# US 2013/0157254 A1 · Method and apparatus for two-step surface-enhanced raman spectroscopy

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- Number: US 2013/0157254 A1
- Document ID: US20130157254A1
- Status: Published application
- Kind: A1
- Application number: 13/374,225
- Filed: 2011-12-16
- Published: 2013-06-20
- Earliest claimed: 2011-12-16
- Assignees: Real-Time Analyzers, Inc.
- Inventors: Atanu SENGUPTA; Frank E. INSCORE; Chetan SHENDE; Michael J. DONAHUE; Stuart FARQUHARSON
- Web page: https://patentgrep.com/patent/US20130157254A1

## CPC

- G01N 21/658: {enhancement Raman, e.g. surface plasmons}
- G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- G01N 33/587: {Nanoparticles}
- 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

A SERS method and apparatus employ a sample device having support structure including a first material containing a SER-active metal functionalized with a binding agent having specific capability for binding a designated target analyte. An analyte sample is introduced upon the functionalized SER-active metal; conditions to effect binding of the target analyte to the binding agent are maintained; unbound chemicals, biochemicals, or biologicals are removed; a second SER-active material is introduced to cause it to attach to the bound target analyte; the support structure is irradiated to generate a SER spectrum, with the first and second SER-active materials acting in concert; and the SER spectrum is detected and analyzed to determine the presence and quantity of the target analyte. Alternatively, the second SER-active material may be functionalized with a binding agent, with the procedure being modified accordingly.

## Claims

### Claim 1

A method for the detection, identification, analysis and quantitation, by surface-enhanced Raman spectroscopy, of at least one designated target analyte, comprising the steps:
providing a support structure comprised of a first, SER-active metal-containing SER-active material;
providing a liquid reagent comprised of a second, SER-active metal-containing SER-active material;
functionalizing the SER-active metal of at least one of said first and second SER-active materials with at least one binding agent that has a specific capability for binding thereto at least one designated target analyte, to provide at least one functionalized SER-active material;
obtaining an analyte sample suspected of containing said at least one designated target analyte;
adding said analyte sample to one of said at least one functionalized SER-active materials;
establishing or maintaining conditions sufficient to effect attachment of said at least one designated target analyte to said at least one binding agent of said one functionalized SER-active material;
effecting the removal of any unbound chemicals, biochemicals, or biologicals from said one functionalized SER-active material to which said at least one target analyte is attached;
adding said liquid reagent to said support structure;
establishing or maintaining conditions effective to cause the other of said first and second SER-active materials to attach to said at least one target analyte already attached to said at least one binding agent of said one functionalized SER-active material;
irradiating said first and said second SER-active materials attached to said at least one target analyte so as to cause said SER-active metals of said SER-active materials to cooperatively generate a SER spectrum;
detecting said cooperatively generated SER spectrum; and
analyzing said detected SER spectrum to determine the presence and quantity of said at least one designated target analyte.

### Claim 2 (depends on 1)

The method of claim 1 wherein said first SER-active material is comprised of at least one chemically synthesized porous material.

### Claim 3 (depends on 1)

The method of claim 1 wherein the effects of said SER-active metals attached to said at least one target analyte, in cooperatively generating said SER spectrum, are synergistic.

### Claim 4 (depends on 1)

The method of claim 1 wherein said support structure comprises a substrate supporting said first SER-active material.

### Claim 5 (depends on 4)

The method of claim 4 where said substrate is fabricated from metal, glass, paper, or plastic, and is in the form of a substantially planar sheet, plate, or membrane.

### Claim 6 (depends on 5)

The method of claim 5 wherein said substrate has a multiplicity of wells formed thereinto for receiving said analyte sample.

### Claim 7 (depends on 4)

The method of claim 4 wherein said substrate is fabricated from glass or plastic and is in the form of a wall of a vial, capillary, or channel.

### Claim 8 (depends on 1)

The method of claim 1 wherein said at least one binding agent is attached to said SER-active metal of said at least one functionalized SER-active material by means of covalent, ionic, or hydrogen bonding, or by van der Waals or electostatic interactions between charged, polar, hydrophobic, or hydrophilic chemical groups on the surface of said at least one binding agent.

