herein is contacted with individual amino acids in an aqueous solution in the presence of a sufficient amount of energy to form a peptide bond. In some embodiments, the peptide(s) of interest as described herein can also be referred to as a template peptide in the present disclosure.

[0095]

[0084] Provided herein is a method of synthesizing a peptide, the method comprising: (a) providing a peptide; (b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in a peptide sequence; (c) contacting the peptide and the mixture of individual amino acids in an aqueous solution; (d) providing of energy to the aqueous solution comprising the peptide and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the peptide amino acid sequence; and (e) performing step (d) for a time period sufficient to synthesize a peptide from the mixture of individual amino acids, wherein the amino acid sequence of the synthesized peptide is the same as the amino acid sequence of the original peptide.

[0096]

[0085] In some embodiments, the methods described herein, wherein the conditions comprise: (i) a pH of about 3.0-10.0; or (ii) a pressure of about 5 mbar to about 20 mbar. In some embodiments, the conditions further comprise a reaction temperature of about 37°C to about 70°C. In some embodiments, the conditions further comprise a reaction temperature of about 40°C. In some embodiments, the pH is about 6.0. In some embodiments, the pressure is about 8 mbar. In some embodiments, the amount of energy provided to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the amount of energy is provided to the aqueous solution in cycles. In some embodiments, the amount of energy provided to the aqueous solution in cycles comprises periodically increased the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light, using an energy applicator. In some embodiments, the energy applicator is a light source or a heating element. In some embodiments, the light source emits full spectrum light. In some cases, the light source is a full spectrum light source (LED). In some embodiments, periodically exposing the aqueous solution to full spectrum light using a LED at a distance of at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, or at least 5 cm from the aqueous solution.

[0097]

[0086] In some embodiments, the amino acids and template peptide may be added to the aqueous solution sequentially or simultaneously. In some embodiments, the method facilitates a peptide synthesis, wherein all the reagents required (e.g., the amino acids and template peptide) are added into the solution at the outset of the synthesis. Consequently, the methods described herein may obviate the need to use complex chemistries and / or multistep processes (such as the need to use protecting group strategies and / or solid phase components). By providing a template peptide in the method, it can be possible to obtain copies of the template peptide in high quantity and quality. Typically, at least 80%, 90%, 95%, 99%, 99.5% or even higher amounts of the peptides produced are identical to the template peptide.

[0098]

[0087] In some embodiments, the progress of the peptide synthesis is monitored. For example, the amount of synthesized peptide in the aqueous solution may be monitored using mass- spectrometry (MS) techniques (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion-trap electrospray MS).

[0088] In some embodiments, the increase in the amount of synthesized peptide is observed initially, as the amino acids react to form copies of the template peptide. In some embodiments, the amount of synthesized peptide plateaus (e.g., as all the starting amino acids are consumed). In some embodiments, after an initial increase, the amount of synthesized peptide falls (e.g., if additional amino acids start to add to the synthesized peptide, increasing the length of the peptide chain). In some embodiments, peptide synthesis is terminated when an observed amount of synthesized peptide plateaus or decreases.

[0099]

[0089] In some embodiments, the duration of peptide synthesis is dependent upon the length of the template peptide and / or the nature of the amino acids within it. For example, shorter template peptides may be copied more rapidly than longer template peptides. Therefore, monitoring the peptide synthesis can be used to experimentally determine the duration of the peptide synthesis reaction that will provide the desired peptide yield.

[0100]

[0090] In some embodiments, peptide synthesis is terminated by removing the energy provided, cooling the aqueous solution (e.g., to a temperature below 10 °C, below about 5 °C or below about 0 °C), removing the mixture of amino acids from the aqueous solution, or combinations thereof.

[0101] Method of Amplifying a Peptide

[0102]

[0091] Provided herein are methods comprising a peptide or peptide(s) of interest. Surprisingly, the methods for peptide synthesis described herein can be performed in an aqueous solution as described herein and without the need for chemical solvents or toxic solvents. Thus, the methods for peptide synthesis described herein result in the desired yield of synthesized peptide without the waste of chemical solvents or toxic solvents common in methods of peptide synthesis known in the art.

[0103]

[0092] In some embodiments, a method of amplifying a peptide(s) of interest from a biological sample, the method comprising: (a) providing a biological sample comprising a peptide(s) of interest for amplification; (b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest; (c) contacting the biological sample and the mixture of individual amino acids in an aqueous solution such that copies of the peptide(s) of interest in the biological sample is produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample; (d) providing of energy to the aqueous solution of the peptide(s) of interest and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and (e) performing step (d) for a time period sufficient to amplify the peptide(s) of interest with the mixture of individual amino acids, wherein the amino acid sequence of the amplified peptide is the same as the amino acid sequence of the peptide(s) of interest. In some embodiments, the peptide(s) of interest comprises about 2 amino acids to about 200 amino acids. In some embodiments, the peptide(s) of interest comprises amino acids that are about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% hydrophobic. In some embodiments, contacting the biological sample and the mixture of individual amino acids in an aqueous solution occurs in conditions comprising: (a) a reaction temperature of about 37°C to about 70°C; (b) a pH of about 3.0-10.0; or (c) a pressure of about 5 mbar to about 20 mbar. In some embodiments, contacting the biological sample and the mixture of individual amino acids in an aqueous solution occurs in conditions comprising: (a) a reaction temperature of about 37°C to about 100 °C; (b) a pH of about 7; or (c) a pressure of about 5 mbar to about 20 mbar. In some embodiments, the reaction temperature is about 40°C. In some embodiments, the pH is about 6.0. In some embodiments, the pressure is 8 about mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by a cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the aqueous solution further comprises the peptide(s) of interest and the mixture of individual amino acids after contacting. In some embodiments, the aqueous solution is sterile. In some embodiments, the mixture of individual amino acids comprises only the amino acids that constitute the peptide(s) of interest. In some embodiments, the mixture of individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the peptide(s) of interest. In some embodiments, the concentration of the mixture of individual amino acids in the aqueous solution prior of to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL. In some embodiments, the time period is from 10 minutes to 5 days. In some embodiments, amplifying a peptide is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution. In some embodiments, the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2. In some embodiments, the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells. In some embodiments, the mixture of individual amino acids comprises natural amino acids. In some embodiments, the mixture of individual amino acids comprises unnatural amino acids. In some embodiments, the unnatural amino acids comprise labeled amino acids. In some embodiments, the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or any synthetic, modified or unnatural amino acid, or combinations thereof. In some embodiments, amplifying a peptide is optimized for maximizing peptide yield and minimizing contaminants. In some embodiments, the biological sample (e.g., a blood sample, a tissue sample, a cell sample) is processed prior to contacting the biological sample and the mixture of individual amino acids in an aqueous solution. In some embodiments, the processing is digesting and / or purifying. In some embodiments, the method described herein further comprising amplification reagents. pH

[0104]

[0093] Provided herein are compositions, systems and methods comprising a peptide or peptide(s) of interest at a controlled pH or a variable pH. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a peptide(s) of interest from a biological sample.

[0105]

[0094] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled pH. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable pH.

[0106]

[0095] In some embodiments, the rate of synthesis and / or amplification changes as a function of pH. In some embodiments, the yield of synthesis and / or amplification changes as a function of pH. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of pH. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction pH. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pH of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0107]

[0096] In some embodiments, the provision of energy to the aqueous solution modulates the pH of the aqueous solution. In some embodiments, the provision of energy to the aqueous solution maintains the aqueous solution at a constant pH. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant pH while being provided energy from full spectrum light.

[0108]

[0097] In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy. In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a pH of about 6 while being provided energy from full spectrum light.

[0109]

[0098] In some embodiments, the pH ranges from about 3.0 to about 10. In some embodiments, the reaction pH ranges from about 3.0 to about 10. In some embodiments, the pH ranges from about 4.0 to about 5.0, from about 4.0 to about 6.0, from about 5.0 to about 7.0, from about 6.0 to about 8.0 from about 7.0 to about 9.0, from about 8.0 to about 10, or from about 5.0 to about 10.

[0110]

[0099] In some embodiments, the pH is about 3.0 to about 10. In some embodiments, the reaction pH is about 3.0 to about 10. In some embodiments, the pH ranges from about 4.0 to about 5.0, from about 4.0 to about 6.0, from about 5.0 to about 7.0, from about 6.0 to about 8.0 from about 7.0 to about 9.0, from about 8.0 to about 10, or from about 5.0 to about 10. In some embodiments, the pH is about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or about 10. In some embodiments, the pH is about 6.0.

[0111] Pressure

[0112]

[0100] Provided herein are compositions, systems and methods comprising a peptide or peptide(s) of interest at a controlled pressure or a variable pressure. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a peptide(s) of interest from a biological sample.

[0113]

[0101] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled pressure. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable pressure.

[0114]

[0102] In some embodiments, the rate of synthesis and / or amplification changes as a function of pressure. In some embodiments, the yield of synthesis and / or amplification changes as a function of pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction pressure. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the pressure of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0115]

[0103] In some embodiments, the aqueous solution is maintained at a variable pressure while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant pressure while being provided energy from full spectrum light.

[0104] In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy. In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a pressure of about 8 mbar while being provided energy from full spectrum light.

[0116]

[0105] In some embodiments, the pressure ranges from about 5 mbar to about 20 mbar. In some embodiments, the reaction pressure ranges from about 5 mbar to about 20 mbar. In some embodiments, the pressure ranges from about 5 mbar to about 10 mbar, from about 6 mbar to about 9 mbar, from about 7 mbar to about 12 mbar, from about 9 mbar to about 14 mbar, from about 11 mbar to about 16 mbar, from about 13 mbar to about 18 mbar, or from about 15 mbar to about 20 mbar.

