# US 2023/0054472 A1 · Sample preparation and microbial analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0054472 A1 - Document ID: US20230054472A1 - Status: Published application - Kind: A1 - Application number: 17/788,137 - Filed: 2020-12-23 - Published: 2023-02-23 - Earliest claimed: 2019-12-23 - Assignees: Miraki Innovation Think Tank LLC - Applicants: Miraki Innovation Think Tank LLC - Inventors: James HILL; Nisha Veronica VARMA; Gregory T. MARTIN; Goossen Jan Bernard BOER; Zhiqian ZHOU; Christopher VELIS - Web page: https://patentgrep.com/patent/US20230054472A1 ## CPC - G01N 33/569: for microorganisms, e.g. protozoa, bacteria, viruses - G01N 33/54326: {Magnetic particles} - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 2333/4724: Lectins - G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods are described for preparing samples including biological, environmental, and food products for microbial analysis. Microbes and microbe components in the sample can be treated with antimicrobial compounds and a matrix solution to permit fast and accurate characterization using analysis techniques such as matrix-assisted laser desorption/ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). ## Claims ### Claim 1 A method of preparing a sample for detecting a microbe or microbe components present in the sample, the method comprising the following steps: adding a substance to a sample suspected of comprising a microbe or microbe components; digesting the sample under conditions promoting digestion of a microbe or microbe components in the sample; and optionally contacting the digested microbe or microbe components with a matrix or matrix solution on a target substrate. ### Claim 2 (depends on 1) The method according to claim 1, wherein the substance comprises one or more of an antimicrobial mixture, an enzyme, a protease, or a carbohydrate-cleaving enzyme. ### Claim 3 (depends on 2) The method according to claim 2, wherein the protease is trypsin. ### Claim 4 (depends on 1) The method according to claim 1, wherein the sample is a patient sample and the detecting a microbe or microbe component comprises detecting a microbial infection in a patient. ### Claim 5 (depends on 1) The method according to claim 1, further comprising, prior to adding the substance, contacting the sample with a microbe-targeting molecule bound substrate and isolating from the sample a microbe or microbe components bound to the microbe-targeting molecule. ### Claim 6 (depends on 5) The method according to claim 5, wherein the substrate is a magnetic substrate, a fiber substrate, a polymer substrate, or ELISA plate. ### Claim 7 (depends on 5) The method according to claim 5, wherein the step of isolating comprises applying a magnet or magnetic field to the sample. ### Claim 8 (depends on 5) The method according to claim 5, wherein the step of isolating comprises washing the substrate with a fluid to remove unbound cells, biomolecules, or chemicals. ### Claim 9 (depends on 8) The method according to claim 8, wherein fluid comprises calcium. ### Claim 10 (depends on 5) The method according to claim 5, wherein the step of isolating is in accordance with a characteristic comprising at least one of size, mass, density, or charge. ### Claim 11 (depends on 5) The method according to claim 5, wherein the step of isolating comprises eluting the microbe or microbe components from the substrate. ### Claim 12 (depends on 11) The method according to claim 11, wherein the step of eluting comprises heating to a temperature of at least 70° C. with or without agitation. ### Claim 13 (depends on 12) The method according to claim 12, wherein the heating to a temperature of at least 70° C. is performed in calcium-free water. ### Claim 14 (depends on 11) The method according to claim 11, wherein the step of eluting comprises a pH treatment. ### Claim 15 (depends on 11) The method according to claim 11, wherein the step of eluting comprises treatment with a chelation agent. ### Claim 16 (depends on 15) The method according claim 15, wherein the chelating agent comprises at least one of ethylenediaminetetraacetic acid (EDTA), calcium disodium edetate (CaNa2EDTA), ethylene glycol-bis(R-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), deferoxamine mesylate salt (DFOM). ### Claim 17 (depends on 5) The method according to claim 5, wherein the step of isolating comprises concentrating the microbe or microbe components in the sample. ### Claim 18 (depends on 17) The method according to claim 17, wherein the isolated volume is less than the volume of the sample. ### Claim 19 (depends on 2) The method according to claim 2, wherein the antimicrobial mixture comprises at least one of an antibiotic mixture, an antifungal mixture, and an antiviral mixture. ### Claim 20 (depends on 19) The method according to claim 19, wherein the antibiotic mixture comprises one or more antibiotics from at least one antibiotic class comprising Cephalosporin, Glycopeptide, Cyclic lipopeptide, Aminoglycoside, Macrolide, Oxazolidinone, Fluoroquinolones, Lincosamides, and Carbapenem. ### Claim 21 (depends on 19) The method according to claim 19, wherein the antifungal mixture comprises one or more antifungals from at least one antifungal class comprising Polyenes, Azoles, Nucleoside Analog, Echinocandin, and Allylamine. ### Claim 22 (depends on 19) The method according to claim 19, wherein the antiviral mixture comprises one or more antivirals from at least one antiviral class comprising CCR5 anatonists, Fusion inhibitors, Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Nucleotide reverse transcriptase inhibitors (NtRTIs), Integrase inhibitors, Protease inhibitors, DNA polymerase inhibitors, Guanosine analogs, Interferon-alpha, M2 ion channel blockers, Nucleoside inhibitors, NS5A polymerase inhibitors, NS3/4A protease inhibitors, Neuraminidase inhibitors, Nucleoside analogs, and Direct acting antivirals (DAAs). ### Claim 23 (depends on 19) The method according to claim 19, wherein the antibiotic mixture comprises one or more of Cefepime, Vancomycin, Daptomycin, Amikacin, Erythromycin, Linezolid, Ciproflaxin, Lincomycin, and Meropenem. ### Claim 24 (depends on 19) The method according to claim 19, wherein the antifungal mixture comprises one or more of Caspofungin and Amphotericin. ### Claim 25 (depends on 2) The method according to claim 2, wherein the antimicrobial mixture comprises one or more of Cefepime, Vancomycin, Daptomycin, Amikacin, Erythromycin, Linezolid, Ciproflaxin, Lincomycin, Meropenem, Caspofungin and Amphotericin. ### Claim 26 (depends on 19) The method according to claim 19, wherein the antimicrobial mixture comprises at least one antibiotic mixture at a concentration from about 0.1 ug/mL to about 100 mg/mL. ### Claim 27 (depends on 19) The method according to claim 19, wherein the antimicrobial mixture comprises at least one antifungal mixture at a concentration from about 0.01 ug/mL to about 100 mg/mL. ### Claim 28 (depends on 1) The method according to claim 1, wherein the conditions promoting digestion include heating the sample. ### Claim 29 (depends on 1) The method according to claim 1, further comprising contacting the digested microbe or microbe components with a composition that is more acidic than the digested microbe or microbe components. ### Claim 30 (depends on 29) The method according to claim 29, wherein the composition that is more acidic than the digested microbe or microbe components is present at a volume equal to or greater than the volume of the digested microbe or microbe components. ### Claim 31 (depends on 29) The method according to claim 29, wherein the composition that is more acidic than the digested microbe or microbe components comprises trifluoroacetic acid (TFA), formic acid, or acetic acid. ### Claim 32 (depends on 1) The method according to claim 1, wherein the target substrate is evenly sprayed with matrix solution prior to analyzing microbe or microbe components to generate a homogenous layer of crystallized matrix on top of the substrate. ### Claim 33 (depends on 1) The method according to claim 1, further comprising analyzing the microbe or microbe components. ### Claim 34 (depends on 33) The method according to claim 33, wherein the analyzing step is performed using a method comprising at least one of volatile organic compound method; Raman spectroscopy; FFT (Fast-Fourier Transform); Fourier-Transform Infrared Spectroscopy (FTIR); infrared spectrometry; Nuclear Magnetic Resonance (NMR) spectrometry; chromatographic method, or mass spectrometric method. ### Claim 35 (depends on 34) The method according