WO2011123386A1 - Procédés pour détecter la diffusion raman en utilisant des composés aromatiques comprenant du phosphate et au moins une source de lumière non laser - Google Patents

Procédés pour détecter la diffusion raman en utilisant des composés aromatiques comprenant du phosphate et au moins une source de lumière non laser Download PDF

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WO2011123386A1
WO2011123386A1 PCT/US2011/030166 US2011030166W WO2011123386A1 WO 2011123386 A1 WO2011123386 A1 WO 2011123386A1 US 2011030166 W US2011030166 W US 2011030166W WO 2011123386 A1 WO2011123386 A1 WO 2011123386A1
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Prior art keywords
raman
aromatic compound
detection
sample
phosphate
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PCT/US2011/030166
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English (en)
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Samar Kumar Kundu
Charles Lester Ginsburgh
Neal Arthur Siegel
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Samar Kumar Kundu
Charles Lester Ginsburgh
Neal Arthur Siegel
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Priority to CA2794130A priority Critical patent/CA2794130C/fr
Priority to EP11714460A priority patent/EP2553117A1/fr
Publication of WO2011123386A1 publication Critical patent/WO2011123386A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the present disclosure generally relates to the field of biological diagnostic equipment and testing methods.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reactions
  • Specificity of detection methods can be enhanced by using immunological techniques.
  • medical diagnostics use antibody-based techniques to provide specificity in the detection of biological components of a sample.
  • Antibodies developed to specific compounds are known to have high affinity and specificity for these components.
  • antibodies are difficult to detect and typically require chemical modification with labels or tags to enhance detection.
  • antibody detection is prone to interference from other material in the sample including the sample matrix, wash components, and other chemical and biological agents.
  • current techniques lack sensitivity at low
  • concentrations or numbers of antibodies i.e., low concentrations or numbers of targeted biological components.
  • Raman light scattering techniques have been used in the past to detect specific chemical components. Raman scattering is a basic property of the interaction of light with molecules. When light hits a molecule it can cause the atoms of the molecule to vibrate. This vibration will then change the energy of additional light scattered from the molecule. This scattered light has characteristics that are measurable and are unique to the structure of the vibrating molecule. Thus, a Raman spectrum can be used to uniquely identify a molecule.
  • Raman spectroscopy has several advantages over existing detection methods, including simple application and production of quantifiable data.
  • Raman spectroscopy by itself lacks specificity and sensitivity for the detection of biological organisms and components.
  • dedicated laser-based Raman detectors are expensive, are not easily available, and are limited to a single wavelength. Therefore, there is a need in the art for reagents and methods that allow for the practical use of Raman scattering in the detection of organisms and biological components.
  • the present disclosure is directed to methods that use the
  • Raman scattering a detector comprising at least one non-laser- based light source, and biological labeling techniques to identify and quantify biological organisms and components with higher sensitivity and specificity than prior art techniques.
  • One embodiment of the disclosure is a method for detecting the activity of at least one enzyme in a sample comprising:
  • Another embodiment is a method for detecting the activity of at least one enzyme in a sample comprising:
  • Another embodiment is a method for detecting the activity of at least one enzyme in a sample comprising:
  • Another embodiment is a method for detecting the activity of at least one enzyme in a sample comprising:
  • Another embodiment is a method for detecting at least one target in a sample comprising:
  • Another embodiment is a method for detecting at least one target in a sample comprising:
  • kits for detecting at least one enzyme activity comprising:
  • kits for detecting at least one enzyme activity comprising:
  • the Raman-active product is detected using resonance Raman spectroscopy. In another embodiment, the Raman-active product is detected using scattered light.
  • the at least one non-laser-based light source is a spectrophotometer capable of detecting Raman scattering using a fluorescence detection channel.
  • the at least one amine-containing compound comprises:
  • X is H, NH 2 , CI, Br, nitro, or benzyl
  • Y is H, CI, Br, or nitro
  • Z is H, benzyl, or NH 2 .
  • X is NH 2 , and Y and Z are H.
  • X is CI, and Y and Z are H.
  • X is Br, and Y and Z are H.
  • X is nitro, and Y and Z are H.
  • X and Z are H and Y is CI.
  • X and Z are H and Y is Br.
  • X and Z are H and Y is nitro.
  • X and Z are benzyl and Y is H.
  • X and Z are NH 2 and Y is H.
  • the at least one amine-containing compound comprises:
  • the at least one amine-containing compound comprises:
  • the at least one aromatic compound comprises:
  • W, X, Y, and Z are H or OH.
  • Y is OH and X, Y and Z are H.
  • W is OH, and X, Y and Z are H.
  • W and X are OH, and Y and Z are H.
  • W and Y are OH, and X and Z are H.
  • W and Z are OH and X and Y are H.
  • the at least one aromatic compound comprises:
  • X, Y, and Z are H or OH.
  • X is OH and Y and Z are H.
  • X and Y are OH and Z is H.
  • X and Z are OH and Y is H.
  • Z is OH and X and Y are H.
  • the at least one aromatic compound comprises:
  • X and Y are H or OH.
  • X is OH and Y is H.
  • X is H and Y is OH.
  • the at least one aromatic compound comprises:
  • X and Y are H or OH.
  • X is OH and Y is H.
  • X is H and Y is OH.
  • the at least one amine-containing compound comprises an aromatic amine.
  • the aromatic amine comprises ortho-phenylenediamine, meta-phenylenediamine, or para- phenyleneamine:
  • the at least one aromatic compound comprises:
  • X is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, P0 4 , or COOH;
  • Y is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, or COOH;
  • the at least one aromatic compound comprises:
  • X is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, P0 4 , or COOH;
  • Y is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, or COOH;
  • Z is H, OH, CI, Br, NH 2 , S0 3 H, P0 4 , or COOH.
  • the at least one aromatic compound comprises:
  • Y is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, or COOH.
  • the at least one aromatic compound comprises:
  • X is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, P0 4 , or COOH;
  • Y is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, or COOH.
  • the at least one aromatic compound comprises:
  • the at least one amine-containing compound is chosen from 4-aminoantipyrene, 5-aminosalicyclic acid, and o-phenylenediamine;
  • the at least one aromatic compound is chosen from 2-hydroxybenzyl alcohol, 4- chloro-3,5-dimethylphenol, 2-naphthol, 4-hydroxy-4-biphenyl-carboxylic acid, 5,7- dichloro-8-hydroxyquinoline, 4-chloro-1-naphthol, phenol, 4-amino-1 -phenyl-1- phosphate, 4-hydroxy-1-naphthyl-1-phosphate, 4-amino-1-naphthyl-1-phosphate, hydroquinone diphosphate, and 4,5 dihydroxy-naphthelene-2,7-disulfonic acid; and the at least one electron-donating compound is chosen from an organic hydrogen peroxide, urea hydrogen peroxide, and hydrogen peroxide (H 2 0 2 ).
  • the at least one electron-donating compound is a hydrogen peroxide.