[0117]

[0106] In some embodiments, the pressure is about 5 mbar to about 20 mbar. In some embodiments, the reaction pressure is about 5 mbar, about 6 mbar, about 7 mbar, about 8 mbar, about 9 mbar, about 10, mbar, about 11 mbar, about 12 mbar, about 13 mbar, about 14 mbar, about 15 mbar, about 16 mbar, about 17 mbar, about 18 mbar, about 19 mbar, or about 20 mbar.

[0118]

[0107] In some embodiments, the pressure is controlled for air or air in the presence of oxygen. In some embodiments, the pressure is controlled in the presence of oxygen, Nitrogen, hydrogen, or CO2.

[0119] Temperature

[0120]

[0108] Provided herein are compositions, systems and methods comprising a peptide or peptide(s) of interest at a controlled temperature or a variable temperature. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a peptide(s) of interest from a biological sample.

[0121]

[0109] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a controlled temperature. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide at a variable temperature.

[0122] [HO] In some embodiments, the rate of synthesis and / or amplification changes as a function of temperature. In some embodiments, the yield of synthesis and / or amplification changes as a function of temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction temperature. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide prior to the provision of energy to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the temperature of the aqueous solution comprising individual amino acids and a template peptide during to the provision of energy to the aqueous solution.

[0123] [Hl] In some embodiments, the provision of energy to the aqueous solution modulates the temperature of the aqueous solution. In some embodiments, the provision of energy to the aqueous solution maintains the aqueous solution at a constant temperature. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a constant temperature while being provided energy from full spectrum light.

[0124]

[0112] In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy. In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is maintained at a temperature of about 40°C while being provided energy from full spectrum light.

[0125]

[0113] In some embodiments, the temperature ranges from about 37°C to about 70°C. In some embodiments, the reaction temperature ranges from about 37°C to about 70°C. In some embodiments, the temperature ranges from about 35°C to about 45°C, from about 35°C to about 40°C, from about 40°C to about 60°C, from about 50°C to about 60°C from about 55°C to about 65°C, from about 60°C to about 70°C, or from about 65°C to about 70°C.

[0126]

[0114] In some embodiments, the temperature is about 37°C to about 70°C. In some embodiments, the reaction temperature is about 37°C to about 70°C. In some embodiments, the temperature is about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 69°C, about 60°C, , about 61 °C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C.

[0127] Energy

[0128]

[0115] Provided herein are compositions, systems and methods comprising a peptide or peptide(s) of interest provided energy. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a template peptide. In some embodiments, a peptide(s) of interest as described herein can also be referred to as a peptide(s) of interest from a biological sample.

[0129]

[0116] In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide provided energy. In some embodiments, the provided energy is a variable energy or a controlled energy. In some embodiments, the provided energy is energy from full spectrum light. In some embodiments, the amount of provided energy is related to the concentration of peptide and / or amino acids. In some embodiments, the methods described herein are methods for synthesizing, detecting, and / or amplifying a peptide provided energy. In some embodiments, the amount of provided energy is related to the rate of synthesizing, detecting, and / or amplifying a peptide.

[0130]

[0117] In some embodiments, the rate of synthesis and / or amplification changes as a function of energy. In some embodiments, the yield of synthesis and / or amplification changes as a function of energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of provided energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of reaction provided energy. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the provided energy of the aqueous solution comprising individual amino acids and a template peptide. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the energy provided to the aqueous solution comprising individual amino acids prior to the providing of a template peptide to the aqueous solution. In some embodiments, the yield of synthesis and / or amplification is optimized as a function of the energy provided to the aqueous solution comprising individual amino acids after the provision of the template peptide to the aqueous solution.

[0131]

[0118] In some embodiments, the provision of energy to the aqueous solution modulates the time for synthesis and / or amplification. In some embodiments, the aqueous solution is provided constant energy for the duration of the peptide synthesis and / or amplification. In some embodiments, the aqueous solution is provided energy from full spectrum light. In some embodiments, a full spectrum light source may provide light having wavelengths between about 300 nm to about 700 nm. The full spectrum light may be configured to mimic the composition of natural light. In some embodiments, the energy provided is sunlight, full spectrum light, UV light, heat, electromagnetic radiation, or any other form of energy appropriate for peptide bond formation. In some embodiments, the energy provided is any form of energy that allows peptide bond formation, for example light, heat, or other electromagnetic radiation.

[0132]

[0119] In some embodiments, the energy provided is the energy sufficient for peptide bond formation. In some embodiments, the energy provided is the energy sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the energy provided is the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the template peptide amino acid sequence. In some embodiments, the energy provided is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the energy is provided for a time period sufficient to synthesize the peptide from the mixture of individual amino acids, wherein the amino acid sequence of the peptide is the same as the amino acid sequence of the template peptide. In some embodiments, the energy is provided for a time period of at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or at least 5 hours. In some embodiments, the energy is provided in dark conditions or in the presence of light.

[0133]

[0120] In some embodiments, the energy provided is constant. In some embodiments, the energy provided is a constant source of full spectrum light for the duration of the peptide synthesis.

[0134]

[0121] In some embodiments, an amount of energy provided is cyclical or repetitive. In some embodiments, the energy is provided for a period of time, and then stopped, before providing the energy again for a further period of time.

[0135]

[0122] In some embodiments, an amount of energy is provided to the aqueous solution in cycles. In some embodiments, the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to the energy applicator at a distance of at least 1 cm from the aqueous solution. In some embodiments, the amount of energy is provided to the aqueous solution in cycles comprising periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises an energy applicator at a distance of at least 1 cm from the aqueous solution. In some embodiments, the energy applicator emits full spectrum light. In some embodiments, the energy applicator comprises a full spectrum light source (LED). In some embodiments, the energy applicator is at a distance of 0.5 cm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, or 3 cm. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the amount of energy is provided in cycles comprising a periodical increasing the heat of the aqueous solution, for example by at least 0.25°C, 0.5°C, 1°C, 5°C, or even 10°C, 15°C, 20°C, or 100 °C. Optionally, the aqueous solution is permitted to cool to ambient temperature, before the application of heat occurs again. In some embodiments, the amount of energy provided to the aqueous solution corresponds to the rise and fall in ambient temperature during a day / night cycle. This process may be repeated many, hundreds or even thousands of times. In some embodiments, the aqueous solution is exposed to a constant source of light (e.g. full spectrum light) whilst also being subjected to cyclical temperature oscillations as described above. Alternatively, the aqueous solution is exposed to a cyclical provision of light (e.g., full spectrum light) under constant temperature conditions.

[0136] Synthesis, Isolation and Assaying

[0137]

[0123] Peptides of the present disclosure are synthesized, using the method described herein. In some embodiments, the peptides are synthesized in vitro. In some embodiments, the peptides are further processed by isolation and / or purification.

[0138]

[0124] In some embodiments, a peptide provided herein is an isolated peptide. In some embodiments, the peptide is isolated and purified as described herein. In some embodiments, methods described here comprise the step of isolating peptides described herein. Any suitable method to provide isolated peptides described herein is used in the present disclosure, for example, precipitation, filtration, ion-exchange, and / or chromatography. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). The methods and conditions for biochemical purification of a peptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

[0139]

[0125] In some embodiments, a peptide provided herein is purified from the aqueous solution. In some embodiments, purification and / or isolation are performed through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some embodiments, purification comprises binding members which can selectively bind the peptide(s) of interest, for example an antibody or fragment thereof. In some embodiments, the peptide(s) of interest provided by the method is provided with a tag to allow it to be purified. In some embodiments, beads with a binding member specific for the peptide(s) of interest or tag are attached.

[0140]

[0126] In some embodiments, purification is monitored by mass-spectrometry (MS) techniques, including matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion-trap electrospray MS. Sequencing by Edman degradation sequence analysis, or in tandem with MS, for example, can be used to confirm peptide homogeneity.

[0141]

[0127] In some embodiments, peptides described herein are isolated from a biological sample. In some embodiments, the compositions described herein comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, 98% or more by weight, or 99.5% or more by weight of the peptide, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages refer to total peptide content relative to contaminants. Thus, in some embodiments, the peptide is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-desired peptides or other macromolecules, etc.).

[0142] Detection Reagents / Components and Reporters

[0143]

[0128] In some embodiments, systems disclosed herein comprise detection reagents to facilitate detection of peptides as described herein. Non-limiting examples of detection reagents include reporter nucleic acids, detection moieties, additional polypeptides, or combinations thereof. Upon the occurrence of the detection event, a signal (e.g., a detectable signal or detectable product) can be generated thereby indicating peptide synthesis or detection of a peptide. In some embodiments, any suitable detection reagent may be used. Any reagents suitable with the detection reactions, events, and signals described herein are useful as detection reagents for the systems, compositions, methods, kits, devices, and solutions provided herein, including a buffer, stain reporter, or combinations thereof.

[0144]

[0129] In some embodiments, detection reagents detect a peptide in a sample. In some embodiments, peptide amplification improves at least one of sensitivity, specificity, or accuracy of the detection assay. Accordingly, in some embodiments, detection assay comprises amplification, providing improved sensitive, specific, or rapid detection. In some embodiments, detection reagents comprise peptides, buffers, and / or signal reagents suitable for a detection reaction.

[0145]

[0130] In some embodiments, detection is performed in a detection region on a support medium, or sample interface. Alternatively, or in combination, the detection is performed in a reagent chamber, and the resulting sample is applied to the support medium, sample interface, or surface within a reagent chamber.

