MASSIVELY MULTIPLEXED RAMAN OPTICAL BARCODING FOR ANALYTE DETECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/424,223, filed November 10, 2022, the contents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant numbers GM128214 and GM132860 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide sequences which are present in the file named “231109_ 91962-A-PCT_Sequence_Listing_AWG.xml”, which is 1,804 bytes in size, and which was created on November 8, 2023 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed November 8, 2023 as part of this application.

BACKGROUND

[0004] The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0005] Detecting and observing the subjects of interest is one of the fundamental approaches to studying biological processes. Yet the biological system is complicated in all aspects, and is challenging at the cellular and subcellular level. Therefore, there is an ongoing need for methods capable of detecting and analyzing a large number of biological molecules simultaneously and accurately.

SUMMARY OF THE INVENTION

[0006] Herein a Raman spectroscopy-based platform for massively multiplexed detection of analytes of interest is disclosed, including massively multiplexed labeling assays, and a hardware platform for economical and high-throughput detection. Non-limiting examples of use of the platform in multiplex detecting nucleic acids or peptides/proteins are demonstrated.

[0007] According to certain exemplary embodiments of the present disclosure, a method is provided for detecting the presence of one or more different analytes present in a sample comprising:

(a) contacting the sample with a plurality of bead types, each bead type comprising:

(1) one, or more, types of Raman-active small molecule(s) (RASMs), each at a predefined concentration in the bead, and

(2) a binding molecule, affixed thereon, specific for one of each of said one or more different analytes; wherein each bead type differs from the remaining bead types of the plurality in (i) concentration of Raman-active small molecule(s), (ii) spectrum or spectra of Raman- active small molecule(s), or (ii) both concentration and spectrum/spectra pattern of Raman-active small molecule(s);

(b) removing unbound analytes or unbound beads; and

(c) determining, with Stimulated Raman Scattering (SRS) or Spontaneous Raman Scattering, which bead type(s) having an analyte bound thereto are present after step (b) so as to thereby determine which type(s) of analyte are bound, thereby detecting which of one or more different analytes are present in the sample.

[0008] Also provided is a system for Raman spectroscopy and fluorescence spectroscopy of a sample of microbeads doped with Raman-active-small-molecules (RASMs), comprising: a spectrometer; a light source comprising a first laser and a second laser, the first laser configured to excite Raman scattering from the sample with signature peaks at a first wavelength and the second laser configured to generate an emission spectrum from the sample with wavelengths higher than the first wavelength; a flowcell through which the sample is passed during spectroscopy of the sample; telescopic lenses configured to collimate and expand laser beams emitted by the first and second lasers; reflective lenses for directing the laser beams to the flowcell; an objective lens configured to focus an emitted Raman signal and an emitted fluorescence frequency spectrum from the sample resulting from illumination of the sample in the flowcell by the laser beams; a dichroic beam splitter configured to direct the emitted Raman signal and the emitted fluorescence frequency spectrum to the objective lens; a pin hole through which the focused Raman signal and the fluorescence frequency spectrum are passed; and relay lenses positioned after the pinhole for directing the Raman signal and the fluorescence frequency spectrum to the spectrometer, the spectrometer comprising a diffraction grating that filters the Raman signal from the fluorescence frequency spectrum so that both the Raman signal and the fluorescence frequency spectrum are detected by the spectrometer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1C: 1A) A large number of Raman-active-small-molecules (RASMs) can be efficiently concentrated in polymer microbeads due to non-covalent interactions; IB) Using two dimensions of optical barcoding - brightness barcoding and spectral barcoding - manifold unique beads can be generated; 1C) Assuming: i) each one of the RASM can be doped into microbeads at M different concentrations, resulting in microbeads that have M brightness levels (brightness barcoding); ii) a combination of n RASMs can be doped together into one microbead and the Raman signal of each RASM in the same microbead can be detected individually (spectral barcoding), then the total possible number of unique “Vibrant MicroBeads” (a laboratory name for the “barcoded” RASM-loaded microbeads disclosed herein) would be Mn-1. If 5 brightness barcodings and 10 spectral barcodings were considered, the resulting unique Vibrant MicroBeads would be 5 ^10-1 = 9,765,624 flavors.

[0010] FIG. 2: shows massive multiplexity is the direct result of the fact that RASMs can have individually resolvable Raman spectra. In comparison, most fluorescence-based techniques only have about three spectrally resolvable fluorescence dyes usable at the same time, greatly limiting the multiplexity. The red alkyne bonds represent the presence of ¹³ C.

[0011] FIGS. 3A-3B: 3A shows an annotated image of a mixture of 96 flavors of Vibrant MicroBeads by Stimulated Raman Scattering microscopy. The image was composed of a stack of images each taken at one of the featuring Raman peaks. 3B indicates a spectral analysis of each Vibrant MicroBead and the density plot was generated by plotting the intensity at each Raman shift in a pairwise manner.

[0012] FIGS. 4A-4C: Exemplary detection of various mock virus nucleic acid sequence detection including A) optional CRSIPR technique to increase specificity; B) indication of optical barcoding for different mock viral sequences (left panel, barcoded RASM microbeads) and positive detection of sequences (right panel; fluorophore); C) predicted detection fidelity - see FIG. 5 for results.

[0013] FIG. 5: Dozens of synthetic targets were synthesized as mock viral genomes across six viral families. Out of 17 mock viral genomes tested, an accuracy of -95% was achieved.

[0014] FIGS. 6A-6B: 6A: Massively multiplexed fluorescence-linked immunosorbent assay for peptide and protein detection. In a non-limiting example, a certain antibody or aptamer is bioconjugated to the surface of Vibrant MicroBeads. Then different Vibrant MicroBeads with different antibodies can be mixed for the multiplex detection of peptides or protein. 6B: Preliminary tests had been carried out and the sensitive detection limit was realized as low as 5pg/ml.

[0015] FIG. 7: Raman spectrum from a single 5 pm Vibrant MicroBead. Even when the exposure time was as short as 3ms, the signal-to-noise ratio of the RASMs peak is as high as about 50, making it possible for high throughput detection.

[0016] FIGS. 8A-8E: 8A) Exemplary spontaneous Raman flow cytometry setup design. 8B) Shows the schematics of the concept. 8C) Shows a combined Raman and fluorescence spectra of a Vibrant MicroBead with a fluorescence signal attached to it. 8D) For the liquid flow cell system, a microfluidic device featuring a single straight channel (300pm rectangular cross section) and acoustic focusing powered by a piezo chip vibrating at 2.49 Mhz was used. 8E) Detection with a microwell array. The microwell array is designed so that each microwell can only hold one microbead at a time. Therefore, by scanning the microwell array where the microbeads have settled down, the signal from each individual bead can be acquired efficiently and maintenance-free.