to claim 34, wherein the analyzing step is performed via a mass spectrometric method. ### Claim 36 (depends on 35) The method according to claim 35, wherein the mass spectrometric method comprises at least one of electron ionization, chemical ionization, electrospray ionization, atmospheric pressure chemical ionization, and matrix-assisted laser desorption ionization (MALDI-TOF MS). ### Claim 37 (depends on 35) The method according to claim 35, wherein the mass spectrometric method is automated. ### Claim 38 (depends on 1) The method according to claim 1, wherein the sample comprises blood, serum, plasma, sputum, urine, joint fluid, or any other tissue or biological sample. ### Claim 39 (depends on 1) The method according to claim 1, further comprising optionally culturing the sample prior to adding the substance. ### Claim 40 (depends on 5) The method according to claim 5, wherein the microbe-targeting molecule comprises a microbe surface-binding domain. ### Claim 41 (depends on 40) The method according to claim 40, wherein the microbe surface-binding domain comprises a mannose-binding lectin (MBL). ### Claim 42 (depends on 41) The method according to claim 41, wherein the microbe surface-binding domain comprises a human mannose-binding lectin (MBL). ### Claim 43 (depends on 40) The method according to claim 40, wherein the microbe surface-binding domain comprises a carbohydrate recognition domain (CRD) of MBL. ### Claim 44 (depends on 43) The method according to claim 43, wherein the CRD is linked to an immunoglobulin or fragment thereof. ### Claim 45 (depends on 43) The method according to claim 43, wherein the CRD is linked to an Fc component of human IgG1 (FcMBL). ### Claim 46 (depends on 6) The method according to claim 6, wherein the magnetic substrate is a superparamagnetic substrate. ### Claim 47 (depends on 6) The method according to claim 6, wherein the magnetic substrate comprises at least one of a magnetic bead, a superparamagnetic bead, or a magnetic microbead. ### Claim 48 (depends on 5) The method according to claim 5, wherein the microbe-targeting molecule is linked to an ELISA plate. ### Claim 49 (depends on 1) The method according to claim 1, wherein the microbe comprises a Gram-positive bacterial species, a Gram-negative bacterial species, a mycobacterium, a fungus, a parasite, a bacterial antigen, a viral antigen, a protozoan, an alga, or a virus. ### Claim 50 (depends on 1) The method according to claim 1, wherein the microbe component comprises a component from a Gram-positive bacterial species, a Gram-negative bacterial species, a mycobacterium, a fungus, a parasite, a bacterial antigen, a viral antigen, a protozoan, an alga, or a virus. ### Claim 51 (depends on 1) The method according to claim 1, wherein the microbe component comprises microbe-associated molecular patterns (MAMPs) and/or microbe-associated proteins. ### Claim 52 (depends on 1) The method according to claim 1, wherein the microbe is a pathogen that affects humans. ### Claim 53 (depends on 1) The method according to claim 1, wherein the microbe or microbe component is a pathogen or component thereof. ### Claim 54 (depends on 53) The method according to claim 53, further comprising identifying the group of the pathogen. ### Claim 55 (depends on 53) The method according to claim 53, further comprising identifying the domain of the pathogen. ### Claim 56 (depends on 53) The method according to claim 53, further comprising identifying the species of the pathogen. ### Claim 57 (depends on 53) The method according to claim 53, further comprising identifying the strain of the pathogen. ### Claim 58 The method according to 53, further comprising identifying the antimicrobial susceptibility of the pathogen. ### Claim 59 A method of preparing a sample for detecting a microbe or microbe components present in the sample, the method comprising the following steps: isolating from a sample a microbe or microbe components bound to a microbe-targeting molecule on a substrate; adding a substance to the isolated microbe or microbe components; digesting the isolated microbe or microbe components under conditions promoting digestion of the microbe or microbe components; and contacting the digested microbe or microbe components with a matrix or matrix solution on a target substrate.