  • the hydrogen peroxide is chosen from an aromatic hydrogen peroxide, urea hydrogen peroxide and hydrogen peroxide (H 2 0 2 ).
  • the at least one enzyme is a peroxidase or a phosphatase.
  • the phosphatase is alkaline phosphatase.
  • the alkaline phosphatase is conjugated to an antibody, an avidin moiety, a streptavidin moiety, a biotin moiety, or a biotin-binding protein.
  • the at least one aromatic compound is 2- hydroxybenzyl alcohol
  • the at least one amine containing compound is 5- aminosalicyclic acid
  • the at least one electron-donating compound is urea hydrogen peroxide
  • the at least one enzyme is a peroxidase
  • the mixture is incubated in the presence of a base.
  • the base is sodium hydroxide.
  • the oxidizing agent is sodium metaperiodate.
  • the mixture further comprises biotin.
  • the ligand is chosen from a receptor and an antibody. In another embodiment, the ligand is an antibody.
  • the at least one target is an organism.
  • the organism is chosen from a bacteriophage, a bacterium, including E. coli, Listeria, Salmonella, Vibrio, Camphelbacter, and Staphylococcus, and a virus such as HIV, Hepatitis, Adenovirus, Rhino virus, and Human papilloma virus.
  • the target is a component of an organism.
  • the component is a protein.
  • the protein is an interleukin.
  • the interleukin is IL-2.
  • the protein is chosen from C-Reactive protein, Tumor Necrosis Factor Receptor II, and Human Cardiac Troponin I.
  • the target is a component of an organism chosen from amino acids, nucleic acids, nucleotides, metabolites, carbohydrates, hormones, and metabolic intermediates.
  • Figure 2 is a diagram of an embodiment of the disclosed apparatus.
  • Figure 3 is a flow chart of an embodiment of the disclosed technique for the detection of biological organisms and/or components.
  • Figure 4 is a block diagram of the enzyme system for converting chemical components to a Raman-active compound.
  • Figure 5 is a flow chart of a technique for choosing light frequencies to excite specific target molecules.
  • Figure 6 is an illustration of a micro-fluidic channel designed to detect Raman-active compounds.
  • Figure 7 is an illustration of an array of micro-fluidic channels such as might be incorporated into a custom integrated circuit.
  • Figure 8 plots Raman spectra from an enzyme-linked immunoassay for a pathogenic bacteria, Listeria, utilizing an antibody linked to peroxidase and with shift numbers (cm "1 ) plotted on the abscissa and signal magnitudes plotted on the ordinate (arbitrary units) for a sample containing Listeria (a) and a sample not containing Listeria (b).
  • Figure 9 A plots Raman spectra measured at 3260 cm '1 produced using Raman Reagent formulation A-1 in three experiments, while Figure 9 B plots SQR Raman spectra measured at 3500-4000 cm "1 produced using Raman Reagent A-1 in the three experiments.
  • Figure 10 plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent A-1 (diamonds), and Raman Reagent A-2 (triangles) and A-3 (squares).
  • Figure 1 1 plots SQR Raman spectra measured at 3500-4000 cm "1 produced using Raman Reagent formulation A-1 (diamonds), and Raman Reagent A-2 (squares and triangles).
  • Figure 12 plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent formulation A-2 (squares) and A-2 with fresh HPRO in BSA diluent (diamonds).
  • Figure 13 plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent B-1 (diamonds), B-2 (squares), B-3 (triangles), and B-4 ("Xs").
  • Figure 4 plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent B-2 (squares) and B-2 with fresh HPRO in BSA diluent (diamonds).
  • Figure 15 A plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent C-1 while Figure 15 B plots the corresponding SQR Raman spectra measured at 3500-4000 cm “1 .
  • Figure 16 A plots Raman spectra measured at 3260 cm “1 produced using Raman Reagent D-1 while Figure 16 B plots the corresponding SQR Raman spectra measured at 3500-4000 cm “1 .
  • Figure 17 plots SQR Raman spectra measured at 3500-4000 cm '1 produced using Biotin-ASA-UP and ASA-UP.
  • Figure 18 is a bar graph showing the relative sensitivity of the reagents tested.
  • Figure 19 is a plot of the SQR Raman spectra measured at 3500- 4000 cm “1 and absorbance spectrum measured at 450 nm in IL-2 immunoassays using BASH-UP and TMB.
  • Figure 20 A is a plot of an absorbance spectrum for a BASH-UP reaction
  • Figure 20 B is an absorbance spectrum for an OPD reaction.
  • Figures 21 A and 21 B are plots of fluorescence spectra of BASH-UP and OPD reactions without peroxidase
  • Figures 21 C and 21 D are plots of fluorescence spectra of BASH-UP reactions with peroxidase.
  • Figures 22 A and 22 B are plots of fluorescence spectra of OPD reactions without peroxidase
  • Figures 22 C and 22 D are plots of fluorescence spectra of OPD reactions with peroxidase.
  • Figures 23 A and 23 B are plots of Raman signals produced by BASH-UP and OPD reactions, respectively.
  • Figure 24 A is a plot of Raman signals over time for an OPD reaction without peroxidase
  • Figures 24 B-E are plots of Raman signals over time for OPD reactions with decreasing amounts of peroxidase.
  • Figures 25 A-D are plots of SQR spectra over time for OPD reactions.
  • Figure 26 A is a plot of Raman signal of benzoquinone and Figure 26 B is a plot of Raman signal of pyrogallol, both figures showing enhanced Raman signal upon adding sodium hydroxide.
  • Figure 26 A (a) is a plot of Raman signal for benzoquinone with NaOH added
  • Figure 26 A (b) is a plot of Raman signal for benzoquinone with no NaOH.
  • Figure 26 B (a) is a plot of Raman signal for pyrogallol with NaOH added
  • Figure 26 B (b) is a plot of pyrogallol with no NaOH.
  • Figure 27 A is a plot of Raman signal of 1 ,4-naphthaquinone
  • Figure 27 B is a plot of Raman signal of 1 ,4-iminonaphthaquinone, both figures illustrating a dependence on periodate and sodium hydroxide.
  • Figure 27 A (a) is a plot of Raman signal of 1 ,4-naphthaquinone with no periodate or NaOH.
  • FIG. 27 (b) is a plot of Raman signal of 1 ,4-naphthaquinone with periodate but no NaOH.
  • Figure 27 A (c) is a plot of Raman signal of 1 ,4-naphthaquinone with no periodate but with NaOH.
  • Figure 27 A (d) is a plot of Raman signal of 1 ,4-naphthaquinone with periodate and NaOH.
  • Figure 27 B (a) is a plot of Raman signal of 1 ,4- iminonaphthaquinone with NaOH.
  • Figure 27 B (b) is a plot of Raman signal of ,4- iminonaphthaquinone in borate buffer.
  • Figure 27 B (c) is a plot of Raman signal of 1 ,4-iminonaphthaquinone with periodate and NaOH.