[0017] FIGS. 9A-9B: 9A & 9B together show a data matrix summarizing the validation results where 124 viral species and 9 control samples (list in description hereinbelow) were subject to the tests described herein (tested species on left, and detected species on bottom).

[0018] FIG. 10: Shows a data matrix summarizing the validation results for an upper respiratory infection panel, tested with inactivated real viruses and bacteria (tested species on left, and detected species on bottom).

[0019] FIG. 11 : Shows the detection sensitivity of the assays described herein. Strong signal was recorded even the sample concentration was down to 2aM. The negligible signal from the control group also indicates high specificity.

[0020] FIG. 12: A fluorescence dye, Alexa647, was diluted to lOOnM in DMSO and subject to signal acquisition described in FIGS. 8. Raman peaks from the solvent are clearly visible, with no elevated background from fluorescence interference. Above 4000 cm-1 (~675nm with 532nm excitation laser wavelength), fluorescence emission from Alexa647 can recorded with very high signal-to-noise ratio. DETAILED DESCRIPTION

[0021] A method is provided for detecting the presence of one or more different analytes present in a sample comprising:

(a) contacting the sample with a plurality of bead types, each bead type comprising: one, or more, types of Raman-active small molecule(s), each at a predefined concentration in the bead, and a binding molecule, affixed thereon, specific for one of each of said one or more different analytes; wherein each bead type differs from the remaining bead types of the plurality in (i) concentration of Raman-active small molecule(s), (ii) spectrum or spectra of Raman-active small molecule(s), or (ii) both concentration and spectrum/ spectra pattern of Raman-active small molecule(s);

(b) removing unbound analytes or unbound beads; and

(c) determining, with Stimulated Raman Scattering (SRS) or Spontaneous Raman Scattering, which bead type(s) having an analyte bound thereto are present after step (b) so as to thereby determine which type(s) of analyte are bound, thereby detecting which of one or more different analytes are present in the sample.

[0022] In embodiments, each bead type comprises a polystyrene particle. In embodiments, the beads are paramagnetic microbeads.

[0023] In embodiments, oil-in-water emulsion droplets are used, in place of beads, where a number of different active reactions in the droplet are to be monitored in parallel, and the barcoding serves as the identifier of the identity of the reactions. In embodiments, any substance of substrate that the RASMs can partition stably into can be used in place of beads in the methods described herein, mutatis mutandis.

[0024] In embodiments, all bead types are of the same size, or are all of about the same size. In embodiments, each bead type of the plurality has an average diameter of at least 1.0 pm. In embodiments, each bead type of the plurality has an average diameter of 3.0 pm. In embodiments, each bead type of the plurality has an average diameter of 5.0 pm. In embodiments, each bead type of the plurality has an average diameter of 10.0 pm. In embodiments, each bead type of the plurality has an average diameter of not less than 1.0 pm. In embodiments, each bead type of the plurality has an average diameter of not less than 750 nm. In regard to about the same size, the term about means up to 10% more or up to 10% less in diameter to the reference diameter.

[0025] In embodiments, the types of bead are further barcoded by size difference. In embodiments, size barcoding is read by taking images and measuring the diameter of each microbead. In embodiments, size barcoding is read quantitatively by measuring the forward and backward scattering (since the scattering strength is dependent on their sizes).

[0026] In embodiments, each bead type of the plurality has peak Raman shift at a predetermined stimulation wavelength at least 10 cm' ¹ less, or 10 cm' ¹ more, than the peak Raman shift of all the other bead types in the plurality.

[0027] Raman-active small molecules are a result of a molecular vibration causing a change in polarizability of the molecule. The symmetrical stretch out and then in can be detected by Raman spectroscopy.

[0028] In embodiments, the Raman-active small molecules are alkyne-containing. In embodiments, the Raman-active small molecules are organic and do not exceed a molecular weight of 350 g/mol. In embodiments, the Raman-active small molecules are organic and do not exceed a molecular weight of 310 g/mol. In embodiments, the RASMs are based on one or more Carbow dyes.

[0029] In embodiments, the Raman-active small molecules comprise one or more of the following, wherein adjacent to an alkyne carbon atom indicates presence of a 13C isotope: [0030] In embodiments, the Raman-active small molecules comprise one or more of the following, wherein adjacent to an alkyne carbon atom indicates presence of a 13C isotope

[0031] In embodiments, the plurality of beads comprises 5 or more different brightness levels. In embodiments, the plurality of beads comprises 10 or more different brightness levels. In embodiments, the plurality of beads comprises 15 or more different brightness levels.

[0032] In embodiments, the plurality of beads comprises at least 90 different bead types. In embodiments, the plurality of beads comprises at least 100 different bead types. In embodiments, the plurality of beads comprises at least 500 different bead types. In embodiments, the plurality of beads comprises at least 1000 different bead types. In embodiments, the plurality of beads comprises at least 10,000 different bead types. In embodiments, the plurality of beads comprises at least 100,000 different bead types.

[0033] In embodiments, wherein the one or more different analytes are nucleic acids.

[0034] In embodiments, the one or more different analytes are peptides.

[0035] In embodiments, the one or more different analytes are proteins.

[0036] In embodiments, the one or more different analytes are antibodies.

[0037] In embodiments, the one or more different analytes are lipids or carbohydrates.

[0038] In embodiments, the binding molecule specific for one of each of said one or more different analytes comprises a nucleic acid. In embodiments, the binding molecule specific for one of each of said one or more different analytes comprises an aptamer. In embodiments, bead types are bound to a surface or a sample holder. In embodiments, a bead type is bound by being affixed to a solid surface. In embodiments, a bead type is bound by being restricted in movement within a spatial location, e.g., within an individual well of a sample holder which has an opening aperture smaller than the bead type diameter.

[0039] In embodiments, the binding molecule specific for one of each of said one or more different analytes comprises a peptide or antibody or antibody fragment.

[0040] In embodiments, the one or more different analytes comprise a nucleic acid sequence found in a pathogen.

[0041] In embodiments, the pathogen is a pathogen of a mammal.

[0042] In embodiments, the pathogen is a virus or a bacteria.

[0043] In embodiments, the analytes are nucleic acids from one or more, or a subset of, the pathogens listed in Fig. 9.

[0044] In embodiments, the analytes comprise nucleic acids from human respiratory viral pathogens. For example: Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, Influenza A/Hl, Influenza A/H3, Influenza A/Hl-2009, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus.

[0045] In embodiments, the predefined nucleic acids are from human respiratory bacterial pathogens. For example, Bordetella pertussis, Chlamydophila pneumoniae, Mycoplasma pneumoniae.