### Claim 9 (depends on 1)

The method of claim 1 wherein a linker chemical or biochemical is interposed for attaching said at least one binding agent to said SER-active metal of said at least one functionalized SER-active material.

### Claim 10 (depends on 9)

The method of claim 9 wherein the molecule of said linker chemical or biochemical contains sulfur.

### Claim 11 (depends on 1)

The method of claim 1 wherein a spacer chemical or biochemical is interposed effectively between said at least one binding agent and said SER-active metal of said at least one functionalized SER-active material to provide sufficient space for the at least one target analyte to attach to said at least one binding agent.

### Claim 12 (depends on 1)

The method of claim 1 wherein a blocking chemical or biochemical is added to said at least one functionalized SER-active material to prevent extraneous SER-active chemicals, biochemicals, or biologicals contained in the analyte sample from interacting with said SER-active metal of said at lest one functionalized SER-active material so as to thereby minimize the production of SER spectra that would interfere substantially with a spectrum of said at least one designated target analyte.

### Claim 13 (depends on 1)

The method of claim 1 wherein said SER-active metal of each of said first and second SER-active materials is selected from the group consisting of copper, gold, silver, nickel, platinum, rhodium, iron, ruthenium, cobalt, nickel, palladium, and alloys and mixtures thereof.

### Claim 14 (depends on 1)

The method of claim 1 wherein said SER-active metal of said first SER-active material is of particulate form or in the form of a surface having a morphology that is functionally equivalent to metal particles for generating a plasmon field when irradiated, and wherein said SER-active metal of said second SER-active material is of particulate form.

### Claim 15 (depends on 13)

The method of claim 13 wherein said SER-active metals of said first and second SER-active materials are different from one another.

### Claim 16 (depends on 2)

The method of claim 2 wherein said at least one binding agent, said liquid reagent comprised of said second SER-active material, said at least one designated target analyte, and any signature chemical that may be present, can readily pass through said at least one chemically synthesized porous material.

### Claim 17 (depends on 16)

The method of claim 16 wherein said chemically synthesized porous material is effective to separate said at least one designated target analyte from other components of said analyte sample.

### Claim 18 (depends on 1)

The method of claim 1 wherein each of said at least one designated target analyte and said at least one binding agent is a chemical, biochemical, or biological substance.

### Claim 19 (depends on 18)

The method of claim 18 wherein said at least one binding agent and said at least one designated target analyte are paired with one another for effective interbonding, such pairs being selected from the group consisting of (a) antibodies and antigens, (b) peptides and biologicals, (c) drug receptors and drugs, (d) enzymes and their specific biochemical substrates, (e) carbohydrates and lectins, and (f) nucleic acid sequences and their complements.

### Claim 20 (depends on 1)

The method of claim 1 where said at least one designated target analyte is attached to said at least one binding agent by means of covalent, ionic, or hydrogen bonding, or by van der Waals or electrostatic interactions between charged, polar, hydrophobic, or hydrophilic chemical groups on the surface of said at least one binding agent.

### Claim 21 (depends on 1)

The method of claim 1 wherein said at least one target analyte sample comprises a biological agent.

### Claim 22 (depends on 21)

The method of claim 21 wherein said biological agent is selected from the group consisting of Bacillus anthraces, Clostridium botulinum A, Dengue fever, Ebola virus, Francisella tularensis, Leishmania genus, Marburg virus, Mycobacterium leprae, Plasmodium genus, Puumala hantavirus, Ricin toxin, Variola virus, and Yersinia pestis.

### Claim 23 (depends on 1)

The method of claim 1 wherein said at least one target analyte sample comprises a foodborne or waterborne pathogen.

### Claim 24 (depends on 23)

The method of claim 23 wherein said foodborne or waterborne pathogen is selected from the group consisting of Aeromonas hydrophilia, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum B and C, Clostridium difficile and perfringens, Cryptosporidium parvum, Escherichia coli, Giardia lamblia, Hepatitis A, Listeria monocytogenes and subspecies thereof, Salmonella enterica, typhimurium and subspecies thereof, Shigella, Yersinia enterocolitica.