  • Figure 27 B (d) is a plot of Raman signal of 1 ,4-iminonaphthaquinone with periodate but no NaOH.
  • Figure 28 A is a logarithmic plot of Raman spectral values at 3300 cm " recorded for 4-aminophenylphosphate as a function of alkaline phosphatase- antibody conjugate concentration with the addition of oxidizing agent, while Figure 28 B shows the linear plot.
  • Figure 29 A shows Raman spectra of 4-aminophenylphosphate as a function of alkaline phosphatase conjugate concentration ranging from 0-1000 ng/mL with the addition of oxidizing agent, while Figure 29 B shows the range 0-10 ng/mL.
  • Figure 29 A (a-f) show Raman spectra of 4-aminophenylphosphate as a function of the concentration of alkaline phosphatase conjugate: (a) 1000 ng/ml, (b) 100 ng/ml,
  • Figure 29 B shows Raman spectra of 4-aminophenylphosphate as a function of the concentration of alkaline phosphatase conjugate: (a) 10 ng/ml, (b) 1 ng/ml, (c) 0 .1 ng/ml, (d) 0.01 ng/ml; and (e) 0 ng/ml.
  • Figure 30 A is a logarithmic plot of Raman spectral values at 3300 cm "1 recorded for 4-aminophenylphosphate as a function of alkaline phosphatase concentration, while Figure 30 B shows the linear plot.
  • Figure 31 A shows Raman spectra of 4-aminophenylphosphate as a function of alkaline phosphatase concentration ranging from 0-2500 mU/mL, while Figure 31 B shows the range 0-25 mU/mL.
  • Figure 31 A (a-f) shows Raman spectra of 4-aminophenylphosphate as a function of the concentration of alkaline
  • phosphatase (a) 2500 mU/mL; (b) 250 mU/mL; (c) 25 mU/mL; (d) 2.5 mU/mL; (e) 0.25 mU/mL; and (f) 0 mU/mL.
  • Figure 31 B (a-e) shows Raman spectra of 4- aminophenylphosphate as a function of alkaline phosphatase concentration: (a) 25 mU/mL; (b) 2.5 mU/mL; (c) 0.25 mU/mL; (d) 0.025 mU/mL; and (e) 0 mU/mL.
  • Figure 32 shows Raman spectra of peroxidase detected using a fluorescent microplate reader.
  • Figure 33 shows Raman spectra of peroxidase detected using a fluorescent microplate reader.
  • Figure 34 shows the Raman spectral response of peroxidase at different emission wavelengths detected using a fluorescent microplate reader.
  • Figure 35 shows dose response curves of peroxidase detected using a single lens Raman optics based reader (Raman SV) and a fluorescent microplate reader at 680 nm.
  • Figure 36 shows dose response curves of peroxidase detected using a single lens Raman optics based reader (Raman SQR) and a fluorescent microplage reader at 680 nm.
  • Figure 37 shows dose response curves of TNF-a detected using a single lens Raman optics based reader (Raman SV) and a fluorescent microplate reader at 680 nm.
  • Figure 38 shows dose response curves of TNF-a detected using a single lens Raman optics based reader (Raman SQR) reader and a fluorescent microplate reader at 690 nm.
  • Figure 41 shows the results of the scan with alkaline phosphatase.
  • Figure 42 shows the results from alkaline phosphatase from calf intestine for 45 minute reading at excitation wavelength 550 nm and emission at 580.
  • Figure 43 shows the results from alkaline phosphatase from E. coli for 45 minute reading at excitation wavelength 550 nm and emission at 580 nm.
  • diagnostics pathogen detection, forensics, and homeland security require the rapid and specific identification of biological organisms, such as contaminating bacteria, and biological components such as proteins, DNA, or other genetic material.
  • biological organisms such as contaminating bacteria, and biological components such as proteins, DNA, or other genetic material.
  • biological components such as proteins, DNA, or other genetic material.
  • a common assay to identify a bacterium in a sample is an
  • immunoassay which relies on detecting an antibody bound to the bacterium.
  • the antibody is labeled and the presence of the antibody is detected by assaying for the presence of the label.
  • the antibody is conjugated to an enzyme, and the presence of the antibody-enzyme conjugate is detected by assaying for enzymatic activity.
  • a commonly used assay that employs an enzyme- antibody conjugate is the enzyme linked immunosorbant assay (ELISA).
  • ELISA enzyme linked immunosorbant assay
  • enzymatic activity can be measured by incubating the enzyme-antibody conjugate in the presence of reactants that are converted by the enzyme into products which can be detected through colorimetric, fluorogenic, and
  • chemiluminescent means suffers from several deficiencies such as limited dynamic range, limited sensitivity, and interference from background.
  • Raman spectroscopy has several advantages over these methods, it generally cannot be used in combination with commonly used colorimetric, fluorogenic, and chemiluminscent reagents because they typically do not produce useful Raman spectra.
  • the colorimetric reagents 3,3', 5,5'- tetramethelene benzidine (TMB), and azinobisethlybenzthiazolinesulfonic acid (ABTS) do not produce Raman spectra useful for detecting organisms.
  • reagents that produce Raman-active products useful for detecting organisms are desired, including reagents that can be used in immunoassay formats employing enzyme-antibody conjugates.
  • Reagents useful for detecting a bacterium in an immunoassay format using Raman scattering have certain desired characteristics.
  • the reagents should produce a Raman signal in an area of the Raman spectrum that does not already have background signal produced by the bacterium.
  • the Raman signal produced by the reagents should be quantifiable, allowing for detection over a wide range of concentrations.
  • Certain embodiments of the present disclosure are also based in part on the discovery that certain combinations and amounts of the reagents of the disclosure produce superior sensitivity. This sensitivity can be further enhanced through use of the Single Quantifiable Result (SQR) method of the disclosure, which employs multiple wavenumber spectroscopic analyses.
  • SQL Single Quantifiable Result
  • Raman-active products may be detected using a non-laser- based light source, such as a fluorescence-detection device, a laser-based light source, or both.
  • a non-laser- based light source such as a fluorescence-detection device, a laser-based light source, or both.
  • Additional embodiments of the present disclosure are based in part on the discovery that compounds having at least one phosphate group can be used as phosphatase substrates to produce Raman-active products, or precursors to Raman- active products.
  • the phosphatase substrates may be aromatic compounds that may be enzymatically dephosphorylated in the presence of a phosphatase to form the corresponding phenols or aminophenols. The phenols and aminophenols may then autooxidize or become oxidized by the addition of an oxidizing agent to generate the corresponding Raman-active quinones or iminoquinones.
  • the phosphatase substrates may be used in an immunoassay format.
  • the phosphatase may be alkaline phosphatase.
  • the precursors to Raman-active products may be converted to Raman-active products by exposure to a base.
  • the base may be NaOH.
  • Antibody means an immunoglobulin or a part thereof, and encompasses any polypeptide comprising an antigen-binding site regardless of the source, method of production, and other characteristics.