[0146]

[0131] In some embodiments, detection reaction of peptides as described herein is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the detection reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the detection reaction is performed at a temperature of around 20-80°C. In some embodiments, the detection reaction is performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 53°C, 55°C, 58°C, 60°C, 62°C, 65°C, 68°C, 70°C, 75°C, 80°C or any value 20 °C to 80 °C. In some embodiments, the detection reaction is performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C, or any value 20°C to 80°C. In some embodiments, the detection reaction is performed at a temperature of 20°C to 45°C, 35°C to 60°C, 40°C to 70°C, or 50°C to 65°C.

[0147]

[0132] In some embodiments, the reagents or components for detecting a nucleic acid are, for example, consistent for use within various fluidic devices disclosed herein for detection of a peptide within the sample, wherein the fluidic device may comprise multiple pumps, valves, reservoirs, and chambers for sample preparation, amplification, mixing, and detection of a detectable signal arising from peptide synthesis within the fluidic system itself. These reagents are compatible with the samples, solutions, compositions, systems, devices, fluidic devices, methods of detection, and support mediums as described herein. In some embodiments, systems disclosed herein comprise a reporter.

[0133] In some embodiments, a reporter comprises a protein that generates a detectable signal or signal. In some embodiments, a reporter is operably linked to the protein that generates a signal. In some embodiments, a signal is a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some embodiments, the reporter comprises a detection moiety. In some embodiments, the reporter is configured to release a detection moiety or generate a signal. In some embodiments, suitable detectable labels and / or moieties provide a signal. In some embodiments, non-limiting example of a suitable detectable label and / or moiety comprises an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; and a quantum dot.

[0148]

[0134] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, P-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).

[0149]

[0135] In some embodiments, the reporter comprises a detection moiety. In some embodiments, the detection moiety comprises a fluorescent dye. Sometimes the detection moiety comprises a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the detection moiety comprises an infrared (IR) dye. In some embodiments, the detection moiety comprises an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety comprises a protein. Sometimes the detection moiety comprises an antigen. Sometimes the detection moiety comprises a biotin. Sometimes the detection moiety comprises at least one of avidin or streptavidin. In some embodiments, the detection moiety comprises a polysaccharide, a polymer, or a nanoparticle. In some embodiments, the detection moiety comprises a gold nanoparticle or a latex nanoparticle.

[0136] In some embodiments, a detection moiety comprises any moiety that generates a detectable product or detectable signal upon peptide synthesis. In some embodiments, the detectable product comprises a detectable unit generated from the detectable moiety and that emits a detectable signal as described herein. In some embodiments, the detectable product further comprises a detectable label, a fluorophore, a reporter, or a combination thereof. Iln some embodiments, the detectable product is configured to generate a signal indicative of the presence or absence of the synthesized peptide.

[0150]

[0137] In some embodiments, a detection moiety comprises any moiety that generates a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A nucleic acid of a reporter, sometimes, is protein-nucleic acid that generates a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc. , or piezo-electric signal upon peptide synthesis. Often a calorimetric signal is heat produced after peptide synthesis. Sometimes, a calorimetric signal is heat absorbed after peptide synthesis. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after peptide synthesis. Sometimes, an optical signal is a change in light absorbance between before and after the peptide synthesis. Often, a piezo-electric signal is a change in mass between before and after the peptide synthesis.

[0151]

[0138] In some embodiments, the detectable signal comprises a colorimetric signal or a signal visible by eye. In some embodiments, the detectable signal may be fluorescent, electrical, chemical, electrochemical, or magnetic. In some embodiments, there is more than one detectable signal. In some embodiments, the detectable signal is generated directly by the peptide bond formation event. Alternatively, or in combination, the detectable signal is generated indirectly by the peptide bond formation event. Sometimes the detectable signal is not a fluorescent signal. In some embodiments, the detectable signal comprises a colorimetric or color-based signal. In some embodiments, the detected peptide is identified based on its spatial location on the detection region of the support medium. In some embodiments, the detectable signal is generated after a peptide yield is reached during peptide synthesis reaction.

[0152] Amplification Reagents / Components

[0153]

[0139] In some embodiments, systems described herein comprise a reagent or component for amplifying a peptide. In some embodiments, peptide amplification is isothermal peptide amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification.

[0154]

[0140] Often, the peptide amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes. In some embodiments, the amplification reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. In some embodiments, the amplification reaction is performed at a temperature of around 20°C to 45°C. In some embodiments, the amplification reaction is performed at a temperature of around 20°C to 70°C. In some embodiments, the amplification reaction is performed at a temperature of around 20°C to 100°C. In some embodiments, the amplification reaction is performed at a temperature no greater than 20°C, 25°C, 27°C, 30°C, 35°C, 37°C, 40°C, 45°C, 47°C, 50°C, 55°C, 57°C, 60°C, 65°C, 67°C, 70°C, 75°C, 77°C, 80°C, 85°C, 87°C, 90°C, 95°C, 97°C, 100°C, or any value 20 °C to 100 °C. In some embodiments, the amplification reaction is performed at a temperature of at least 20°C, 25°C, 27°C, 30°C, 35°C, 37°C, 40°C, 45°C, 47°C, 50°C, 55°C, 57°C, 60°C, 65°C, 67°C, 70°C, 75°C, 77°C, 80°C, or any value 20 °C to 80 °C. In some embodiments, the amplification reaction is performed at a temperature of 20°C to 45°C, 25°C to 40°C, 30°C to 40°C, 35°C to 40°C, 40°C to 45°C, 45°C to 50°C, 50°C to 55°C, 55°C to 60°C, 35°C to 40°C, 50°C to 65°C, 65°C to 70°C, 70°C to 80°C, or 75°C to 80°C.

[0155] Additional System Components

[0156]

[0141] Provided herein are systems comprising a peptide or peptide(s) of interest. In some embodiments, the systems described herein are systems for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the systems described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the systems described herein comprise a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a peptide that is the product of synthesis or amplification. In some embodiments, the systems described herein are used for the methods of synthesizing, detecting, and / or amplifying a peptide described herein.

[0157]

[0142] In some embodiments, systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, or tubes, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, test wells, bottles, vials, syringes, and test tubes. In some embodiments, the containers are formed from a variety of materials such as glass, plastic, or polymers. In some embodiments, the system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.

[0158]

[0143] In some embodiments, systems described herein include labels listing contents and / or instructions for use, or package inserts with instructions for use. In some embodiments, the systems include a set of instructions and / or a label is on or associated with the container. In some embodiments, the label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container (e.g., as a package insert). In some embodiments, the label is used to indicate that the contents are to be used for a specific therapeutic application. In some embodiments, the label indicates directions for use of the contents, such as in the methods described herein. In some embodiments, after packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing.

[0159] Devices

[0160]

[0144] Provided herein are devices capable of amplifying a peptide(s) of interest from a biological sample. In some embodiments, a device comprises a sample interface configured to receive a biological sample comprising a peptide(s) of interest ', one or more of a temperature regulator, a pH regulator, and a pressure regulator, an energy applicator, a chamber fluidically connected to the sample interface, and a processor and a computer readable memory. In some embodiments, the chamber is configured to receive an aqueous solution that comprises a mixture of all individual amino acids in the peptide(s) of interest, thereby admixing the peptide(s) of interest with the mixture of individual amino acids in the aqueous solution. In some embodiments, the processor and the computer readable memory at least transiently stores instructions. In some embodiments, the processor and the computer readable memory permanently stores instructions. In some cases, when the processor is executed, it causes the device to maintain one of more conditions in the chamber. In some embodiments, the one or more conditions in the chamber comprise a temperature of from about 37°C to about 70°C using the temperature regulator, a pH of from about 3.0 to about 10.0 using the pH regulator, or a pressure of from about 5 mbar to about 1000 mbar using the pressure regulator. In some embodiments, when the one or more conditions in the chamber is maintained, the device amplifies the peptide(s) of interest using the mixture of all individual amino acids by providing an amount of energy to the chamber using the energy applicator for a time period sufficient to amplify the peptide(s) of interest using the mixture of individual amino acids. In some embodiments, the amplified peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample. In some instances, the device further comprises a filter for purifying the amplified peptide from the aqueous solution by solid phase extraction.

[0161]

[0145] Also provided herein are devices comprising a peptide or peptide(s) of interest. In some embodiments, the devices described herein are devices for synthesizing, detecting, and / or amplifying a peptide. In some embodiments, the devices described herein comprise a template peptide which serves as a reference for the synthesis or amplification of a peptide. In some embodiments, the template peptide is a peptide from a biological sample. In some embodiments, the devices described herein comprise a template peptide, an aqueous solution, a mixture of individual amino acids, and optionally a peptide that is the product of synthesis or amplification. In some embodiments, the devices described herein are used for the methods of synthesizing, detecting, and / or amplifying a peptide described herein.

[0162]

[0146] In some embodiments, devices comprise components comprising one or more of: compositions described herein; systems described herein; other components or appurtenances as described herein; or combinations thereof. In some embodiments, devices comprise compositions or systems described herein. In some embodiments, devices perform methods described herein.

[0163]

[0147] In some embodiments, a device comprising: a sample interface configured to receive a biological sample comprising a peptide(s) of interest; a chamber fluidically connected to the sample interface; wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide(s) of interest is amplified with the mixture of individual amino acids in aqueous solution within the chamber; and wherein the aqueous solution comprising the amplified peptide(s) of interest is filtered through a membrane to separate the amplified peptide(s) of interest from the aqueous solution after peptide amplification is terminated. In some embodiments, amplifying a peptide within the chamber comprises: (a) contacting the biological sample comprising the peptide(s) of interest and the mixture of individual amino acids in the aqueous solution within the chamber; and (b) providing of energy to the aqueous solution for a time period sufficient to produce copies of the peptide(s) of interest with the mixture of individual amino acids, wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample. In some embodiments, the peptide(s) of interest is amplified in conditions comprising: (a) a reaction temperature of about 37°C to about 70°C; (b) a pH of about 3.0-10.0; or (c) a pressure of about 5 mbar to about 20 mbar. In some embodiments, the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. In some embodiments, the providing of energy to the aqueous solution is by cyclical provision. In some embodiments, the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light. In some embodiments, periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution. In some embodiments, the aqueous solution comprises pure or substantially pure water. In some embodiments, the aqueous solution comprises a phosphate buffered saline solution. In some embodiments, the aqueous solution comprises an acid or a base. In some embodiments, the acid or the base is HC1, Formic Acid, or NaOH. In some embodiments, the device further comprises a pump for vacuum.