[0046] In embodiments, the analytes comprise nucleic acids from human gastrointestinal viral pathogens. For example: Adenovirus F40/41, Astrovirus, Norovirus GVGII, Rotavirus A, Sapovirus (I, II, IV, and V).

[0047] In embodiments, the analytes comprise nucleic acids from human gastrointestinal bacterial pathogens. For example: Campylobacter (jejuni, coli and upsaliensis), Clostridium difficile (Toxin A/B), Plesiomonas shigelloides, Salmonella, Yersinia enterocolitica, Vibrio (parahaemolyticus, vulnificus and cholerae), Vibrio cholerae, Diarrheagenic E.coli/Shigella, Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC) It/st, Shiga-like toxin-producing E. coli (STEC) stxl/stx2, E. coli 0157, Shigella/Enteroinvasive E. coli (EIEC). [0048] In embodiments, the analytes comprise nucleic acids from human gastrointestinal parasitic pathogens. For example: Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia.

[0049] In embodiments, the analytes comprise nucleic acids from human blood pathogens. For example: gram negative bacteria or gram positive bacteria which are pathogenic. For example: Enterococcus, Listeria monocytogenes, Staphylococcus, Streptococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes. For example: Acinetobacter baumannii, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Enterob act eriaceae, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, Serratia marcescens. In embodiments, the predefined nucleic acids are from human blood pathogens which are yeast. For example: Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis.

[0050] In embodiments, the analytes comprise nucleic acids from CNS pathogens, e.g., as found in CSF. For example, bacterial CNS pathogens such as: Escherichia coli KI, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae. For example, viral CNS pathogens such as: Cytomegalovirus (CMV), Enterovirus, Epstein-Barr virus (EBV), Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), Human herpesvirus 6 (HHV-6), Human parechovirus, Varicella zoster virus (VZV). For example, yeast CNS pathogens such as: Cryptococcus gattii, Cryptococcus neoformans.

[0051] The analyzed sample can be any analyte of interest from a sample including, for example, DNA, RNA, various mixtures of DNA and RNA. The sample can be, or be derived from, biofluids, blood, serum, urine, dried blood, cell growth media, lysed cells, beverages or food, and can include environmental samples such as water, air or soil. Samples include, without limitation, saliva, blood, CSF, mucus, nasal discharge, GI samples/stool samples, plasma, urine, sweat, and swabbed fluids from, e.g., mouth, lung, vagina, colon.

[0052] In embodiments, the analytes comprise nucleic acids from pathogens. In embodiments, the pathogen(s) are human pathogens.

[0053] Embodiments of human bacterial pathogens:

[0054] Actinomyces israelii

[0055] Bacillus anthracis [0056] Bacteroides fragili

[0057] Bordetella pertussis

[0058] Borrelia

[0059] B. burgdorferi

[0060] B. garinii

[0061] B. afzelii

[0062] B. recurrentis

[0063] Brucella

[0064] B. abortus

[0065] B cards

[0066] B. melitensis

[0067] B suis

[0068] Campylobacter jejuni

[0069] Chlamydia

[0070] C. pneumoniae

[0071] C. trachomatis

[0072] Chlamydophila psittaci

[0073] Clostridium

[0074] C. botulinum

[0075] C. difficile

[0076] C. perfringens

[0077] C. tetani

[0078] Corynebacterium diphtheriae

[0079] Ehrlichia

[0080] E. canis

[0081] E. chaffeensis

[0082] Enterococcus

[0083] E. faecalis

[0084] E. faecium

[0085] Escherichia

[0086] E. coli (generally) [0087] Enterotoxigenic E. coli (ETEC)

[0088] Enteropathogenic E. coli

[0089] Enteroinvasive E.coli (EIEC)

[0090] Enterohem orrhagic (EHEC), including E. coli O157:H7

[0091] Francisella tularensis

[0092] Haemophilus influenzae

[0093] Helicobacter pylori

[0094] Klebsiella pneumoniae

[0095] Legionella pneumophila

[0096] Leptospira species

[0097] Listeria monocytogenes

[0098] Mycobacterium

[0099] M. leprae

[00100] M. tuberculosis

[00101] Mycoplasma pneumoniae

[00102] Neisseria

[00103] N. gonorrhoeae

[00104] N. meningitidis

[00105] Pseudomonas aeruginosa

[00106] Nocardia asteroides

[00107] Rickettsia rickettsii

[00108] Salmonella

[00109] S typhi

[00110] S. typhimurium

[00111] Shigella

[00112] S. sonnei

[00113] S. dysenteriae

[00114] Staphylococcus aureus

[00115] epidermidis

[00116] saprophyticus

[00117] Streptococcus agalactiae [00118] pneumoniae

[00119] pyogenes

[00120] viridans

[00121] Treponema pallidum subspecies pallidum

[00122] Vibrio cholerae

[00123] Yersinia pestis.

[00124] Embodiments of human viral pathogens:

[00125] Adeno-associated virus

[00126] Aichi virus

[00127] Australian bat lyssavirus

[00128] Banna virus

[00129] Barmah forest virus

[00130] BK polyomavirus

[00131] Bunyamwera virus

[00132] Bunyavirus La Crosse

[00133] Bunyavirus snowshoe hare

[00134] Cercopithecine herpesvirus

[00135] Chandipura virus

[00136] Chikungunya virus

[00137] Cosavirus A

[00138] Cowpox virus

[00139] Coxsackievirus

[00140] Crimean-Congo hemorrhagic fever virus

[00141] Dengue virus

[00142] Dhori virus

[00143] Dugbe virus

[00144] Duvenhage virus

[00145] Eastern chimpanzee simian foamy virus

[00146] Eastern equine encephalitis virus

[00147] Ebolavirus

[00148] Echovirus [00149] Encephalomyocarditis virus

[00150] Epstein-Barr virus

[00151] European bat lyssavirus

[00152] GB virus C/Hepatitis G virus

[00153] Hantaan virus

[00154] Hendra virus

[00155] Hepatitis A virus

[00156] Hepatitis B virus

[00157] Hepatitis C virus

[00158] Hepatitis delta virus

[00159] Hepatitis E virus

[00160] Horsepox virus

[00161] Human adenovirus

[00162] Human astrovirus

[00163] Human coronavirus

[00164] Human cytomegalovirus

[00165] Human enterovirus

[00166] Human herpesvirus 1

[00167] Human herpesvirus 2

[00168] Human herpesvirus 6

[00169] Human herpesvirus 7

[00170] Human herpesvirus 8

[00171] Human immunodeficiency virus

[00172] Human papillomavirus 1

[00173] Human papillomavirus

[00174] Human papillomavirus 2

[00175] Human parainfluenza

[00176] Human parvovirus B 19

[00177] Human respiratory syncytial virus

[00178] Human rhinovirus

[00179] Human SARS coronavirus [00180] Human T-lymphotropic virus

[00181] Human torovirus

[00182] Influenza A virus

[00183] Influenza B virus

[00184] Influenza C virus

[00185] Isfahan virus

[00186] Japanese encephalitis virus

[00187] JC polyomavirus

[00188] Junin arenavirus

[00189] KI Polyomavirus

[00190] Lagos bat virus

[00191] Lake Victoria marburgvirus

[00192] Langat virus

[00193] Lassa virus

[00194] Louping ill virus

[00195] Lymphocytic choriomeningitis virus

[00196] Machupo virus

[00197] Mammalian orthorubulavirus 5 (Simian virus 5)