  • the term includes for example, polyclonal, monoclonal, monospecific, polyspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies.
  • a part of an antibody can include any fragment which can still bind antigen, for example, an Fab, F(ab') 2 , Fv, scFv.
  • the origin of the antibody is defined by the genomic sequence irrespective of the method of production.
  • amino acids of any length
  • polymeric form of amino acids of any length can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones.
  • the term includes single chain protein as well as multimers.
  • amino acid refers to monomeric forms of amino acids, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides.
  • polynucleotide refers to polymeric forms of nucleotides of any length.
  • the polynucleotides can comprise deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
  • nucleotide refers to monomeric nucleotides and includes deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
  • Light source refers to any source of energy that falls within the electromagnetic spectrum.
  • Light sources include, but are not limited to, light bulbs, lasers, diodes, masers, and gas-discharge lamps including neon lamps, xenon lamps, xenon flash lamps, and mercury-vapor lamps.
  • the present disclosure can be practiced in various formats.
  • the format is an immunoassay.
  • a target biologic is first bound to an antibody that is attached to a solid surface.
  • Unbound components of the test sample are then optionally washed away leaving only the bound biologic/antibody combinations, which can be detected by Raman light scattering.
  • a target biologic is first bound to an antibody, or an antibody-enzyme conjugate. This biologic/antibody or
  • biologic/antibody-enzyme combination reacts with a substrate compound, such as an aromatic organic compound having at least one phosphate group, via the antibody portion of the biologic/antibody or biologic/antibody-enzyme combination.
  • a substrate compound such as an aromatic organic compound having at least one phosphate group
  • the substrate compound then further oxidizes into a Raman-active product.
  • quantification of the target biologic is thus achieved by detection of the Raman-active product.
  • the combination of the new reactant(s) with the biologic/antibody combination can now be detected using Raman scattering of light.
  • examples of such reactants include, but are not limited to the reagents listed in Table 1.
  • the Raman detection methods can use chemicals that interact with the biologic without the antibody.
  • the Raman-based methods can be applied to many immunoassays including, but not limited to, the detection of Human IL-1 1 , Rat C- reactive Protein, Soluble Tumor Necrosis Factor Receptor II, and Human Cardiac Troponin I.
  • the Raman-based methods can be applied to the detection of variety of organisms and components.
  • bacteriophage are detected.
  • bacteria including E. coli, Listeria, Salmonella, Vibrio, Camphelbacter, and Staphylococcus and detected.
  • viruses such as HIV, Hepatitis, Adenovirus, Rhino virus, Human papilloma virus are detected.
  • components including proteins, amino acids, nucleic acids, nucleotides, metabolites, hormones, and metabolic intermediates are detected.
  • SQL Single Quantifiable Result
  • Raman spectra can be analyzed by obtaining a Single Quantifiable Result (SQR).
  • SQR number is the difference between a Raman spectra corresponding to a targeted analyte measured in a sample, and any background Raman signal/spectra observed in the measurement process.
  • the steps of the SQR process are shown in Table 2.
  • spectra for the background of the sample (Negative Control) and for the samples being investigated (Test Samples) are measured.
  • the square root of the "Sum of the Squares of the Differences” is calculated ("Square Root of the Sum of the Squares of the Differences"). This value is designated as the SQR value.
  • the SQR process can include an assessment of whether the Raman signals from the sample and background are appropriate (i.e., "valid") and sufficient to indicate the presence of the targeted analyte in the sample (i.e., "positive value”).
  • the SQR process may be performed manually or with designed computer software.
  • the Raman signals for multiple wave numbers are tabulated for the background and test spectra. In one embodiment, every 2 nd wave number is tabulated for both the background and test spectra. In another embodiment, every wave number is tabulated for both the background and test spectra. In one embodiment the range of wave numbers is from 2000 to 4000 cm "1 .
  • the range of wave numbers is from 3500 to 4000 cm ⁇
  • the difference between the test signal and background signal is determined for a range of wave numbers and the square of this difference is stored.
  • the sum of the squares is determined, and the square root of this sum is the SQR value.
  • the Raman value of the background sample ("Negative Control") at a wave number, for example, 3260 cm "1 should run as expected (above a minimum and below a maximum value). This determination will aid in ensuring that a correct sample was run as the negative control, and that the assay was run correctly.
  • the SQR value of the positive control should not run below an expected value. This will aid in ensuring that a correct sample was run as the positive control, and that the assay was run correctly.
  • the "Sum of the Differences" for each test sample should not run below an expected value.
  • the SQR method can be carried out manually or with the aid of a computer.
  • One embodiment of the disclosure is a computer bearing machine operable language for the calculation of the SQR.
  • the Raman-active compounds of the invention may be detected using an instrument with a traditional laser-based light source.
  • Such instruments include, for example, a Raman Systems INC QE 65000 Raman Detector and a Lambda Solutions model PS-1 detector.
  • the Raman- active compounds may be detected using an instrument with a non-laser-based light source.
  • the non-laser-baesd light source may comprise a high-intensity light source.
  • the high-intensity light source may comprise a xenon lamp, a xenon flash lamp, a neon lamp, or a mercury-vapor lamp.
  • Suitable instruments comprising a non-laser-based light source include, for example, fluorescence detectors such as, for example, Tecan Infinite 200, Tecan Infinite ® 200 PRO, and Tecan Infinite ® M1000.
  • embodiments of the present disclosure can be implemented on a micro-fluidic channel (or well) integrated circuit using micro or nano-fabrication technology in which the binding partner is immobilized in one or more micro-fluidic channels in a custom integrated circuitry which would also include equipment to detect the Raman spectra generated by the methods of the invention.
  • Such an implementation could detect single biological components such as pathological bacteria, proteins or genetic material.
  • an object of certain embodiments of the present disclosure is to have a system for the detection of target biological organisms or components that utilizes a combination of chemical interactions including binding with a final step of Raman light scattering.
  • Another object of certain embodiments of the present disclosure is to have a system for the detection of target inorganic or organic components that utilizes a combination of chemical interactions including binding with a final step of Raman light scattering.
  • Another object of certain embodiments of the present disclosure is to combine an immunoassay with detection using Raman light scattering.
  • Still another object of certain embodiments of the present disclosure is to increase sensitivity of detection by the use of chemical reactants that produce resonance Raman light scattering.
  • Yet another object of certain embodiments of the present disclosure is to have an integrated circuit design with micro-fluidic channels or wells which can perform the combination of binding and Raman light scattering measurements.
  • FIG. 1 is a flow chart of a typical prior art immunoassay technique (ELISA) ( 0) for the detection of biological organisms or components.
  • the process begins by step (11) of preparing the liquid sample that includes the target biologic.
  • the sample can be prepared by pre-enrichment in a growth medium such as half-Frasier's broth or other suitable microbial growth medium.
  • a liquid sample for testing may be obtained from any liquid source. Solid material may be immersed in an appropriate liquid solution and potential target organism or molecules placed in solution and then sampled in the liquid.