[0164]

[0148] In some embodiments, the device comprises a component for reaction temperature regulation. In some embodiments, the device comprises a temperature regulator. In some embodiments, the temperature regulator is a thermoelectric cooler and heater. In some embodiments, the temperature regulator is configured to adjust a temperature in the chamber of the device. In some embodiments, the device comprises a pH regulator. In some embodiments, the pH regulator detects changes in pH in the aqueous solution. In some embodiments, the pH regulator maintains the desired pH in the chamber. In some embodiments, the device comprises a pressure regulator. In some embodiments, the pressure regulator is a pump, or a vacuum. In some embodiments, the pump comprise a vacuum pump. In some embodiments, the device comprise an energy applicator. In some embodiments, the energy applicator is a light source or a heating element. In some embodiments, the light source emits full spectrum light. In some cases, the light source is a full spectrum light source (LED).

[0165]

[0149] In some embodiments, the device further comprises a component for evaporation and / or condensation regulation. In some embodiments, the device further comprises a component for addition and removal of reagents. In some embodiments, the device further comprises a component for purification of amplified peptide. In some embodiments, the device further comprises a component for removal of amplified peptide. In some embodiments, the device further comprises a membrane to separate the peptide from the aqueous solution after peptide amplification is terminated. In some embodiments, the membrane is a hydrophilic membrane. In some embodiments, the membrane is a size-specific membrane. In some embodiments, the device further comprises tubes with high optical transmission. In some embodiments, the device further comprises tubes and / or containers in non-reactive or absorptive material such as glass or stainless steel.

[0166]

[0150] In some embodiments, a device comprises an in-line sampler that can be used to extract a small amount from the reaction mixture for characterization or other purposes. In some embodiments, characterization comprises an activity assay. In some embodiments, characterization comprises a peptide activity assay. In some embodiments, characterization comprises characterization of pH, temp, amino acid concentration, template peptide, synthesized peptide, waste or contaminants, detection reagents, amplification reagents, or combinations thereof. In some embodiments, sampling comprises in-line sampling comprising receiving a sample, pumping the sample down a monolithic column, drying the sample in drying gas, characterizing the sample, or combinations thereof. In some embodiments, sampling the reaction mixture to assess the progress of peptide amplification indicates whether to add individual amino acids or remove individual amino acids from the aqueous solution.

[0167]

[0151] In some embodiments, the device further comprises a component for monitoring and documenting the progress of peptide amplification. In some embodiments, the device further comprises a component for monitoring and documenting the contamination profile during peptide amplification and / or after peptide amplification is terminated.

[0168]

[0152] In some embodiments, the device comprises a component for monitoring the progress of peptide amplification with visual detection, pH detection, mass spectrometer (MS), high- performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device further comprises a component for sampling the reaction mixture to assess the progress of peptide amplification. In some embodiments, the device is programmed to adhere to a method of amplifying a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. In some embodiments, the device comprises multiple detection components. In some embodiments, the device comprises a detector for measuring the peptide(s) of interest, aqueous solution, individual amino acids, amplified peptide, or combinations thereof. In some embodiments, the detector is capable of visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof. In some embodiments, the device comprises touch screen user interface. In some embodiments, the device is a bench top instrument. In some embodiments, the device is capable of amplifying 10-100 mg of peptide. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a microfluidic device.

[0169]

[0153] In some embodiments, a device for performing the method of amplifying a peptide(s) of interest from a biological sample as described herein, the device comprising a structural component as well as a composition component, wherein the composition component is a reaction mixture comprising a peptide(s) of interest, an aqueous solution, a mixture of individual amino acids, and optionally an amplified peptide. In some embodiments, the composition component is also referred to as a sample component.

[0170] Device Components

[0171]

[0154] In general, device components comprise a structural component as well as sample components, including compositions, solutions, and systems described herein. Often, a sample component comprises or consists essentially of compositions, or systems described herein. Additional device components may comprise one or more hydrogels or surfaces with immobilized reporters. In some embodiments, a device’s sample component may be contained in at least one structural device component, such as a sample interface, which may be in fluid communication with a chamber. In some embodiments, the sample interface is fluidically connected to a chamber. By way of non-limiting example, a device’s sample component may be simultaneously contained in a sample interface and a chamber. In some embodiments, by being in fluid communication with a chamber, a device’s sample component may flow from the sample interface to the chamber. In some embodiments, a device’s sample component may flow from the sample interface into a chamber by way of the fluid connection. In some embodiments, a reporter is immobilized to a surface or support medium within the chamber, which may be a hydrogel. In some embodiments, a chamber comprises more than one peptide or more than one peptide type. In some embodiments, the devices described herein comprise a plurality of hydrogels each comprising reporter molecules (e.g., in order to facilitate multiplexing and / or improve signal).

[0155] Any of the devices described herein comprise one or more compartments, chambers, channels, or locations. In some embodiments, two or more of the compartments or chambers are in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, two or more compartments or chambers are arranged in a sequence. In some embodiments, two or more compartments or chambers are arranged in parallel. In some embodiments, two or more compartments or chambers are arranged in sequence, parallel, or both. In some embodiments, one or more compartments or chambers comprise a well. In some embodiments, one or more compartments or chambers comprise a flow strip. In some embodiments, one or more compartments or chambers comprise a heating element. In some embodiments, one or more compartments or chambers comprise a vacuum element. In some embodiments, one or more compartments or chambers comprise a vacuum pump.

[0172]

[0156] Any of the devices described herein comprise a sample interface, which are in fluid communication with a valve and / or a chamber or comprising configuration to be fluidically connected to a valve and / or a chamber. In some embodiments, a chamber comprises configuration to comprise compositions, systems, one or more reagents for amplification (z.e., amplification reagents), one or more reagents for detection (z.e., detection reagents), or combinations thereof. In some embodiments, a chamber and / or a valve comprises configuration to be thermally connected to a heating element. In some embodiments, each of the valves of the plurality of valves is thermally connected to a heating element. In some embodiments, each of the valves is filled with a material configured to change between liquid and solid phases when heated by a heating element.

[0173]

[0157] In general, the buffers described herein are compatible for use in the devices described herein. In some embodiments, the device is a microfluidic device. In some embodiments, the device is a handheld device. In some embodiments, the device is a point-of-need device. In some embodiments, the device comprises any one of the device configurations described herein. In some embodiments, the device comprises one or more parts of any one of the device configurations described herein.

[0174]

[0158] Generally, a sample comprises one or more peptides and a chamber (e.g., a reaction chamber) comprises one or more of: aqueous solution, individual amino acids, or combinations thereof. In some embodiments, a sample flows from a sample interface into a chamber by way of the fluid connection wherein the sample interacts with the components of the compositions, systems, and solutions contained therein. In some embodiments, contacting the template peptide with the mixture of amino acids in aqueous solution, provided energy sufficient to form a peptide bond, results in a peptide is synthesized.

[0175]

[0159] Any of the devices described herein are compatible with any of the compositions, systems, kits, or methods disclosed herein, including methods of synthesizing, detecting, and / or amplifying a peptide.

[0176] Kits

[0177]

[0160] In some embodiments, compositions and / or system components are assembled in an kit, wherein the kit is an amplification kit. Accordingly, disclosed herein are amplification kits for synthesizing a peptide or detecting a peptide. In some embodiments, kits are compatible with any methods disclosed herein, including methods used for synthesizing or detection, which may be related to a treatment, and / or diagnosis of a disease or disorder.

[0178]

[0161] Any of the kits described herein are compatible with any of the compositions, systems, kits, or methods disclosed herein, including methods used for synthesizing or detection, which may be related to a treatment, and / or diagnosis of a disease or disorder. By way of non-limiting example, in some embodiments, the kits described herein are used in synthesis of peptide. In some embodiments, the kits described herein are used in synthesis of peptide described in TABLE 1. By way of non-limiting example, in some embodiments, the kits described herein are used in synthesis of a peptide having a therapeutic target described in TABLE 2. In some embodiments, the kits described herein are used in detection of peptide described in TABLE 1. In some embodiments, the kits described herein are used in detection of peptide associated with any one of the diseases or disorders recited in TABLE 3.

[0179]

[0162] In some embodiments, kits are compatible with methods of detection as disclosed herein, wherein a kit further comprises a detectable label or a nucleic acid encoding a detectable label. In some embodiments, the components of the kit are in the same container. In some embodiments, the components of the kit are in separate containers.

[0180] Kit Components

[0181]

[0163] In general, kit components comprise structural components as well as sample components, including compositions and systems described herein. Often, kits comprise one or more containers compatible for containing the samples, compositions, and systems described herein. In some embodiments, components of the samples, compositions, and systems are contained in the same container or in separate containers. In some embodiments, a container is a syringe, test wells, bottles, chambers, channels, vials, or test tubes. In one embodiment, the containers are formed from a variety of materials such as glass, plastic, or polymers. The system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.

[0182]

[0164] In some embodiments, a kit comprises components, compositions, systems, and / or reagents for performing any methods disclosed herein. In some embodiments, a kit comprises components, compositions, and / or reagents for performing an assay disclosed herein. In some embodiments, a kit comprises other therapeutic agents, carriers, buffers, containers, and / or devices for administration. In some embodiments, kits described herein comprise a solid support. In some embodiments, a peptide is attached to a solid support. For example, in some embodiments, the solid support is an electrode or a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the peptide flows through a chamber into a mixture comprising a substrate. When the peptide meets the substrate, a reaction occurs, such as a colorimetric reaction, which is then detected.