### Claim 25 (depends on 1)

The method of claim 1 wherein said at least one target analyte is a disease-causing pathogen.

### Claim 26 (depends on 25)

The method of claim 25 wherein said disease-causing pathogen is selected from the group consisting of Burkholderia mallei, coronaviruses, Corynebacterium diphtheriae, Enteric viruses, Enterobacter aerogenes, Equine encephalitis, hemorrhagic fevers, Hepatitus A through E, herpes simplex viruses 1 and 2, human immunodeficiency virus, Legionella, Lyme borreliosis, measles, meningitis, mumps, MRSA, Mycobacterium tuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Norwalk virus, members of the Orthomyxoviridae family, rabies virus, rhinoviruses, Rubella virus, saxitoxin, sepsis, Shigella subspecies, and dysenteriae, Staphylococcal, Streptococcus pneumonia, Swine disease, Treponema pallidum, Vibrio cholerae, Varicella zoster virus, West Nile virus, and Yellow fever.

### Claim 27 (depends on 1)

The method of claim 1 including the further step of adding to said analyte sample at least one reactive reagent that is effective to react, under the conditions existing or established, with a constituent of said analyte sample for releasing a signature chemical, a signature biochemical, or a signature biological, said signature chemical, biochemical, or biological constituting said at least one designated target analyte.

### Claim 28 (depends on 27)

The method of claim 27 wherein said at least one target analyte is a released signature biochemical selected from the group consisting of amino and nucleic acids, nucleotides, nucleosides, peptides, proteins, lipids, polysaccharides, haptans, antibodies, antigens, biomarkers, enzymes, steroids, hormones, lectins, aptamers, and fragments and polymers thereof.

### Claim 29 (depends on 1)

The method of claim 1 wherein said support structure is constructed to effectively enable irradiation of said at least one designated target analyte and said first and second SER-active materials, and detection of said cooperatively generated SER spectrum.

### Claim 30 (depends on 1)

The method of claim 1 wherein a chemometric technique is applied to augment at least one of detection, identification, analysis, and quantitation of the at least one target analyte.

### Claim 31 (depends on 30)

The method of claim 30 wherein said chemometric technique applied augments analysis for the determination of pathogenicity, potency, toxicity or viability.

### Claim 32

Apparatus, in the form of a kit of components, for use in the detection, identification, analysis, and quantitation of at least one designated target analyte in an analyte sample, by surface-enhanced Raman spectroscopy, comprising:
packaging means for the containment of a multiplicity of components;
a SER-active device component constructed for receiving the at least one designated target analyte, and for operative assembly with a Raman spectrometer to enable detection, identification, analysis, and quantitation, by SERS, of the at least one designated target analyte, said SER-active device providing a support structure comprised of a first, SER-active metal-containing SER-active material;
at least one collection component constructed for analyte sample collection and discharge;
at least one container component containing a liquid reagent comprised of a second SER-active metal-containing SER-active material; and
a component for introducing said liquid reagent into said SER-active device; at least one of said first and second SER-active materials being functionalized with at least one binding agent that has the specific capability of binding thereto the at least one designated target analyte, and being accessible for the deposit thereupon of liquid analyte samples, said SER-active device being constructed for receiving, by way of said component for introducing, said liquid reagent, and for enabling irradiation, and collection of Raman scattered radiation cooperatively generated by the SER-active metals of said first and second SER-active materials, by and from a Raman spectrometer, all of said components being normally contained by said packaging means.

### Claim 33 (depends on 32)

The apparatus of claim 32 where said SER-active device component comprises a substrate supporting said first SER-active material.

### Claim 34 (depends on 32)

The apparatus of claim 32 where said substrate is fabricated from metal, glass, paper, or plastic, and is in the form of a substantially planar sheet, plate, or membrane.

### Claim 35 (depends on 34)

The apparatus of claim 34 wherein said substantially planar sheet, plate, or membrane has a multiplicity of wells formed thereinto for receiving an analyte sample.