  • the prepared liquid sample is combined (or mixed) with a binding partner that has been attached to a solid surface.
  • Typical binding partners include antibodies,
  • bacteriophage and bacteriophage proteins.
  • plastic microtiter plates latex beads or magnetic microparticles may be used.
  • Other solid supports such as nitrocellulose, filter paper, nylon and other plastics may also be used.
  • the antibody/biologic combination is then incubated in step (13) to allow time for the biologic and antibody to bind together. Once this has occurred the combined binding partner/biologic is decanted (poured off) and washed to remove unbound biologies and other unwanted materials. New reactants are added in step (15) to enhance the sensitivity of the mixture to detection of signal molecules by various methods.
  • step (13) The mixture containing the bound binding partner/biologic and new reactants is the incubated in step (13) to allow time for the reaction to occur.
  • the reaction part of the process (10) is complete and step (16) of measuring the molecules produced or included in steps (11 ) through (15) inclusive can be performed. If additional reactants are required, steps (14), (15) and (13) may be repeated one or more times in succession until the appropriate signal molecules are present.
  • step (17) The measurement of the signal molecule(s) provides a quantitative result that can then be analyzed and compared in step (17) to a known set of calibrated responses of known concentrations of the target biologic. This comparison results in step (18) which is the quantified result and associated report of the concentration of the target biologic in the sample prepared in step (1 1).
  • process (10) of FIG. 1 has been associated with the detection of a biological organism or component, the process (10) is also applicable to the detection of many types of molecules to which antibodies or other binding partners can react.
  • FIG. 2 is a diagram of an embodiment of a laser-based Raman detection sub-system (20).
  • a laser (21) produces a laser beam (22) which is focused by the focusing optics (23) into a focused laser beam (24) which hits the target sample (25).
  • the backscattered light (26) from the sample (25) is focused into the beam (28) by the focusing optics (27).
  • the beam (28) is directed into the
  • the laser (21 ) is typically a continuous wavelength (CW) laser with output in the visible range.
  • CW continuous wavelength
  • Focusing optics (23) and (27) include mirrors, lenses, irises, shutters, diffraction gratings, and/or polarizers.
  • the target sample (25) may be liquid, gas or solid and in certain embodiments, the target sample would use a liquid or precipitated solid.
  • the spectrometer (30) spatially separates the scattered light based on wavelength.
  • An example of a usable spectrometer for the present disclosure is the Lambda Solutions model PS-1.
  • the detector (31) measures the amplitude of the light spatially separated by the spectrometer (30) and converts this into an electrical signal (analog or digital). In certain embodiments, the detector would provide the electrical signal using a standardized computer interface such as RS-232, USB, parallel, IEEE 1394.
  • An example of a usable detector (30) for the present disclosure is a Raman Systems INC QE 65000 Raman Detector or a Lambda Solutions model PS-1 detector.
  • the personal computer (40) can be any desktop or laptop PC with an appropriate interface to the detector (31) and software designed to analyze, store and/or print the spectrum of the backscattered light (26) received by the spectrometer (30).
  • FIG. 3 is a flow chart of an embodiment of the present disclosure (30) for the detection of biological organisms and/or components.
  • the process begins by step (31 ) of preparing the liquid sample that includes the target biologic.
  • the sample may be prepared by pre-enrichment in a growth medium such as half-Frasier's broth or other suitable microbial growth medium.
  • a liquid sample for testing may be obtained from any liquid source. Solid material may be immersed in an appropriate liquid solution and potential target organism or molecules placed in solution and then sampled in the liquid.
  • the prepared liquid sample is combined (or mixed) with an antibody that has been attached to a solid surface.
  • plastic microtiter plates, latex beads or magnetic microparticles may be used.
  • step (33) The antibody/biologic combination is then incubated in step (33) to allow time for the biologic and antibody to bind together. Once this has occurred the combined antibody/biologic is decanted (poured off) and washed to remove unbound biologies and other unwanted materials. New reactants are added in step (35) to enhance the sensitivity of the mixture for detection of the Raman light scattering. Examples of such reactants are listed in Table 1.
  • step (33) The mixture containing the bound antibody/biologic and new reactants is the incubated in step (33) to allow time for the reaction to occur.
  • step (36) of measuring Raman light scattering from Raman-active molecules produced by steps (31 ) through (35) inclusive can be performed. If additional reactants are required, steps (34), (35) and (33) may be repeated one or more times in succession until the appropriate Raman-active molecules are present.
  • step (37) The measurement of Raman light scattering can then be analyzed and compared in step (37) to a known set of calibrated responses of known concentrations of the target biologic. This comparison results in step (38) which is the quantified result and associated report of the concentration of the target biologic in the sample prepared in step (31 ).
  • Listeria may be measured in an (enzyme-linked immunosorbant assay) ELISA format. 100 microliters of various concentrations of bacteria; 100,000, 50,000, 25,000, 12,500, 6,250 and 0 colony forming units (cfu) per ml are added to microwells coated with anti-Listeria antibodies. After an incubation period between 30 and 60 minutes at 37°C, the wells are decanted and washed with a mild detergent solution three times. 100 ⁇ of peroxidase-conjugated anti- Listeria antibodies are added to the well and incubated for 1 to 4 hours at 37°C. The wells are decanted and washed with a mild detergent solution three times.
  • FIG 4 is a block diagram for a chemical conversion system (40) which uses an enzyme for converting chemical components to a Raman-active compound.
  • one or more reactants designated (41), (42) and (43) are mixed with a biological catalyst (44).
  • the biological catalyst (44) may be an enzyme specific for metabolizing the reactants provided or RNA structures designed to interact with the one or more reactants (41 ), (42), and (43).
  • a conversion or combination of the reactants occurs in the reaction (45) and a measurable product (46) is formed.
  • the reactants and those in Table 4 are mixed together in the presence of peroxidase (44) and urea hydrogen peroxide (UP) (43).
  • Additional reactants that may produce Raman-active products can be used in the disclosed methods, such as compounds comprising a least one hydroxyl group and one amino group at positions 1 and 4 in a benzene or naphthalene.
  • Such compounds include:
  • X is H, NH 2 , CI, Br, nitro, or benzyl
  • Y is H, CI, Br, or nitro
  • Z is H, benzyl, or NH 2 .
  • X is NH 2 , ancTY and Z are H.
  • X is CI, and Y and Z are H.
  • X is Br, and Y and Z are H.
  • X is nitro, and Y and Z are H.
  • X and Z are H and Y is CI.
  • X and Z are H and Y is Br.
  • X and Z are H and Y is nitro.
  • X and Z are benzyl and Y is H.
  • X and Z are NH 2 and Y is H.
  • Such compounds also include:
  • X is H, OH, CI, Br, or nitro (N0 2 ).
  • Such compounds also include:
  • X is H, CI, Br, or N0 2 .
  • Additional compounds that may produce Raman-active products in the disclosed methods include compounds comprising at least two hydroxyl functions in 1 , 2 or 1 , 4 positions in a benzene or naphthalene ring.