[0183]

[0165] In some embodiments, the kit comprises labels and / or instructions for synthesizing or detecting a peptide. In some embodiments, the kit comprises labels and / or instructions for use. In some embodiments, labeling and / or instructions includes, for example, information concerning the amount, frequency and method of introduction and / or administration of the compositions, systems, and / or nucleic acid constructs described herein. In some embodiments, a label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. After packaging the formed product and wrapping or boxing to maintain a sterile barrier, in some embodiments, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing.

[0184]

[0166] In some embodiments, the instructions for practicing the methods are recorded on a suitable recording medium. In some embodiments, the instructions are printed on a substrate, such as paper or plastic, etc. In some embodiments, the instructions are present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g. via the Internet), are provided. In some embodiments, the kit includes a web address where the instructions are viewed and / or from which the instructions are downloaded.

[0185] Detection of a Peptide

[0186]

[0167] Described herein are various methods of sample amplification and detection in a single reaction volume. In some embodiments, any of the devices described herein are configured to perform amplification and detection in a single well, chamber, channel, or volume in the device. In some embodiments, methods include simultaneous amplification and detection in the same volume and / or in the same reaction. In some embodiments, methods include sequential amplification and detection in the same volume. In some embodiments, amplification and detection occur in a single reaction or in a single volume.

[0187]

[0168] In some embodiments, a detection reaction is used for detecting the presence of a peptide in the same. In some embodiments, the detection reaction produces a detectable signal, as described elsewhere herein, in the presence of a peptide. In some embodiments, a detection reaction is used for detecting the concentration of a peptide in the same. In some embodiments, the detection reaction produces a detectable signal, as described elsewhere herein, in the presence of a concentration of a peptide.

[0188]

[0169] In some embodiments, a detection reaction is used for detecting the presence of a template peptide in a reaction volume or a sample. In some embodiments, a detection reaction is used for detecting the presence of a peptide(s) of interest in a biological sample.

[0189] Disease or Disorder

[0190]

[0170] In some embodiments, the peptide described herein has a therapeutic target associated to a disease or disorder.

[0191]

[0171] In some embodiments, treating, preventing, or inhibiting disease or disorder in a subject comprises contacting a peptide described herein to a therapeutic target associated to a disease or disorder in a patient in need thereof. In some embodiments, the therapeutic target is a therapeutic target described in TABLE 2. In some embodiments, the disease or disorder is a disease or disorder described herein in TABLE 3. In some embodiments, the methods of treating, preventing, or inhibiting a disease or disorder involves administration of a peptide described herein. In some embodiments, a peptide described herein is used for therapy.

[0192]

[0172] In some embodiments, the use of the peptide described herein in the manufacture of a medicament. In some embodiments, the use of the peptide described herein in the manufacture of a medicament for therapeutic and / or prophylactic treatment of a disease or condition described herein.

[0193] Cancer

[0194]

[0173] In some embodiments, the disease comprises cancer. Non-limiting examples of cancers include: acute lymphoblastic leukemia; acute lymphoblastic lymphoma; acute lymphocytic leukemia; acute myelogenous leukemia; acute myeloid leukemia (adult / childhood); adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix cancer; astrocytoma; atypical teratoid / rhabdoid tumor; basal-cell carcinoma; bile duct cancer; extrahepatic (cholangiocarcinoma); bladder cancer; bone osteosarcoma / malignant fibrous histiocytoma; brain cancer (adult / childhood); brain tumor; cerebellar astrocytoma (adult / childhood); brain tumor, cerebral astrocytoma / malignant glioma brain tumor; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumors; brain tumor, visual pathway and hypothalamic glioma; brainstem glioma; breast cancer; bronchial adenomas / carcinoids; bronchial tumor; Burkitt lymphoma; cancer of childhood; carcinoid gastrointestinal tumor; carcinoid tumor; carcinoma of adult, unknown primary site; carcinoma of unknown primary; central nervous system embryonal tumor; central nervous system lymphoma, primary; cervical cancer; childhood adrenocortical carcinoma; childhood cancers; childhood cerebral astrocytoma; chordoma, childhood; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small round cell tumor; emphysema; endometrial cancer; ependymoblastoma; ependymoma; esophageal cancer; Ewing sarcoma in the Ewing family of tumors; extracranial germ cell tumor; extragonadal germ cell tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric (stomach) cancer; gastric carcinoid; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor; germ cell tumor: extracranial, extragonadal, or ovarian gestational trophoblastic tumor; gestational trophoblastic tumor, unknown primary site; glioma; glioma of the brain stem; glioma, childhood visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver cancer); Hodgkin’s lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi Sarcoma; kidney cancer (renal cell cancer); Langerhans cell histiocytosis; laryngeal cancer; lip and oral cavity cancer; liposarcoma; liver cancer (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; male breast cancer; malignant fibrous histiocytoma of bone / osteosarcoma; medulloblastoma; medulloepithelioma; melanoma; melanoma, intraocular (eye); Merkel cell cancer; Merkel cell skin carcinoma; mesothelioma; mesothelioma, adult malignant; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides, myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer of the bone-marrow); myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma, non-small cell lung cancer; non-Hodgkin’s lymphoma; oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer (surface epithelial-stromal tumor); ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pituitary tumor, islet cell; papillomatosis; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; pituitary adenoma; plasma cell neoplasia / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell carcinoma (kidney cancer); renal pelvis and ureter, transitional cell cancer; NUT midline carcinoma; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin cancer (melanoma); skin cancer (non-melanoma); small cell lung cancer; small intestine cancer soft tissue sarcoma; soft tissue sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; subependymal glioma; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); testicular cancer; throat cancer; thymoma; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, childhood; transitional cell cancer of the renal pelvis and ureter; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and Wilms Tumor.

[0195] TABLES

[0196]

[0174] TABLE 1 provides exemplary peptides of interest that are useful in the compositions, systems, devices, kits, and methods described herein.

[0197] TABLE 1. EXEMPLARY PEPTIDES

[0198]

[0175] TABLE 2 provides exemplary peptide therapeutic targets that are useful in the compositions, systems, devices, kits, and methods described herein.

[0199] TABLE 2. EXEMPLARY THERAPEUTIC TARGETS

[0200]

[0176] TABLE 3 provides illustrative diseases and syndromes associated with the exemplary peptide therapeutic targets described herein.

[0201] TABLE 3. EXEMPLARY DISEASES AND SYNDROMES

[0202] EMBODIMENTS

[0203]

[0177] Embodiment 1. A method of amplifying a peptide(s) of interest from a biological sample, the method comprising:

[0204] (a) providing a biological sample comprising a peptide(s) of interest for amplification;

[0205] (b) providing a mixture of individual amino acids in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest;

[0206] (c) contacting the biological sample and the mixture of individual amino acids in an aqueous solution such that copies of the peptide(s) of interest in the biological sample is produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample;

[0207] (d) providing of energy to the aqueous solution of the peptide(s) of interest and the mixture of individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and

[0208] (e) performing step (d) for a time period sufficient to amplify the peptide(s) of interest with the mixture of individual amino acids, wherein the amino acid sequence of the amplified peptide is the same as the amino acid sequence of the peptide(s) of interest.

[0209]

[0178] Embodiment 2. The method of embodiment 1, wherein the peptide(s) of interest comprises about 2 amino acids to about 200 amino acids.

[0210]

[0179] Embodiment 3. The method of embodiment 1, wherein the peptide(s) of interest comprises amino acids that are about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% hydrophobic.

[0211]

[0180] Embodiment 4. The method of embodiment 1, wherein contacting the biological sample and the mixture of individual amino acids in an aqueous solution occurs in conditions comprising:

[0212] (a) a reaction temperature of about 37°C to about 70°C;

[0213] (b) a pH of about 3.0-10.0; or

[0214] (c) a pressure of about 5 mbar to about 1000 mbar.

[0181] Embodiment 5. The method of embodiment 4, wherein the reaction temperature is about 40°C.

[0215]

[0182] Embodiment 6. The method of embodiment 4, wherein the pH is about 6.0.

[0216]

[0183] Embodiment 7. The method of embodiment 4, wherein the pressure is 8 about mbar.

[0217]

[0184] Embodiment 8. The method of embodiment 1, wherein the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

[0218]

[0185] Embodiment 9. The method of embodiment 1, wherein the providing of energy to the aqueous solution is by a cyclical provision.

[0219]

[0186] Embodiment 10. The method of embodiment 9, wherein the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light.

[0220]

[0187] Embodiment 11. The method of embodiment 10, wherein periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution.

[0221]

[0188] Embodiment 12. The method of embodiment 1, wherein the aqueous solution comprises pure or substantially pure water.

[0222]

[0189] Embodiment 13. The method of embodiment 1, wherein the aqueous solution comprises a phosphate buffered saline solution.

[0223]

[0190] Embodiment 14. The method of embodiment 1, wherein the aqueous solution comprises an acid or a base.

[0224]

[0191] Embodiment 15. The method of embodiment 14, wherein the acid or the base is HC1, Formic Acid, or NaOH.

[0225]

[0192] Embodiment 16. The method of embodiment 1, wherein the aqueous solution further comprises the peptide(s) of interest and the mixture of individual amino acids after contacting.

[0226]

[0193] Embodiment 17. The method of embodiment 1, wherein the aqueous solution is sterile.

[0227]

[0194] Embodiment 18. The method of embodiment 1, wherein the mixture of individual amino acids comprises only the amino acids that constitute the peptide(s) of interest.