[00198] Mayaro virus

[00199] Measles virus

[00200] Merkel cell polyomavirus

[00201] MERS coronavirus

[00202] Mokola virus

[00203] Molluscum contagiosum virus

[00204] Monkeypox virus

[00205] Mumps virus

[00206] Murray valley encephalitis virus

[00207] New York virus

[00208] Nipah virus

[00209] Norwalk virus

[00210] O'nyong-nyong virus [00211] Orf virus

[00212] Oropouche virus

[00213] Pichinde virus

[00214] Poliovirus

[00215] Punta toro phlebovirus

[00216] Puumala virus

[00217] Rabies virus

[00218] Rift valley fever virus

[00219] Rosavirus A

[00220] Ross river virus

[00221] Rotavirus A

[00222] Rotavirus B

[00223] Rotavirus C

[00224] Rubella virus

[00225] Sagiyama virus

[00226] Salivirus A

[00227] Sandfly fever Naples phlebovirus (Toscana virus)

[00228] Sandfly fever Sicilian virus

[00229] Sapporo virus

[00230] SARS coronavirus 2

[00231] Semliki forest virus

[00232] Seoul virus

[00233] Simian foamy virus

[00234] Sindbis virus

[00235] Southampton virus

[00236] St. louis encephalitis virus

[00237] Tick-borne powassan virus

[00238] Torque teno virus

[00239] Uukuniemi virus

[00240] Vaccinia virus

[00241] Varicella-zoster virus [00242] Variola virus

[00243] Venezuelan equine encephalitis virus

[00244] Vesicular stomatitis virus

[00245] West Nile virus

[00246] Western equine encephalitis virus

[00247] WU polyomavirus

[00248] Yaba monkey tumor virus

[00249] Yaba-like disease virus

[00250] Yellow fever virus

[00251] Zika virus.

[00252] In embodiments, the binding molecules specific for one of each of said one or more different analytes comprises a nucleic acid and the different analytes comprise singlenucleotide polymorphisms (SNP) of one or more loci.

[00253] In embodiments, the one or more different analytes are nucleic acids and the binding molecule specific for one of each of said one or more nucleic acids to be detected comprises a complementary nucleic acid capable of hybridizing with the analyte nucleic acid.

[00254] In embodiments, the method further comprises performing one or more cycles of polymerase chain reaction (PCR) on the analyte nucleic acids of the sample prior to step (a) with one or more primer sequence pairs comprising a forward primer and a reverse primer, the sequence of each of which primers is adjacent to, or flanks, a sequence of the target nucleic acid to which the hybridizing nucleic acid hybridizes.

[00255] In embodiments, the method further comprises denaturing double-stranded amplicons resulting from the PCR into single-stranded nucleic acids.

[00256] In embodiments, the forward and reverse primers are ended with a repeating 5’ phosphorothioate and the method further comprises contacting the PCR products with a CRISPR cas9 nuclease so as to cleave off 5’ phosphorothioate, thus permitting digestion by lambdaexonuclease so as to form single-stranded DNA.

[00257] In embodiments, the reverse primer, or forward primer, of each pair comprises additionally a nucleic acid sequence capable of binding a nucleic acid detection probe that comprises a fluorophore. [00258] In embodiments, the method further comprises contacting the single-stranded nucleic acids with the plurality of bead types and a nucleic acid detection probe comprising a fluorophore, under conditions permitting hybridization of single-stranded nucleic acids to complementary nucleic acid binding molecules affixed on the bead, and hybridization of the nucleic acid detection probes to the single-stranded nucleic acids, and then washing to remove nucleic acids not bound to a bead, wherein the presence of the fluorophore co-located with a type of bead indicates the presence of the nucleic acid analyte in the sample and wherein the type of that bead determined in step (c) indicates the specific analyte present.

[00259] In embodiments, the nucleic acid analytes are nucleic acids specific to a viral pathogen or bacterial pathogen.

[00260] In embodiments, the one or more different analytes are proteins, wherein the binding molecule specific for one of each of said one or more proteins to be detected comprises a peptide, aptamer, antibody or antibody fragment capable of hybridizing with the analyte protein.

[00261] In embodiments, prior to step (c) the bound analyte is contacted with a second peptide, second aptamer, second antibody or second antibody fragment, labelled with a biotin or streptavidin, capable of hybridizing with the analyte protein, under conditions permitting the said second peptide, second aptamer, second antibody or second antibody fragment to bind the analyte, and further comprising contacting the biotin or streptavidin with a fluorophore-labelled streptavidin, or fluorophore-labelled biotin, respectively, and then washing to remove unbound reagents.

[00262] In embodiments, the presence of the fluorophore co-located with a type of bead indicates the presence of a protein analyte in the sample and wherein the type of that bead determined in step (c) indicates the specific protein analyte present.

[00263] In embodiments, the SRS is performed using a 532 nm laser.

[00264] In embodiments, the fluorophore is a far-red fluorophore. In embodiments, the fluorophore is a Cy5. In embodiments, the fluorophore, with an excitation peak at 651 nm and an emission peak at 670 nm.

[00265] In embodiments, the fluorophore has an emission maxima greater than 660 nm.

[00266] In embodiments, the fluorophore is excited with a 660 nm laser.

[00267] In embodiments, presence of the fluorophore is detected by an imaging device. [00268] In embodiments where the analytes are nucleic acids, analyte levels as low as 20aM, 5aM or 2aM can be detected. In embodiments where the analytes are peptides or proteins, analyte levels as low as 20pg/ml, lOpg/ml or 5pg/ml can be detected.