### Claim 36 (depends on 33)

The apparatus of claim 33 wherein said substrate is fabricated from glass or plastic and is in the form of a capillary, vial, disc, or lab-on-a-chip channel, said first SER-active material being supported on a surface provided by said substrate.

### Claim 37 (depends on 32)

The apparatus of claim 32 wherein a linker chemical or biochemical is interposed for attaching said at least one binding agent to said SER-active metal of said at least one of said first and second SER-active materials.

### Claim 38 (depends on 32)

The apparatus of claim 32 wherein said SER-active metal of each of said first and second SER-active materials is selected from the group consisting of copper, gold, silver, nickel, platinum, rhodium, iron, ruthenium, cobalt, nickel, palladium, and alloys and mixtures thereof.

### Claim 39 (depends on 32)

The apparatus of claim 32 wherein said SER-active metal of said first SER-active material is of particulate form or in the form of a surface having a morphology that is functionally equivalent to metal particles for generating a plasmon field when irradiated, and wherein said SER-active metal of said second SER-active material is of particulate form.

### Claim 40 (depends on 38)

The apparatus of claim 38 wherein said SER-active metals of said first and second SER-active materials are different from one another.

### Claim 41 (depends on 32)

The apparatus of claim 32 wherein said support structure of said SER-active device component comprises a chemically synthesized porous structure through which said liquid reagent comprised of said second SER-active material, said at least one designated target analyte, and any signature chemical that may be present, can readily pass.

### Claim 42 (depends on 41)

The apparatus of claim 41 wherein said chemically synthesized porous material is effective to separate said at least one designated target analyte from other substances normally contained in an analyte sample.

### Claim 43 (depends on 32)

The apparatus of claim 32 wherein each of the at least one designated target analyte and said at least one binding agent is a chemical, biochemical, or biological substance.

### Claim 44 (depends on 43)

The apparatus of claim 43 wherein said at least one binding agent and said at least one designated target analyte are paired with one another for effective interbonding, such pairs being selected from the group consisting of (a) antibodies and antigens, (b) peptides and biologicals, (c) drug receptors and drugs, (d) enzymes and their specific biochemical substrates, (e) carbohydrates and lectins, and (f) nucleic acid sequences and their complements.

### Claim 45 (depends on 32)

The apparatus of claim 32 wherein said apparatus comprises a plurality of collection components, contained by said packaging means, selected from the group consisting of eyedroppers, syringes, pipettes, micropipetters, swabs, and various combinations thereof.

### Claim 46 (depends on 32)

The apparatus of claim 32 additionally including a water-supply component, contained by said packaging means, comprised of a container containing distilled, deionized water.

### Claim 47 (depends on 32)

The apparatus of claim 32 additionally including at least one mixing vial component contained by said packaging means.

### Claim 48 (depends on 32)

The apparatus of claim 32 additionally including a filtering component, contained by said packaging means, for filtering an analyte sample.

### Claim 49 (depends on 32)

The apparatus of claim 32 additionally including at least one second reagent supply component comprised of a container containing a second reagent, said second reagent being selected from the group consisting of buffered solutions, digesting agents, and agents that are reactive to degrade biological materials so as to effect release of signature analytes.

### Claim 50 (depends on 49)

The apparatus of claim 49 wherein said second reagent is an agent that is reactive with constituents of analyte samples to degrade biological materials and release at least one signature chemical, signature biochemical, or signature biological so as to provide a signature analyte constituting the at least one designated target analyte.

### Claim 51 (depends on 50)

The apparatus of claim 50 wherein said agent is selected from the group consisting of acetic acid, adipic acid, ascorbic acid, citric acid, formic acid, fumaric acid, lactic acid, malic acid, palmitic acid, peracetic acid, propionic acid, salicylic acid, sorbic acid, succinic acid, trihaloacetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, cyclohexane, dichloromethane, diethyl ether, dimethylsulfoxide, ethyl acetate, ethylene glycol, isopropyl ether, methyl ethyl ketone, n-hexane, phenolic derivatives, tetrahydrofuran, toluene, and mixtures thereof.