[0195] Embodiment 19. The method of embodiment 1, wherein the mixture of individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 or 10 to 1, with respect to the total weight of the mixture of individual amino acids and the weight of the peptide(s) of interest.

[0228]

[0196] Embodiment 20. The method of embodiment 1, wherein the concentration of the mixture of individual amino acids in the aqueous solution prior to the providing of energy is from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL.

[0229]

[0197] Embodiment 21. The method of embodiment 1, wherein the time period is from 10 minutes to 5 days.

[0230]

[0198] Embodiment 22. The method of embodiment 1, wherein amplifying a peptide is terminated by separation of the peptide from the mixture of individual amino acids in the aqueous solution.

[0231]

[0199] Embodiment 23. The method of embodiment 1, wherein the method is carried out in the presence of oxygen, Nitrogen, hydrogen, or CO2.

[0232]

[0200] Embodiment 24. The method of embodiment 1, wherein the method is performed in the absence of nucleic acids, enzymes, co-enzymes, other cellular material or cells.

[0233]

[0201] Embodiment 25. The method of embodiment 1, wherein the mixture of individual amino acids comprises natural amino acids.

[0234]

[0202] Embodiment 26. The method of embodiment 1, wherein the mixture of individual amino acids comprises unnatural amino acids.

[0235]

[0203] Embodiment 27. The method of embodiment 1, wherein the unnatural amino acids comprise labeled amino acids.

[0236]

[0204] Embodiment 28. The method of embodiment 1, wherein the mixture of individual amino acids comprises: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, or combinations thereof.

[0237]

[0205] Embodiment 29. The method of embodiment 1, wherein amplifying a peptide is optimized for maximizing peptide yield and minimizing contaminants.

[0206] Embodiment 30. The method of embodiment 1, wherein the biological sample is processed prior to contacting the biological sample and the mixture of individual amino acids in an aqueous solution.

[0238]

[0207] Embodiment 31. The method of embodiment 30, wherein the processing is digesting and / or purifying.

[0239]

[0208] Embodiment 32. The method of embodiment 30, further comprising amplification reagents.

[0240]

[0209] Embodiment 33. A device comprising:

[0241] (a) a sample interface configured to receive a biological sample comprising a peptide(s) of interest;

[0242] (b) a chamber fluidically connected to the sample interface; wherein the chamber comprises an aqueous solution, and a mixture of individual amino acids such that a peptide(s) of interest is amplified with the mixture of individual amino acids in aqueous solution within the chamber; and wherein the aqueous solution comprising the amplified peptide(s) of interest is enriched from the reaction utilizing solid phase extraction to separate the amplified peptide(s) of interest from the aqueous solution after peptide amplification is terminated.

[0243]

[0210] Embodiment 34. The device of embodiment 33, wherein amplifying a peptide within the chamber comprises:

[0244] (a) contacting the biological sample comprising the peptide(s) of interest and the mixture of individual amino acids in the aqueous solution within the chamber; and

[0245] (b) providing of energy to the aqueous solution for a time period sufficient to produce copies of the peptide(s) of interest with the mixture of individual amino acids, wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample.

[0246]

[0211] Embodiment 35. The device of embodiment 33, wherein the peptide(s) of interest is amplified in conditions comprising:

[0247] (a) a reaction temperature of about 37°C to about 70°C;

[0248] (b) a pH of about 3.0-10.0; or (c) a pressure of about 5 mbar to about 1000 mbar.

[0249]

[0212] Embodiment 36. The device of embodiment 34, wherein the providing of energy to the aqueous solution is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

[0250]

[0213] Embodiment 37. The device of embodiment 34, wherein the providing of energy to the aqueous solution is by cyclical provision.

[0251]

[0214] Embodiment 38. The device of embodiment 37, wherein the cyclical provision of energy comprises periodically increasing the heat of the aqueous solution or periodically exposing the aqueous solution to full spectrum light.

[0252]

[0215] Embodiment 39. The device of embodiment 38, wherein periodically exposing the aqueous solution to full spectrum light comprises a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution.

[0253]

[0216] Embodiment 40. The device of embodiment 33, wherein the aqueous solution comprises pure or substantially pure water.

[0254]

[0217] Embodiment 41. The device of embodiment 33, wherein the aqueous solution comprises a phosphate buffered saline solution.

[0255]

[0218] Embodiment 42. The device of embodiment 33, wherein the aqueous solution comprises an acid or a base.

[0256]

[0219] Embodiment 43. The device of embodiment 42, wherein the acid or the base is HC1, Formic Acid, or NaOH.

[0257]

[0220] Embodiment 44. The device of embodiment 33, wherein the device further comprises a pump for vacuum.

[0258]

[0221] Embodiment 45. The device of embodiment 33, wherein the device further comprises a component for reaction temperature regulation.

[0259]

[0222] Embodiment 46. The device of embodiment 33, wherein the device further comprises a component for evaporation and / or condensation regulation.

[0260]

[0223] Embodiment 47. The device of embodiment 33, wherein the device further comprises a component for addition and removal of reagents.

[0261]

[0224] Embodiment 48. The device of embodiment 33, wherein the device further comprises a component for purification of amplified peptide.

[0225] Embodiment 49. The device of embodiment 33, wherein the device further comprises a component for removal of amplified peptide.

[0262]

[0226] Embodiment 50. The device of embodiment 33, wherein the device further comprises a solid phase extraction apparatus to separate the peptide from the aqueous solution after peptide amplification is terminated.

[0263]

[0227] Embodiment 51. The device of embodiment 50, wherein the solid phase extraction apparatus comprises a membrane.

[0264]

[0228] Embodiment 52. The device of embodiment 51, where the membrane is a hydrophilic membrane.

[0265]

[0229] Embodiment 53. The device of embodiment 51, wherein the membrane is a sizespecific membrane.

[0266]

[0230] Embodiment 54. The device of embodiment 33, wherein the device further comprises tubes with high optical transmission.

[0267]

[0231] Embodiment 55. The device of embodiment 33, wherein the device further comprises tubes and / or containers in non-reactive or absorptive material such as glass or stainless steel.

[0268]

[0232] Embodiment 56. The device of embodiment 33, wherein the device further comprises a component for monitoring and documenting the progress of peptide amplification.

[0269]

[0233] Embodiment 57. The device of embodiment 33, wherein the device further comprises a component for monitoring and documenting the contamination profile during peptide amplification and / or after peptide amplification is terminated.

[0270]

[0234] Embodiment 58. The device of embodiment 33, wherein the device comprises a component for monitoring the progress of peptide amplification with visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof.

[0271]

[0235] Embodiment 59. The device of embodiment 33, wherein the device further comprises a component for sampling the reaction mixture to assess the progress of peptide amplification.

[0272]

[0236] Embodiment 60. The device of embodiment 59, wherein sampling comprises in-line sampling comprising receiving a sample, pumping the sample down a chromatographic column analyzing the material comprising the reaction with UV and / or visible spectrum and potentially mass spectrometry, characterizing the sample, or combinations thereof.

[0237] Embodiment 61. The device of embodiment 59, wherein sampling the reaction mixture to assess the progress of peptide amplification indicates whether to add individual amino acids or remove individual amino acids from the aqueous solution.

[0273]

[0238] Embodiment 62. The device of embodiment 33, wherein the device is programmed to adhere to a method of amplifying a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof.

[0274]

[0239] Embodiment 63. The device of embodiment 33, wherein the device comprises multiple detection components.

[0275]

[0240] Embodiment 64. The device of embodiment 33, wherein the device comprises a detector for measuring the peptide(s) of interest, aqueous solution, individual amino acids, amplified peptide, or combinations thereof.

[0276]

[0241] Embodiment 65. The device of embodiment 64, wherein the detector is capable of visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof.

[0277]

[0242] Embodiment 66. The device of embodiment 33, wherein the device comprises touch screen user interface.

[0278]

[0243] Embodiment 67. The device of embodiment 33, wherein the device is a bench top instrument.

[0279]

[0244] Embodiment 68. The device of embodiment 33, wherein the device is capable of amplifying 10-100 mg of peptide.

[0280]

[0245] Embodiment 69. The device of embodiment 33, wherein the sample is a blood sample.

[0281]

[0246] Embodiment 70. The device of embodiment 33, wherein he sample is a microfluidic device.

[0282]

[0247] Embodiment 71. A device for performing the method of amplifying a peptide(s) of interest from a biological sample of any one of embodiments 1-32, the device comprising a structural component as well as a composition component, wherein the composition component is a reaction mixture comprising a peptide(s) of interest, an aqueous solution, a mixture of individual amino acids, and optionally an amplified peptide. EXAMPLES

[0283]

[0248] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0284] Example 1: Amplifying a Peptide(s) of interest from a Biological Sample

[0285]

[0249] An in vitro method is carried out to amplify a peptide(s) of interest from a biological sample (e.g., a blood sample, a tissue sample, a cell sample), the method comprising contacting a peptide(s) of interest (e.g., a peptide comprising about 2 amino acids to about 200 amino acids) with a mixture of individual amino acids in an aqueous solution. Briefly, the peptide(s) of interest is contacted with the mixture of individual amino acids in the aqueous solution in the presence of variable conditions (e.g., a reaction temperature of about 37°C to about 70°C, a pH of about 3.0-10.0, a pressure of about 5 mbar to about 1000 mbar, or combinations thereof). The aqueous solution comprising the peptide or peptides of interest and the mixture of individual amino acids was provided sufficient energy (e.g., at least about 1.2 kcal / mol of energy) to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the peptide(s) of interest amino acid sequence. The mixture of individual amino acids was provided sufficient energy for a time period (e.g., 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 1 / 2 day, 1 day, 2 days, 3 days, 4 days, or 5 days) sufficient to amplify or create copies of the peptide(s) of interest from the mixture of individual amino acids, such that the copy of the peptide(s) of interest has the same amino acid sequence as the peptide(s) of interest from the biological sample. Peptide amplification is optimized as a factor of the reaction conditions (e.g., temperature, pH, pressure controlled by a vacuum) and / or the hydrophobicity of the peptide(s) of interest. Without being bound by theory, peptide amplification involving a peptide(s) of interest with high hydrophobicity (e.g., peptide is about 65%, 70%, 75%, 80%, 85%, 90%, or 100% hydrophobic) results in a higher yield of amplified peptide (i.e., peptide product).