[00269] Also provided is a system for Raman spectroscopy and fluorescence spectroscopy of a sample of microbeads doped with Raman-active-small-molecules (RASMs), comprising: a spectrometer; a light source comprising a first laser and a second laser, the first laser configured to excite Raman scattering from the sample with signature peaks at a first wavelength and the second laser configured to generate an emission spectrum from the sample with wavelengths higher than the first wavelength; a sample holder through which the sample is passed during spectroscopy of the sample; telescopic lenses configured to collimate and expand laser beams emitted by the first and second lasers; reflective lenses for directing the laser beams to the sample holder; an objective lens configured to focus an emitted Raman signal and an emitted fluorescence frequency spectrum from the sample resulting from illumination of the sample in the sample holder by the laser beams; a dichroic beam splitter configured to direct the emitted Raman signal and the emitted fluorescence frequency spectrum to the objective lens; a pin hole through which the focused Raman signal and the fluorescence frequency spectrum are passed; and relay lenses positioned after the pinhole for directing the Raman signal and the fluorescence frequency spectrum to the spectrometer, the spectrometer comprising a diffraction grating that filters the Raman signal from the fluorescence frequency spectrum so that both the Raman signal and the fluorescence frequency spectrum are detected by the spectrometer.

[00270] In embodiments, the sample holder comprises a microfluidic device. In embodiments, the sample holder comprises a flowcell, a microarray or a glass substrate. In embodiments, the glass substrate is a glass slide, such as a glass microscope slide.

[00271] In embodiments, the system further comprises an inverted microscope that comprises the dichroic beam splitter and the objective lens. [00272] In embodiments, the system further comprises relay lenses between the pinhole and the spectrometer.

[00273] In embodiments, the system further comprises at least one of a long-pass fdter and a notch filter between the relay lenses.

[00274] In embodiments, the system further comprises a photodiode configured to detect forward scattering of the laser beams to trigger the spectrometer.

[00275] In embodiments, the methods described herein are performed using a system described herein.

[00276] In embodiments, the plurality of bead types is affixed to a surface. In embodiments, the analytes to be analyzed are affixed to a surface

[00277] A method is provided for detecting the presence of one or more different analytes present in a sample comprising:

(a) contacting the sample with a plurality of types of discrete portions of a substrate, wherein Raman-active small molecule(s) can stably partition into said discrete portions of a substrate, each discrete portion of a substrate comprising: one, or more, types of Raman-active small molecule(s), each at a predefined concentration in each type of discrete portion, and a binding molecule, affixed thereon or present therein, specific for one of each of said one or more different analytes; wherein each type of discrete portion differs from the remaining types of discrete portions of the plurality in (i) concentration of Raman-active small molecule(s), (ii) spectrum or spectra of Raman-active small molecule(s), or (ii) both concentration and spectrum/spectra pattern of Raman-active small molecule(s);

(b) removing unbound analytes; and

(c) determining, with Stimulated Raman Scattering (SRS) or Spontaneous Raman Scattering, which type(s) of discrete portion having an analyte bound thereto are present after step (b) so as to thereby determine which type(s) of analyte are bound, thereby detecting which of one or more different analytes are present in the sample.

[00278] As has been demonstrated in earlier work (See world wide web at doi.org/10.1038/s41467-021-21570-0) a large number of Raman-active-small-molecules (RASMs) can be efficiently doped into polymer nanoparticles due to non-covalent interactions (FIG. 1 A). The Raman scattering signal from doped particles is therefore increased as a function of the intra-particle concentration of those RASMs. The signal enhancement factor is proportional to the volume of the particles as larger particles can contain a greater number of RASMs. Also, the Raman spectra of RASMs remain unchanged when doped into particles, making them ideal for scaling up as one only needs to develop RASMs to expand the multiplexity, which is a much easier task than, for example, developing new polymer chemistry.

[00279] Herein, to greatly expand the multiplexity from the currently available dozens of RASMs, a combinatorics strategy was adopted to generate millions of flavors of “Vibrant MicroBeads” (a laboratory name for the disclosed barcoded RASM beads) each having a unique Raman optical barcode (FIGS. 1B-1C). This strategy was achieved by using two dimensions of optical barcoding: brightness barcoding and spectral barcoding. Assuming: 1) each one of the RASM can be doped into microbeads at M different concentrations, resulting in microbeads that have M brightness levels (brightness barcoding); and 2) a combination of n RASMs can be doped together into one microbead and the Raman signal of each RASM in the same microbead can be detected individually (spectral barcoding), then the total possible number of unique Vibrant MicroBeads would be M ¹¹ ' ¹ . Thus, if 5 brightness barcoding and 10 spectral barcoding were considered, the resulting unique Vibrant MicroBeads would be 5 ¹⁰ ' ¹ = 9,765,624 flavors (FIG. IB). This massive multiplexity is the direct result of the fact that these 10 RASMs have individually resolvable Raman spectra (FIG. 2), whereas, for most fluorescence-based techniques, only about 3 spectrally resolvable fluorescence dyes can be used at the same time, greatly limiting the multiplexity.

[00280] In addition to the brightness barcoding and spectral barcoding mechanism, size barcoding can also be used in contexts where various sizes of microbeads can be used together to further increase the multiplexity. These size barcoding can either be read out by taking images and directly measuring the diameter of each microbead or by quantitatively measuring the forward and backward scattering as the scattering strength is dependent on their sizes. When size barcoding is made use of, the total multiplexity would be S*(Mn-l) where S indicates the number of different sizes.

[00281] Since the Raman signal enhancement factor is proportional to the volume of the particles, relatively large micron-sized polystyrene beads (for example, 3 pm; 5 pm, and 10pm diameter) were chosen as the particle-matrix for maximizing the Raman signal intensity and increasing the throughput. To generate Vibrant MicroBeads, one or combinations of spectrally distinguishable few RASMs of various concentrations were doped into polystyrene beads through a swell-and-diffusion mechanism. For example, in a 5 ml tube, 320 pl 4% w/v polystyrene microbeads and 320 pl RO water were mixed. In another 1.5 ml tube, 40pl 20x Carbow dye(s) DMSO stock solution and 120 pl THF (Sigma, 401757) were mixed. Then the THF/dye(s) mixture was added to the diluted microbeads to swell the microbeads and incorporate the dyes, followed by brief vortexing and gentle agitation with a rotary wheel for 30 min at room temperature. Dye concentration level and dye identity /mixture for each set of beads being manufactured was chosen beforehand so as to leverage the brightness barcoding and spectral barcoding concepts, respectively. Next, 3 ml 20 mM phosphate buffer (pH 7.3) was slowly added to shrink and trap the dyes in the microbeads. The mixture was then centrifuged at max speed for 2 min to remove any insoluble materials, and the supernatant was collected to yield Vibrant MicroBeads. Washing 3 times using Amicon 30 kDa MWCO filters (Millipore, UFC9030) with at least 10 ml RO water removes excessive organic solvents.