  • Such compounds include:
  • W, X, Y, and Z are H or OH.
  • Y is OH and X, Y and Z are H.
  • W is OH, and X, Y and Z are H.
  • W and X are OH, and Y and Z are H.
  • W and Y are OH, and X and Z are H.
  • W and Z are OH and X and Y are H.
  • Such compounds include polyphenols, such as:
  • X, Y and Z are H or OH.
  • X is OH and Y and Z are H.
  • X and Y are OH and Z is H.
  • X and Z are OH and Y is H.
  • Z is OH and X and Y are H.
  • Additional compounds that may produce Raman-active products in the disclosed methods include compounds comprising hydroxymethlene (-CH 2 OH) group in a benzene or naphthalene. Inclusion of additional hydroxyl groups at positions 1 , 4, and 6 may enhance the Raman scattering.
  • Such compounds include:
  • X and Y are H or OH.
  • X is OH and Y is H.
  • X is H and Y is OH.
  • Such compounds also include:
  • X and Y are H or OH.
  • X is OH and Y is H.
  • X is H and Y is OH.
  • Such compounds also include aromatic amines, including compounds comprising ortho-phenylenediamine, meta-phenylenediamine, and para- phenyleneamine:
  • Such compounds also include 2,4-diaminobenzyl alcohol, 2-amino- 1-naphthol, and 4-aminoantipyrene.
  • the product of the reaction (45) may be used as a quantitative or qualitative reporting molecule for the reaction and as such may be used as a probe for the presence of specific biological targets if conjoined with, for example, specific antibodies or biological or chemical binding partners.
  • Certain compounds may spontaneously form Raman-active products upon exposure to air ("auto-oxidation"). Such compounds are ill-suited for use in certain assay formats, such as ELISA, because they exhibit Raman signals without being acted on by an enzyme.
  • the present disclosure provides modified versions of these reactants that allow for their use in Raman scattering-based assays.
  • hydroxyl groups present in compounds of the disclosure which may spontaneously oxidize, can be modified with phosphate groups to prevent spontaneous oxidation.
  • these compounds further expand the types of compounds that can be used in the methods presently disclosed.
  • reactants comprising phosphate gropus may be oxidized by the addition of an oxidizing agent.
  • the present disclosure also provides methods for using Raman scattering based on detecting phosphatase activity.
  • Additional reactants that produce Raman-active products can be used in the presently-disclosed methods, such as compounds comprising at least one phosphate group.
  • Such compounds include aromatic organic compounds
  • phosphate group for example compounds comprising benzene or naphthalene rings having at least one phosphate group as a substituent.
  • additional substituent groups such as carboxyl, amine, chlorine, bromine, nitro and/or other functional groups may enhance the Raman scattering of the Raman-active product.
  • Such compounds according to the present disclosure may, for example, have functional groups that are ortho (1 ,2) and/or para (1 ,4) to each other.
  • the aromatic organic compounds comprising at least one phosphate group have the following structure:
  • X is H, OH, CI, Br, N0 2 , NH 2 , S0 3 H, or COOH; Y is H, OH, CI, Br, N0 2 , S0 3 H or NH 2 ; W is OH or P0 4 and Z is H, OH, CI, Br, S0 3 H, P0 4 or NH 2 .
  • X, Y, and Z are H.
  • X is OH, and Y and Z are H.
  • X is N0 2
  • Y and Z are H.
  • X is CI, and Y and Z are H.
  • X is Br, and Y and Z are H.
  • X is COOH, Y is OH, and Z is NH 2 .
  • X is CI, Y is OH, and Z is NH 2 .
  • X is S0 3 H, Y is OH, and Z is NH 2 .
  • such compounds may undergo catalytic dephosphorylation by reaction with alkaline phosphatase (ALP), and then oxidize to form a Raman-active quinone compound.
  • ALP alkaline phosphatase
  • Such compounds oxidized in the ortho (1 ,2) or para (1 ,4) position including, for example, catechol (1 , 2-dihydroxy-benzene), hydroquinone (1, 4- dihydroxybenzene), and pyrogallol (1 , 2, 3- trihydroxybenzene), may undergo rapid oxidation in air to generate the corresponding quinone.
  • the Raman scattering of the quinone product is enhanced upon treatment with a base such as strong sodium hydroxide (NaOH) solution.
  • a base such as strong sodium hydroxide (NaOH) solution.
  • This signal enhancement may be pH-dependent (i.e., NaOH dependent) such that the Raman scattering decreases upon addition of an acid, and increases upon addition of a base (restoring Raman scattering enhancement).
  • NaOH sodium hydroxide
  • auto-oxidation and Raman scattering enhancement upon addition of NaOH has been observed for catechol, pyrogallol, and 1 ,2,4-benezenetriol, which have the following structures:
  • the aromatic organic compounds comprising at least one phosphate group further comprise at least one amine group, and have the following structure:
  • X is H, OH, CI, Br, N0 2 , S0 3 H, P0 4 or NH 2
  • Y is H, OH, CI, Br, N0 2 , S0 3 H or NH 2
  • Z is H, OH, CI, Br, S0 3 H, P0 4 or NH 2 .
  • X, Y, and Z are H.
  • X is OH, and Y and Z are H.
  • such compounds may undergo catalytic dephosphorylation by reaction with alkaline phosphatase (ALP), then oxidize to form the corresponding Raman-active iminoquinone compound.
  • ALP alkaline phosphatase
  • the iminoquinones may generate enhanced Raman scattering that may be quantitated.
  • the aromatic organic compounds comprising at least one phosphate group further comprise at least one hydroxyl group and have the following structure:
  • X is H, OH, CI, Br, N0 2 , S0 3 H, P0 4 or NH 2 and Y is H, OH, CI, Br, N0 2 , S0 3 H or NH 2 .
  • X and Y are H.
  • X is OH and Y is H.
  • X is N0 2 and Y is H.
  • X is CI and Y is H.
  • X is Br and Y is H.
  • the aromatic organic compounds comprising at least one phosphate group further comprise at least one amine group and have the following structure:
  • X is H, OH, CI, Br, N0 2 , S0 3 H or P0 4 ; and NH 2 and Y is H, OH, CI, Br, N0 2 , S0 3 H or NH 2 .
  • X and Y are H.
  • X is OH and Y is H.
  • X is N0 2 and Y is H.
  • X is CI and Y is H.
  • X is Br and Y is H.
  • such compounds may undergo catalytic dephosphorylation to yield the corresponding amino-naphthol, which can further oxidize to form a Raman-active iminonaphthaquinone.
  • the reaction is exemplified below:
  • the aromatic organic compounds comprising at least one phosphate group have the following structure: wherein X, and Z are each H ,0H, S0 3 H, NH 2 , P0 4 or Y and W are each H , OH, SO3H, or NH 2 .
  • X, Y, Z, and W are H.
  • X is H
  • Y, Z and W are OH.