[0286]

[0250] In some instances, the method of amplifying a peptide comprises at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. In brief, the reaction mixture is sampled during the sampling step to assess the progress of amplifying a peptide. The yield of amplified peptide may be optimized after sampling by adding or removing individual amino acids from the aqueous solution. After peptide amplification has resulted in the desired yield of amplified peptide, the reaction mixture is filtered using a membrane to separate the amplified peptide from the aqueous solution and contaminants, such that the amplified peptide (e.g., insulin) is packaged and the aqueous solution is removed as waste.

[0287] Example 2: Amplifying a Peptide(s) of interest from a Biological Sample in a Device

[0288]

[0251] An in vitro method is carried out to amplify a peptide(s) of interest from a biological sample, the method comprising contacting a peptide or peptides of interest (e.g., a peptide comprising about 2 amino acids to about 200 amino acids) with a mixture of individual amino acids in an aqueous solution using a microfluidic device. Briefly, the peptide(s) of interest is contacted with the mixture of individual amino acids in the aqueous solution within a chamber (e.g., a chamber of non-reactive or absorptive material such as stainless steel or glass with high optical transmission) of the microfluidic device in the presence of variable conditions (e.g., a reaction temperature of about 37°C to about 70°C, a pH of about 3.0-10.0, a pressure of about

[0289] 5 mbar to about 1000 mbar, or combinations thereof). The aqueous solution comprising the peptide(s) of interest and the mixture of individual amino acids was provided sufficient energy (e.g., at least about 1.2 kcal / mol of energy) to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the peptide(s) of interest amino acid sequence. The mixture of individual amino acids was provided sufficient energy for a time period (e.g., 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours,

[0290] 6 hours, 1 / 2 day, 1 day, 2 days, 3 days, 4 days, or 5 days) sufficient to amplify or create copies of the peptide or peptides of interest from the mixture of individual amino acids, such that the copy of the peptide or peptides of interest has the same amino acid sequence as the peptide(s) of interest from the biological sample. Peptide amplification is optimized as a factor of the variable reaction chamber conditions (e.g., temperature, pH, pressure controlled by a vacuum) and / or the hydrophobicity of the peptide(s) of interest. Without being bound by theory, peptide amplification involving a peptide(s) of interest with high hydrophobicity (e.g., peptide is about 65%, 70%, 75%, 80%, 85%, 90%, or 100% hydrophobic) results in a higher yield of amplified peptide (i.e., peptide product).

[0291]

[0252] The device is programmed to adhere to a method of amplifying a peptide comprising at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof. Overall, the device monitors the progress of amplifying a peptide with detection (e.g., visual detection, pH detection, mass spectrometer (MS), high-performance liquid chromatography (HPLC), spectrophotometer, or combinations thereof) and documents the progress of amplifying a peptide.

[0253] The reaction mixture is sampled (e.g., in-line sampling comprising receiving a sample, pumping the sample down a chromatographic column, analyzing the material comprising the reaction with UV and / or visible spectrum and potentially mass spectrometry.) to assess the progress of amplifying a peptide. The yield of amplified peptide may be optimized after sampling by adding or removing individual amino acids from the aqueous solution.

[0292]

[0254] After peptide synthesis has resulted in the desired yield of amplified peptide, the reaction mixture is filtered using Solid Phase Extraction to separate the amplified peptide from the aqueous solution and contaminants, such that the amplified peptide (e.g., insulin) is packaged and the aqueous solution is removed as waste.

[0293] Example 3: Amplifying a Peptide(s) of interest from a Biological Sample in with a Bench Top Device and Consumables Kit

[0294]

[0255] An in vitro method is carried out to amplify a peptide(s) of interest from a biological sample, the method comprising contacting a peptide or peptides of interest (e.g., a peptide comprising about 2 amino acids to about 200 amino acids) with a mixture of individual amino acids in an aqueous solution using a bench top device with consumables kit. The workflow is illustrated in FIG. 1A. Briefly, biological samples (e.g., single cells, clinical samples, forensic samples) containing low abundance proteins of interest are collected for processing (e.g., cell lysis, centrifugation) and proteins are solubilized. The proteins are then reduced, alkylated, and digested with protease (e.g., trypsin) into peptides for amplification. The amplification is performed in a bench top device with consumables kit shown in FIGS. 1B-1C. Optionally, the amino acids can be isotope labeled for tracking the peptide synthesis. After amplification, the amplified peptides and old peptides are analyzed with spectroscopic technologies such as mass spectrometry.

[0295] Example 4: Exemplary Biological Pathway Proteomic Analysis using Peptide Amplification Methods Disclosed Herein

[0296]

[0256] The insulin signaling pathway is a critical biological process to regulate metabolism of glucose, fats, and proteins. It plays an important role in maintaining blood sugar homeostasis and has been implicated in diabetes, metabolism, and cell growth. Therefore, it can be important to identify and quantify proteins involved in insulin signaling pathway. To amplify and identify proteins involved in insulin pathway in a biological sample, crude proteins from a biological sample was collected, processed, and amplified as described in Example 3. Briefly, Hela cells were grown to 80% confluency, and they were then serum starved for 14 hours. Cells were then treated with 100 ng / mL of insulin and incubated for 60 mins. The treated cells, and an untreated control, were washed and lysed. The proteins were reduced and alkylated and digested with trypsin. This step ensured than only peptides, and not functional proteins, remained in the resulting peptide solution, which was then used as a template for the amplification process according to the methods disclosed herein. First, a solution of individual amino acids in the ratios found in the UniProt database was completely solubilized in 0.1% formic acid to a final concentration of 0.025 g / ml. Notably, the individual amino acids were isotope labeled, this labelling was then used to identify newly synthesized peptides. The solution was then added to the template solution, and the mixture was then incubated at 37 °C for 24 hours. The resulting peptide mixtures were analysed using LC MS / MS. The raw files were searched against the UniProt human database using MaxQuant. The contaminant and reverse hit protein were removed, and the remaining proteins analyzed.

[0297]

[0257] The amount of peptides after insulin treatment identified for each insulin pathway protein was compared with that in control (without insulin treatment) (FIGS. 2A-2B). FIG. 2A is a bar plot showing the ratio of both light and heavy proteins after insulin treatment to both light and heavy proteins without insulin treatment after peptide amplification for proteins involved in insulin pathway proteins. As shown in FIG. 2A, the proteins identified were all involved in the insulin pathway. FIG. 2B is a bar plot with three variables that show the ratio of amplified proteins after insulin treatment versus amplified proteins without insulin treatment, ratio of native proteins with insulin treatment versus amplified proteins without insulin treatment, and ratio of proteins (including both light and heavy peptides) with insulin treatment versus proteins without insulin treatment after peptide amplification for proteins involved in insulin pathway proteins. Y-axis represents relative abundance. X-axis shows protein identified. As shown in FIG. 2B, most insulin pathway proteins that were in low abundance were successfully detected, identified and quantified after amplification according to the methods disclosed herein. Collectively, these results demonstrated that the peptide amplification according to the methods described herein was able to identify, and analyze upregulation of proteins involved in a representative biological pathway (e.g., the insulin pathway).

[0298] Example 5: Exemplary Protein / Peptides Analysis in Biological Samples After Rapamycin Treatment

[0258] Rapamycin is a potent mTOR (mechanistic Target of Rapamycin) inhibitor with wide- ranging implications in medicine and aging research. Given the extensive research using rapamycin in biological samples, it was used to amplify and identify changes of protein levels affected by rapamycin according to the methods disclosed herein. Briefly, Hela cells were grown to 80% confluency and then serum starved for 14 hours. 100 nM of rapamycin was added to the cells and incubated for 24 hours. The treated cells, and an untreated control, were washed and lysed. The proteins were reduced and alkylated and digested with trypsin. This step ensured than only peptides, and not functional proteins, remained in the resulting peptide solution, which was then used as a template for the amplification process according to the methods disclosed herein. The resulting peptide solution was used then used as a template for the amplification process at a starting concentration of 1 pg, 2 pg and 10 pg for template concentrations, in triplicate. One ml of a solution of all 20 amino acids in the ratios found in the UniProt database, completely solubilised in 0.1% formic acid to a final concentration of 0.025 g / ml. were added. The samples were then incubated at 37 °C for 24 hours. The resulting peptide mixtures were analysed using LC MS / MS. The raw files were searched against the UniProt human database using MaxQuant. The contaminant and reverse hit protein were removed, and the remaining proteins analysed.