[00282] FIG. 3A shows an annotated image of a mixture of 96 flavors of Vibrant MicroBeads by Stimulated Raman Scattering microscopy. The image was composed of a stack of images each taken at one of the featuring Raman peaks. FIG. 3B indicated a spectral analysis of each Vibrant MicroBead and the density plot was generated by plotting the intensity at each Raman shift in a pairwise manner.

[00283] Although in the preliminary data herein set only 5 brightness barcoding levels were demonstrated, more levels are possible. The total possible levels are determined by both the intensity distribution of each single Vibrant MicroBead as well as the dynamic range of the detection system.

[00284] Besides the polystyrene as the particle-matrix which was demonstrated, it is possible to utilize other materials as long as RASMs can be doped or dissolved in those materials. For example, beads with paramagnetic microbeads can be used as the matrix to generate magnetic Vibrant MicroBeads for easy separation. Oil-water emulsion droplets can also be used if a large number of different active reactions in the droplet are to be monitored in parallel, and the barcoding can serve as the identifier of the identity of the reactions.

[00285] The Vibrant MicroBeads were tested in actual use situations, described as follows. [00286] Human virome detection: Infectious diseases are one of the major threats to human health, yet only a small portion of pathogens can be effectively monitored. Among over 1000 viruses that infect vertebrates, only dozens of them can be detected in clinical practice. In addition, most common methods suffer from low throughput (qPCR for instance), or high cost and long turnover (such as sequencing methods). Therefore, a rapid high-throughput method is in demand for viral surveillance, and quick diagnosis. Here such a method capable of diagnosing certain viral infections among all known human virome was designed and tested.

[00287] First, a set of DNA hybridization probes, each one of them targeting a viral species, were designed for all viral species with known genomes. Then, each of these hybridization probes was bioconjugated to a predetermined Vibrant MicroBead, respectively. To improve specificity, maximum sequence diversity was ensured among these probes. Also designed were a set of DNA primers that flank these hybridization regions in the viral genome for each viral species. For the reverse primers, a unique sequence overhang was added to the 5’ end (TATCTTAAGTCTTCGCGTG) (SEQ ID NO: 1) to serve as the binding site of the fluorescence detection probe (this could also be attached alternatively to the forward primer). Second, the viral genome was extracted (and reverse transcribed for RNA virus) and then amplified with the primer set by PCR. Third, after denaturing with NaOH, the resulting singlestranded PCR products were mixed with the bioconjugated Vibrant MicroBeads as well as the detection probe, and then acid was added to obtain neutral pH and permit hybridization between the amplified PCR product and the Vibrant MicroBeads. The Vibrant MicroBeads unbound DNA was then washed away. Lastly, the Vibrant MicroBeads were imaged by SRS or detected using a spontaneous Raman flow cytometer. When a virus species was present in the sample, it would be efficiently amplified and hybridized to its corresponding Vibrant MicroBead, and a fluorescence signal could be detected from the surface of the beads, thus detecting both the presence of and the identity of the specific viral nucleic acid in the sample.

[00288] To further increase the specificity, a CRISPR technique combined with exonuclease can be used to generate single-stranded hybridizable DNA in place of the alkaline denaturing. For this, both the forward and reverse primers are ended with repeating 5’ phosphorothioate. Consequently, only the PCR products that are recognized by the CRISPR cas9 nuclease (which cleaves away the 5’ protective phosphorothioate) can be digested by lambdaexonuclease and form single-stranded DNA which can then hybridize to their Vibrant MicroBeads. By doing this method, another checkpoint is realized by the specificity intrinsic to CRISPR to further increase the detection fidelity. See FIGS. 4A-4C.

[00289] Synthetic viral genomes were used to demonstrate the performance. In the preliminary test, dozens of synthetic targets were synthesized to mock viral genomes across 6 viral families. Out of 17 mock viral genomes tested, an accuracy of -95% accuracy was achieved (FIG. 5).

[00290] Another application for Vibrant MicroBeads is massively multiplexed fluorescence linked immunosorbent assay for peptide and protein detection. Briefly, a certain antibody or aptamer is bioconjugated to the surface of Vibrant MicroBeads. Then different Vibrant MicroBeads with different antibodies can be mixed for the multiplex detection of peptides or proteins (FIG. 6A-6B). Preliminary tests had been carried out and the sensitive detection limit was realized as low as 5pg/ml (FIG. 6C).

[00291] As an information carrier that contains the identity of the analyte in a more generalized term, Vibrant MicroBeads can be coupled with any kinds of affinity binders such as antibodies and nucleic acids to detect their corresponding ligand in a parallel and multiplexed way. For example, SNP phenotyping is also possible when specific oligo sequences that bind SNP flanking region can be coupled to Vibrant MicroBeads and single nucleotide extension can be introduced to readout the SNP.

[00292] Raman flow cytometer: Currently, coherent Raman techniques such as Coherent anti-Stokes Raman spectroscopy (CARS) and Stimulated Raman Scattering spectroscopy (SRS) are widely used in high-speed applications for their orders of magnitude stronger signal compared to spontaneous Raman. However, coherent Raman techniques usually employ ultrafast pulse laser sources which are not readily accessible to most users both economically and technically.

[00293] Since the Vibrant MicroBeads possess strong Raman scattering intensity as a result of concentrated RASMs, it is possible to acquire a sufficiently good signal with spontaneous Raman. FIG. 7 shows the Raman spectrum from a single 5 pm Vibrant MicroBead. Even when the exposure time was as short as 3ms, the signal-to-noise ratio of the RASMs peak is as high as about 50, making it possible for high throughput detection. Assuming the desirable SNR is about 5, then the minimum exposure time can be as short as 0.3ms, and therefore the throughput can be more than 3000 events per second, which is close to fluorescence-based flow cytometry techniques.

[00294] If the same number of events would be acquired with SRS, where laser wavelength needs to be tuned to excite each one of the barcoding Raman peaks, it would usually take 1 min to accumulate 3000 events for each frame of wavelength and thus 7 min in total for 7 peaks for example for the actual acquisition time. Yet the wavelength tuning takes about 2 min every time, so the total time consumed for the same 3000 events would take no less than 21 min. This makes sense since SRS provides high spatial resolution in the sacrifice of throughput, however, in certain scenarios such as barcoding detection discussed here, the spatial information is useless because only the spectral barcoding information of each bead is of interest, the shapes as well as the positions of the beads are not of concern.

[00295] Given that the Raman signal of Vibrant MicroBeads can be detected using spontaneous Raman in high fidelity, a custom spontaneous Raman flow cytometry setup was designed and built (FIG. 8A). In order to read barcoding information and identity information at the same time, the goal of this setup was to acquire Raman scattering signal and fluorescence signal simultaneously on the same detector. This would allow for simpler design as complicated electronics to synchronize Raman and fluorescence signal can be then eliminated. However, this is not common practice because usually the fluorescence signal overwhelms the Raman signal and many techniques such as fluorescence quencher, shifted excitation, FT-Raman, and so on were developed to suppress the fluorescence background so that the Raman signal can be reliably readout.