  • X and Y are H
  • Z and W are OH.
  • FIG. 5 is a flow chart of the technique (50) for choosing one or more light frequencies to excite specific target molecules for detection of the Raman-active products.
  • a Raman-active product (51) such as the product (46) produced by the reaction (45) of FIG. 4, is a chemical that possesses a structure which is Raman- active.
  • the absorbance spectrum of the product (51) is measured in step (52) using a technique such as absorbance or transmittance spectrophotometry.
  • step (53) one or more wavelengths are identified at which the product (51) absorbs light as seen in the spectrum measured in step (52).
  • a light source that emits light at a wavelength corresponding to one of the one or more wavelengths identified in step (53) is then selected.
  • Such wavelengths can be in the visible range, ultraviolet range or infra-red range. For example, for the Listeria detection reaction (30) described for FIG. 3, the wavelength selected is 532 nm.
  • step (55) the light source chosen in step (54) is used to irradiate the Raman-active product created in step (51). This will confirm that there is significant Raman scattering of the Raman-active product created in step (51) to provide adequate signal for detection.
  • the invention may be practiced using detectors other than non-single lens Raman optics detectors, such as fluorescence detectors.
  • FIG. 6 is an illustration of a micro-fluidic channel (60) designed to detect Raman-active compounds.
  • a source liquid (or gas) sample (61 ) including the target biological organisms or components flows through the channel (62).
  • the target biological organisms or components will react and be bound to the reactant(s) attached to the active surface (64).
  • Light (68) from a light source (65) produces Raman scattered light (69) which is detected by the photodetector (66).
  • the photodetector is designed to measure one or more specific wavelengths which correspond to the Raman scattering of the combined reactant(s) and biological organism or component. It is also envisioned that instead of binding the biological organism or component to the surface (64), the reactant(s) may be released from the surface and the Raman-scattering light source (65) and detector (66) may be located downstream from the surface.
  • FIG 7 is an illustration of an array of micro-fluidic channels (70) designed to detect Raman-active compounds.
  • One or more source liquid (or gas) samples (71 A), (71 B) through (71 N) which include the target biological organisms or components flow through the channels (72A), (72B) through (72N).
  • the target biological organisms or components will react and be bound to the reactant(s) attached to the active surfaces (74A), (74B) through (74N).
  • Light, (78A) through (78N) from the light sources, (75A) through (75N), produce Raman-scattered light, (79A) through (79N), which is detected by the photodetectors (76A) through (76N).
  • the photodetectors are designed to measure one or more specific wavelengths which correspond to the Raman scattering of the combined reactant(s) and biological organisms or components bound to the surfaces.
  • Figure 8 depicts Raman spectra obtained from an enzyme-linked immunoassay for the pathogenic bacteria Listeria utilizing the two-component BASH- UP chemistry, an enzyme-linked antibody, and Raman detection procedure described below utilizing the following buffers and reagents:
  • the Raman signal was generally stable for ⁇ 1 hour or longer.
  • the first component in the chemistry (BASH) contained 2-hydroxy benzyl alcohol (0.02 mg/ml), 5-amino salicylic acid (0.1 mg/ml), 0.1 % Tween-20, and ascorbic acid (1 ⁇ g/ml) in the Working Saline Buffer (pH 6.0).
  • the second component (UP) contained urea peroxide adduct (1 mg/ml) the working Saline Buffer (pH 6.0) including EDTA (1 mM). These formulations maintained activity when refrigerated out of direct light for more than one month. Mixing the two components at a ratio of 1 UP to 10 BASH created a working solution of BASH-UP that was generally stable for one working day.
  • Method A HRPO dilutions were made to measure 1000 pg to 0.0125 pg per 50 ⁇ sample in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at pH 7.4. 50 ⁇ HRPO sample per dilution was added to 200 ⁇ TMB reagent and allowed to react for 15 or 30 minutes at which time 200 ⁇ stop-solution (KPL Laboratories) was added. Absorbance was measured at 450 nm for each sample.
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Method B HRPO dilutions were made to allow 1000 pg to 0.0125 pg per 50 ⁇ sample in PBS at pH 7.4. 50 ⁇ HRPO sample per dilution was added to 200 ⁇ ABTS reagent and allowed to react for 15 or 30 minutes at which time 200 ⁇ stop solution (1% SDS in water) was added. Absorbance was measured at 405 nm for each sample.
  • the limit of detection of HRPO for TMB was 8 pg/ml and the dynamic range was 5 to 5000 pg/ml.
  • the limit of detection was 32 pg/ml and the dynamic range was 32 to 5000 pg/ml.
  • EXAMPLE 3 FLUOROGENIC AND CHEMILUMINESCENT ASSAYS OF HRPO
  • AnaSpec Fluorogenic kit utilizes ADHP (10-acetyl-3,7- dihydroxyphenoxazine) to analyze peroxidase in solution whereby ADHP is oxidized in the presence of peroxidase and hydrogen peroxide.
  • the oxidized product of ADHP (resozufin) gives pink fluorescence that can be measured at the emission wavelength of 590 nm with the excitation wavelength of 530-560 nm.
  • An overdose of peroxidase in the assay will further convert the fluorescent resorufin to non- fluorescent resozurin to yield reduced fluorescent signal.
  • HRPO dilutions were made to allow detection of 1 ,000,000 pg to 0.0625 pg per 50 ⁇ sample were prepared in PBS at pH 7.4. The procedure was the same as described earlier for TMB and ABTS assays, and two methods were used.
  • Method A ADHP Reagent and Hydrogen Peroxide were prepared per manufacturer's instructions. 500 ⁇ of peroxidase solution was added to 500 ⁇ ADHP reagent in a 1.5 ml plastic microcuvette. The reaction mixture was gently mixed, and incubated at room temperature for 30 min without light exposure. The fluorescent signal was measured for emission at 590 nm with excitation at 550 nm on an Ocean Optics Fluorescent Spectrometer.
  • Method B Similar to Method A except 400 ⁇ of each of peroxidase and ADHP reagents were used.
  • the sensitivity (lowest limit of detection) of the AnaSpec ADHP fluorescent assay was found to be 12.5 pg/ml of HRPO.
  • the assay range was linear from 250 pg/ml to 0 pg/ml of HRPO.
  • Molecular Probes Fluorogenic assay kit employs Amplex Red (10- acetyl-3,7-dihydroxyphenoxazine), which is similar to AnaSpec ADHP assay.
  • Amplex Red (10- acetyl-3,7-dihydroxyphenoxazine)
  • the oxidized end product of the assay with peroxidase and hydrogen peroxide is resorufin.
  • the assay claim is 1 X 10-5 U/ml, equivalent to 10 pg/ml (1 X 10-5 ml).