[0299]

[0259] The amounts of light peptides and heavy peptides for each protein identified were quantified and analyzed (FIG. 3, FIGS. 4A-4B, and FIG. 5). As shown in FIG. 3, the relative abundance of proteins was higher at a 1 ratio of template peptide to amino acids, as compared to 0.9, 1.1, 1.2, 1.3, and 1.4 in both control and treated conditions. FIG. 4A is a Venn diagram showing the number of light peptides identified (left circle), and the number of heavy peptides identified (right), and the number of peptides that overlap (intersection of the two sets). Thus, as shown in FIG. 4A, 12918 peptides were light or unlabeled peptides were identified across both treatment groups, and there were 10983 heavy -labelled peptides identified. The intersection between the left circle and the right circle represents peptides that were identified both as light and heavy labelled. FIG. 4B is a Venn diagram showing the number of light proteins identified (left circle), and the number of heavy proteins identified (right), and the number of proteins that overlap (intersection of the two sets). Using the peptide data retrieved, the peptides were mapped onto their respective proteins and the comparison analysis was also conducted at protein levels (FIG. 4B). As shown in FIG. 4B, 547 proteins were identified only using light peptides, and 590 proteins were identified only with heavy peptides. Notably, 2300 proteins were identified using both light and heavy peptides, which suggests that many of the amplified heavy peptides were made using a template light peptide in the mixture. As an example, Septin 11 was a protein identified in the present example. As shown above and in FIG. 5, the bold peptide fragment indicates the peptide fragment was present only as heavy peptide after amplification. The three underlined only peptide fragments indicate those three peptide fragments were present only as light peptide after amplification. The peptide fragment that is both italicized and underlined suggests this peptide was present both as light peptide and heavy peptide after amplification. Thus, based on this representative example, and others (data not shown), the 2300 proteins identified indicated that approximately 17.2% of newly synthesized proteins were generated according to the methods disclosed herein.

[0300]

[0260] A volcano plot was generated for analyzing the population of proteins enriched in control cells versus in cells after rapamycin treatment. Proteins upregulated in the control treatment were to the left of the Y-axis, and proteins upregulated in the rapamycin treatment were to the right of the Y-axis. As shown in FIG. 6, there was statistically significant proteins upregulated in each protein group. These upregulated proteins were then input into STRING DB to analyze protein-protein interactions (PPI) network. As shown in FIG. 7A - 7B, proteins that were upregulated in control were found to be associated with extracellular exosome component with 20 proteins identified and a false discovery rate of 4.63e-05. FIG. 7B demonstrates the biological processes upregulated in control group. As shown in FIG. 7C - 7D, proteins that were upregulated in control were found to be associated with extracellular exosome component with 90 proteins identified and a false discovery rate of 7.03e-32. FIG. 7D demonstrates the biological processes upregulated in control group.

[0301]

[0261] Alternatively, peptide quantification was carried out prior to amplification and post amplification using the Pierce™ Quantitative Peptide Assays & Standards (http : / / w w.ther ofisher co / order / catalog / product / 23275). Briefly, the quantities of peptides prior to amplification and post amplification were assessed for the two treatment groups (control, C; rapamycin, R), and the different amounts of starting material (1 pg, 2 pg and 10 pg) for four biological replicates each (BR1-BR4). As shown in FIG. 8, the majority of conditions resulted in an increase in peptide material after amplification, as compared to the level of peptide material.

Claims

CLAIMSWhat is claimed is:

1. A method of amplifying a peptide(s) of interest from a biological sample, the method comprising:(a) providing a biological sample comprising a peptide(s) of interest for amplification;(b) providing a mixture of all individual amino acids present in the peptide(s) of interest in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest;(c) contacting the biological sample and the mixture of all individual amino acids in an aqueous solution such that copies of the peptide(s) of interest in the biological sample is produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample;(d) providing an amount of energy to the aqueous solution of the peptide(s) of interest and the mixture of all individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and(e) performing step (d) for a time period sufficient to amplify the peptide(s) of interest with the mixture of all individual amino acids, wherein the amino acid sequence of the amplified peptide is the same as the amino acid sequence of the peptide(s) of interest, wherein the peptide(s) of interest has a length of from about 2 amino acids to about 200 amino acids.

2. The method of claim [84], wherein contacting the biological sample and the mixture of all individual amino acids in an aqueous solution occurs in conditions comprising:(a) a reaction temperature of from about 30°C to about 100°C;(b) a pH of from about 3.0-10.0; or(c) a pressure of from about 5 mbar to about 1000 mbar.

3. The method of claim 2, wherein the reaction temperature is about 40°C, the pH is about 6.0 and the pressure is about 8 mbar.

4. The method of claim [84], wherein the amount of energy is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

5. The method of claim [84], wherein the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to a full spectrum light source (LED) at a distance of at least 1 cm from the aqueous solution.

6. The method of claim [84], wherein the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of from about 20,000: 1 to about 1 : 1, or from about 10,000: 1 to about 10: 1, with respect to a total weight of the mixture of all individual amino acids and the peptide(s) of interest.

7. The method of claim [84], wherein the mixture of all individual amino acids is provided at a concentration of from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL.

8. The method of claim [84], wherein the time period is from 10 minutes to 5 days.

9. The method of claim [84], wherein the method is performed in the absence of nucleic acids, enzymes, co-enzymes, cellular material or cells.

10. The method of claim [84], wherein the mixture of all individual amino acids comprises unnatural amino acids.I L A device comprising:(a) a sample interface configured to receive a biological sample comprising a peptide(s) of interest;(b) one or more of a temperature regulator, a pH regulator, and a pressure regulator;(c) an energy applicator;(d) a chamber fluidically connected to the sample interface, wherein the chamber is configured to receive an aqueous solution that comprises a mixture of all individual amino acids present in the peptide(s) of interest, thereby admixing the peptide(s) of interest with the mixture of all individual amino acids in the aqueous solution; and(e) a processor and a computer readable memory at least transiently storing instructions that, when executed by the processor, causes the device to: i. maintain one of more of the following conditions in the chamber:(a) a temperature of from about 30°C to about 40°C using the temperature regulator;(b) a pH of from about 3.0 to about 10.0 using the pH regulator; or(c) a pressure of from about 5 mbar to about 1000 mbar using the pressure regulator; and ii. amplify the peptide(s) of interest using the mixture of all individual amino acids by providing an amount of energy to the chamber using the energy applicator for a time period sufficient to amplify the peptide(s) of interest using the mixture of all individual amino acids, wherein each amplified copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample;(f) optionally wherein the device further comprises a filter for purifying the amplified peptide from the aqueous solution by solid phase extraction.

12. The device of claim 11, wherein the amount of energy is provided is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol, and wherein the amount of energy is provided to the aqueous solution in cycles.

13. The device of claim 11, wherein the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to the energy applicator at a distance of at least 1 cm from the aqueous solution.

14. The device of claim 11, wherein the amount of energy is provided is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol, and wherein the amount of energy is provided to the aqueous solution in cycles.

15. The device of claim 11, wherein the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution or periodically exposing the aqueous solution to the energy applicator at a distance of at least 1 cm from the aqueous solution.

16. A system for amplifying a peptide(s) of interest from a biological sample, the system comprising:(a) a mixture of all individual amino acids present in the peptide(s) of interest in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest, wherein the mixture of all individual amino acids is configured to be contacted with the biological sample in an aqueous solution such that copies of the peptide(s) of interest in the biologicalsample are produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample;(b) an energy generating component configured to provide an amount of energy to the aqueous solution of the peptide(s) of interest and the mixture of all individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and wherein the system is configured to amplify the peptide(s) of interest upon provision of the amount of energy.

17. The device of claim 11, wherein the instructions further comprise at least one cleaning step, a preparation step, a binding step, a sampling step, a filtering step, a packaging step, a waste disposal step, or combinations thereof.

18. A system for amplifying a peptide(s) of interest from a biological sample, the system comprising:(a) a mixture of all individual amino acids present in the peptide(s) of interest in an amount that is at least equal to the stoichiometric amount of each amino acid in the sequence of the peptide(s) of interest, wherein the mixture of all individual amino acids is configured to be contacted with the biological sample in an aqueous solution such that copies of the peptide(s) of interest in the biological sample are produced wherein each copy of the peptide(s) of interest comprises the same amino acid sequence as the peptide(s) of interest in the biological sample;(b) an energy generating component configured to provide an amount of energy to the aqueous solution of the peptide(s) of interest and the mixture of all individual amino acids, wherein the energy is sufficient to result in sequence-specific peptide bond formation of individual amino acids from the mixture of individual amino acids based on the amino acid sequence of the peptide(s) of interest; and wherein the system is configured to amplify the peptide(s) of interest upon provision of the amount of energy.

19. The system of claim 16, wherein contacting the biological sample and the mixture of all individual amino acids in an aqueous solution occurs in conditions comprising:(a) a reaction temperature of from about 30°C to about 100°C;(b) a pH of from about 3.0-10.0; or(c) a pressure of from about 5 mbar to about 1000 mbar; or(d) the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio between 5 : 1 and 500: 1.

20. The system of claim 17, wherein the reaction temperature is about 40°C, the pH is about 6.0 and the pressure is about 8 mbar.

21. The system of claim 16, wherein the peptide(s) of interest comprises a length of from about 2 amino acids to about 200 amino acids.

22. The system of claim 16, wherein the amount of energy is at least 0.1 kcal / mol, at least 0.2 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol.

23. The system of claim 16, wherein the amount of energy is provided to the aqueous solution in cycles by increasing the heat of the aqueous solution.

24. The system of claim 16, wherein the mixture of all individual amino acids and the peptide(s) of interest are contacted at a w / w (weight by weight) ratio of from about 20,000:1 to about 1 : 1, or from about 10,000: 1 to about 10:1, with respect to a total weight of the mixture of all individual amino acids and the peptide(s) of interest.

25. The system of claim 16, wherein the mixture of all individual amino acids is provided at a concentration of from about 0.001 g / mL to about 10 g / mL, from about 0.005 g / mL to about 5 g / mL, or from about 0.01 g / mL to about 1 g / mL.

26. The system of claim 16, wherein the time period is from 10 minutes to 5 days.

27. The system of claim 16, wherein the system is configured to perform peptide synthesis in the absence of nucleic acids, enzymes, co-enzymes, cellular material or cells.

28. The system of claim 16, wherein the mixture of all individual amino acids comprises unnatural amino acids.

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