[00296] To solve this problem, a few strategies were adopted herein. Firstly, a 532 laser was used to excite the Raman scattering so that signature peaks from RASMs (-2000 cm-1) were around 600nm. Secondly, a red dye (such as Cy5) was used in fluorescence having emission maximum redder than 660nm. This way, the emission spectrum is well separated from the Raman peaks (that were around 600nm) and thus avoiding interfering with them. Thirdly, a diffraction grating with low groove density (600 1/cm) was chosen so that while a good spectral resolution was preserved, both the Raman spectra and fluorescence emission spectra could fit into the same camera frame. It should be noted that this design was especially suitable to be coupled with Vibrant MicroBeads because the Raman peaks were so strong that the relatively weak anti-stokes emission of the fluorescence dye would not affect the detection of Raman scattering signals.

[00297] FIG. 8B shows the schematics of the concept. FIG. 8C shows a combined Raman and fluorescence spectra of a Vibrant MicroBead with a fluorescence signal attached to it. Judging from the spectrum, Raman peaks themselves were well resolvable, and they were also well separated from the fluorescence emission signal. The Raman peaks were also background free, as discussed above so the weak anti-stokes emission from the fluorescence dye would not affect Raman signals.

[00298] The schematics of an exemplary home-built confocal Raman microscope is shown in FIG.8A. A 532nm laser (Samba 532 nm, 400mW, Cobolt Inc.) and a 660nm laser (Obis 660 nm lOOmW, Coherent) were used as the light source. The laser beam was first collimated and expanded by the telescope lenses (LI, L2/LT, L2’, Thorlabs). The expanded beam was then directed to the inverted microscope (1X71, Olympus) installed with a dichroic beamsplitter (LPD1, LPD02-532RU-25, Semrock). The emitted Raman signal first passed a pinhole (PH, 300um, Thorlabs) for background suppression and was relayed by two lenses (L3, L4, Thorlabs) before being projected to the spectrometer (Kymera 328i with 600 lines/mm grating blazed at 500nm, Andor). A long-pass filter (LP, LP03-532RU-25, Semrock) and notch filter (NF658-26, Thorlabs) were installed between the relay lenses to block laser light from Rayleigh scattering. Raman signal was then collected by an EMCCD (Newton970, Andor). For brightfield imaging, a long-pass dichroic (LPD2, FF511-DiOl, Semrock) was installed in front of the pinhole and a set of relay lenses (L5, L6) were used to project the brightfield images to the CMOS camera (DCC1645C, Thorlabs). A short-pass filter (SP) was installed to suppress ghost images. A photodiode (PD, SM05PD3A, Thorlabs) was mounted on a swivel mount installed above the sample stage, and was used to detect forward scattering to trigger the EMCCD acquisition.

[00299] Although a microscope base was used in this design, it is not necessary to include the microscope per se. For a simpler design, only the objective would be needed and the microscope base can be replaced by a customized frame that can hold the objective, which is the core of the detection system.

[00300] For the liquid flow cell system, a microfluidic device featuring a single straight channel (300 pm rectangular cross section) and acoustic focusing powered by a piezo chip vibrating at 2.49 Mhz was used (FIG. 8D). Other chips or capillary tubes can also be used as long as they provide the capability that can align the particles in the channel. 8E) Detection with a microwell array. The microwell array is designed so that each microwell can only hold one microbead at a time. Therefore, by scanning the microwell array where the microbeads have settled down, the signal from each individual bead can be acquired efficiently and maintenance free.

[00301] FIG. 9: shows confirmation validation of the technique. The following viral species (and control samples) are shown, with the numbered rows starting at the top of the left hand side of the display and increasing by number consecutively as one travels down the chart:

1 Adenoviridae_Mastadenovirus_Human_mastadenovirus_B

2 Adenoviridae_Mastadenovirus_Human_mastadenovirus_C

3 Adenoviridae Mastadenovirus Human mastadenovirus D

4 Adenoviridae_Mastadenovirus_Human_mastadenovirus_E

5 Adenoviridae_Mastadenovirus_Human_mastadenovirus_F

6 Anelloviridae_Betatorquevirus_TTV_like_mini_virus

7 Anelloviridae_Gyrovirus_Avian_gyrovirus_2

8 Anelloviridae Gyrovirus Chicken anemia virus

9 Anelloviridae_Iotatorquevirus_Torque_teno_sus_virus_l a

10 Anelloviridae_lotatorquevirus_Torque_teno_sus_virus_lb

11 Anelloviridae_Torque_teno_Leptonychotes_weddellii_virus_l

12 Anelloviridae_Torque_teno_Leptonychotes_weddellii_virus_2

13 Anelloviridae Torque teno virus

14 Arenaviridae_Arenavirus_Mopeia_Lassa_virus_reassortant_29_L

15 Arenaviridae Mammarenavirus Argentinian mammarenavirus L

16 Arenaviridae Mammarenavirus Cali mammarenavirus S

17 Arenaviridae Mammarenavirus Guanarito mammarenavirus L

18 Arenaviridae_Mammarenavirus_Lassa_mammarenavirus_L

19 Arenaviridae_Mammarenavirus_Lymphocytic_choriomeningitis_mam marenavirus_L

20 Arenaviridae_Mammarenavirus_Whitewater_Arroyo_mammarenavirus _S A st rovi ri d ae_M am astrovi ru s_M am astrovi ru s_ 1

Caliciviridae Sapovirus Sapporo virus

Coronaviridae_Alphacoronavirus_Human_coronavirus_229E

Coronaviridae_Alphacoronavirus_Human_coronavirus_NL63

Coronaviridae_Betacoronavirus_Human_coronavirus_HKUl

Coronaviridae_Betacoronavirus_Middle_East_respiratory_syn drome_related_coronavirus

Coronaviridae Betacoronavirus Severe acute respiratory syndrome related coronavirus

Deltavirus Hepatitis delta virus

Filoviridae Ebolavirus Sudan ebolavirus

Filoviridae Ebolavirus Zaire ebolavirus

Filoviridae_Marburgvirus_Marburg_marburgvirus

Flaviviridae Flavivirus Bagaza virus

Flaviviridae Flavi virus Cul ex flavi virus

Flavi viridae_Flavivirus_Dengue_virus

Flaviviridae Flavivirus Japanese encephalitis virus

Flaviviridae_Flavivirus_Kyasanur_Forest_disease_virus

Flavi viridae_Flavivirus_Murray_Valley_encephalitis_virus

Flaviviridae_Flavivirus_Saint_Louis_encephalitis_virus

Flavi viridae_Flavivirus_Tick_borne_encephalitis_virus

Flaviviridae_Flavivirus_Usutu_virus

Flaviviridae_Flavivirus_West_Nile_virus

Fl avi vi ri dae_F 1 avi vi rus_Y el 1 ow_fever_vi rus

Flaviviridae_Flavivirus_Zika_virus

Flaviviridae Hepacivirus Hepacivirus C

Flavi viridae_Pegivirus_Pegivirus_A

Flaviviridae Pegivirus Pegivirus C

Flavi viridae_Pegivirus_Pegivirus_H

Hantaviridae Orthohantavirus Andes orthohantavirus S Hantaviridae_Orthohantavirus_Dobrava_Belgrade_orthohantaviru s_L

Hantaviridae_Orthohantavirus_Imjin_orthohantavirus_M

Hantaviridae_Orthohantavirus_Nova_orthohantavirus_S

Hantaviridae_Orthohantavirus_Puumala_orthohantavirus_M

Hantaviridae_Orthohantavirus_Seoul_orthohantavirus_L

Hantaviridae_Orthohantavirus_Sin_Nombre_orthohantavirus_M

Hantaviridae Orthohantavirus Thottapalayam orthohantavirus S

Hantaviridae_Orthohantavirus_Tula_orthohantavirus_S

Hepeviridae Orthohepevirus Orthohepevirus A

Herpesviridae_Cytomegalovirus_Human_betaherpesvirus_5

Herpesviridae_Lymphocryptovirus_Human_gammaherpesvirus_4

Herpesviridae_Simplexvirus_Human_alphaherpesvirus_2

Herpesviridae_Varicellovirus_Human_alphaherpesvirus_3

Nairoviridae_Orthonairovirus_Crimean_Congo_hemorrhagic_fe ver_orthonairovirus_L

Nairoviridae_Orthonairovirus_Nairobi_sheep_disease_orthon airovirus_S

Orthomyxoviridae_Alphainfluenzavirus_Influenza_A_virus_l

Orthomyxoviridae_Betainfluenzavirus_Influenza_B_virus_l

Orthomyxoviridae_Gammainfluenzavirus_Influenza_C_virus_3

Papillomaviridae_Alphapapillomavirus_Alphapapillomavirus_ 4

Papillomaviridae Betapapillomavirus Betapapillomavirus l

Paramyxoviridae_Avulavirus_Avian_avulavirus_l

Paramyxoviridae Henipavirus Hendra henipavirus

Paramyxoviridae_Henipavirus_Nipah_henipavirus

Paramyxoviridae Morbillivirus Canine morbillivirus

Paramyxoviridae Morbillivirus Feline morbillivirus

Paramyxoviridae_Morbillivirus_Measles_morbillivirus

Paramyxoviridae Morbillivirus Rinderpest morbillivirus

Paramyxoviridae Respirovirus Bovine respirovirus 3 Paramyxovi ri dae Rubul avi rus Hum an rubul avi ru s_2

Peribunyaviridae_Orthobunyavirus_Akabane_orthobunyavirus_ M

Peribunyaviridae_Orthobunyavirus_Bunyamwera_orthobunyavir us_L

Peribunyaviridae_Orthobunyavirus_Califomia_encephalitis_o rthobunyavirus_L

Peribunyaviridae_Orthobunyavirus_Oropouche_orthobunyaviru s_L

Peribunyaviridae_Orthobunyavirus_Sathuperi_orthobunyaviru s_M

Peribunyaviridae Orthobunyavirus Shuni orthobunyavirus M

Phenuiviridae_Phlebovirus_Candiru_phlebovirus_L

Phenuiviridae_Phlebovirus_Rift_Valley_fever_phlebovirus_L

Phenuiviridae_Phlebovirus_SFTS_phlebovirus_L

Phenuiviridae_Phlebovirus_Sandfly_fever_Naplesjjhleboviru s_L

Phenuiviridae_Phlebovirus_Uukuniemi_phlebovirus_L

Picomaviridae Aphthovirus Foot and mouth disease virus

Pi comavi ri d ae_C ardi ovi ru s_C ar di ovi ru s_ A

Picomaviridae Enterovirus Enterovirus A

Picomaviridae Enterovirus Enterovirus D

Picomaviridae Enterovirus Enterovirus E

Picomaviridae Enterovirus Rhinovirus B

Picomaviridae Enterovirus Rhinovirus C

Picomaviridae Hepatovirus Hepatovirus A

Picomaviridae_Kobuvirus_Aichivirus_A

Picomaviridae Parechovirus Parechovirus A

Pneumoviridae_Metapneumovirus_Avian_metapneumovirus

Pneumoviridae Orthopneumovirus Human orthopneumovirus

Pneum oviri dae Respi ratory sy ncy ti al vi rus

Polyomaviridae_Alphapolyomavirus_Human_polyomavirus_5

Polyornaviridae_Betapolyomavirus_Macaca_mulatta_polyomavi rus_l

Poxviridae Orthopoxvirus Cowpox virus Poxviridae Parapoxvirus Orf virus

Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_L2 Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_L3 Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_Ml Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_M2 Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_M3 Reoviridae Orthoreovirus Mammalian orthoreovirus SI Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_S3 Reoviridae_Orthoreovirus_Mammalian_orthoreovirus_S4 Reoviridae_Rotavirus_Rotavirus_A_10 Reoviridae_Rotavirus_Rotavirus_B_l Reoviridae_Rotavirus_Rotavirus_C_3 Reoviridae_Seadomavirus_Banna_virus_12

Retroviridae_Deltaretrovirus_Primate_T_lymphotropic_virus _l Retroviridae_Lentivirus_Human_immunodeficiency_virus_l Retroviridae Lentivirus Simian immunodeficiency virus Rhabdoviridae_Lyssavirus_European_bat_l_lyssavirus Rhabdoviridae_Lyssavirus_Rabies_lyssavirus Rhabdoviridae Vesiculovirus Indiana vesiculovirus Rhabdoviridae_Vesiculovirus_New_Jersey_vesiculovirus control control control control control control control control [00302] FIG. 10 shows a data matrix summarizes validation results for upper respiratory infection panel, tested with inactivated real viruses and bacteria.

[00303] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[00304] The term "alkynyl" herein refers to a hydrocarbon radical straight or branched, containing at least 1 carbon-to-carbon triple bond - an “alkyne” - and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cn alkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkynyl ¹ ' means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C2-Cn alkynyl. An embodiment can be C2-C12 alkynyl or C3-C8 alkynyl.

[00306] The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not limited to, for example, data and information. It should be understood that, while these words, and/or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.