  • HRPO dilutions made to allow detection of 1 ,000,000 pg to 0.0625 pg per 50 ⁇ sample were prepared in PBS, pH 7.4. Amplex Red Reagent and
  • Hydrogen Peroxide were prepared per Manufacturer's instructions. 400 ⁇ of peroxidase solution was added to 400 ⁇ ADHP reagent in a 1.5 ml plastic microcuvette. The reaction mixture was gently mixed and incubated at room temperature for 30 min in the dark. The fluorescent signal was measured at 590 nm with excitation at 550 nm on an Ocean Optics Fluorescent spectrometer at 30 min and 35 min.
  • the sensitivity (lowest limit of detection) of the Molecular Probes Amplex Red Fluorescent assay was found to be 25 pg/ml of HRPO.
  • the assay range was linear from 250 pg/ml to 0 pg/ml of HRPO.
  • LumiGLO is a luminol-based chemiluminescent substrate designed for use with peroxidase-labeled reporter molecules.
  • HRPO converts luminol to an excited intermediate dianion. This dianion emits light on return to its ground state. After reaction with HRPO conjugate, the light emission from LumiGLO reaches maximum intensity within 5 minutes and is sustained for approximately 1 - 2 hours.
  • Table 7 compares the detection limits from different Raman Reagent A formulations, showing the increase in sensitivity provided by the SQR method.
  • Table 9 compares the detection limits from several different Raman reagent B formulations, showing the increase in sensitivity provided by the SQR method.
  • Raman Reagent A-1 500 pg/ml ASA; 20 pg/ml HBA; 20 pg/ml AA
  • Raman Reagent B-3 250 pg/ml ASA; 25 pg/ml CDMP
  • HRPO dilutions made to allow 1000 pg to 0.0125 pg per 50 pi sample were prepared in PBS at pH 7.4. 50 pi HRPO sample per dilution was added to 150 ⁇ reagent and allowed to react for 30 minutes. 50 ⁇ of 0.5 N NaOH was then added. After incubation for 30 minutes, Raman spectra were recorded using a Sword Diagnostics Raman Systems INC QE 65000 Raman Detector. Data were analyzed using SQR. Results from representative experiments appear in Tables 14- 18.
  • Results in Figure 17 show that the Biotin-ASA-UP combination provides a sensitive assay that can detect as low as 0.00625 pg sample.
  • ASA-UP without HBA also enables detection as low as 2 pg of HRPO.
  • Raman Reagent A increasing ASA from 100 to 250 or 500 ⁇ g/ml
  • Reagent B increasing ASA from 100 to 250 or 500 ⁇ g/ml
  • Biotin-ASA provide ultra sensitive peroxidase assays, compared to Reagent A-1 and Reagent C formulations.
  • Raman-based assays provide highly sensitive detection of Peroxidase in solution, which is shown graphically in Figure 18.
  • ASA by itself provides very good sensitivity, which is increased by the addition of CDMP, Biotin and even NAP.
  • the limit of detection of peroxidase was 3.9 and 4.4 pg/ml when 500 ⁇ g/ml of ASA was used and Raman signal analyzed with wave number 3,300 cm "1 and SQR, respectively.
  • the limit of detection was 2.3 and 1.9 when the Raman signal was analyzed with wave number 3,300 cm "1 and SQR, respectively
  • the Amplex Read Peroxidase assay is linear between 25 and 250 pg/50 ⁇ of sample (per vendor's claim) and the assay is able to detect as low as 1x10-5 U/ml.
  • the Sigma HRPO used in the current study had an activity of 1080 U/mg solid. On this basis, 1x10 "5 U/ml HRPO is equivalent to 10 pg/ml (0.5 pg/50 ⁇ ).
  • Table 18 summarizes a representative comparison of Raman-based detection and detection by absorbance, chemiluminescence, and fluorescence.
  • Urea-Peroxide 1000 ug/mL which contains 360 ug/mL Hydrogen Peroxide
  • the reagent was dissolved in 10 mM phosphate buffered saline with 2 mM EDTA, pH 6.0 (PBS-EDTA) and filtered through a sterile 0.45 micron cellulose nitrate filter and was stored in an amber colored polyethylene bottle at 2-8 °C.
  • Raman substrate was prepared by mixing Reagent A and Reagent B in a volume ratio of 9:1 prior to use. The substrate should be used in the same of preparation.
  • EXAMPLE 11 ABSORBANCE. FLUORESCENCE, AND RAMAN DETECTION OF
  • OPD o-phenylenediamine
  • Emission spectra were collected using 12 second integration and a box width of 30. Emission spectra are shown in Figures 21 A-D.
  • the fluorescence emission spectra of both the negative (0 pg/ml peroxidase) and reactive (2,000 pg/ml peroxidase) BASH reactions were similar ( Figures 21 A and B), with a low level of inherent fluorescence.
  • the OPD reaction fluorescence spectra were similar ( Figures 22 A-D).
  • EXAMPLE 12 RAMAN SENSITIVITY OF OPD-PEROXIDASE REACTIONS AND MEASUREMENTS OF ENZYME KINETICS
  • Raman spectra were collected in the range 0-4000 cm "1 with a Sword Diagnostics Raman Systems INC QE 65000 Raman Detector equipped with a 532 nm laser.
  • the compounds examined were benzoquinone, pyrogallol, 1 ,4- naphthaquinone, and 1 ,4-iminonaphthaquinone.
  • Figure 26 A shows Raman spectra of benzoquinone as a function of adding strong NaOH solution 0.5 N where the added NaOH causes enhanced Raman signal. This enhancement was found to be reversible, where addition of an acid decreased the signal and re-addition of NaOH again increased the signal.
  • Figure 26 B shows Raman spectra of pyrogallol (1 ,2,3-trihydroxybenzene) also as a function of added NaOH. Pyrogallol exemplifies an aromatic (phenyl) structure hydroxylated in the ortho (1 ,2) position.
  • Figure 27 A shows Raman spectra of 1 ,4-naphthaquinone as a function of both NaOH and periodate.
  • Figure 27 B similarly shows Raman spectra of 1 ,4-iminonaphthaquinone. These plots indicate that such compounds undergo rapid auto-oxidation to generate Raman signal.
  • 1 ,4-naphthaquinone (Figure 27 A) shows very high signal with or without periodate without the presence of NaOH. The spectral pattern changes with the addition of NaOH and showed reduced signal.
  • 1 ,4- iminonaphthaquinone shows enhanced Raman signal without periodate in the presence of NaOH.
  • This compound shows reduced signal with periodate in the presence of NaOH, possibly due to further oxidation of imino function in this compound.
  • the Raman signal of 1 ,4-iminoquinone could not be generated without NaOH ( Figure 27 B (d and e).
  • EXAMPLE 14 EXEMPLARY PHOSPHATASE-BASED RAMAN IMMUNOASSAY REAGENTS AND PROCEDURES
  • Goat anti-human IgG H+L alkaline phosphatase conjugate (KPL) (contains protein stabilizer and
  • Enzyme Storage Buffer 10 mM TRIS buffer, 50 mM KCI, 1 mM MgCI 2 , 0.1 mM ZnCI 2 , 50% glycerol, pH 8.2.
  • Coating Buffer 50 mM sodium carbonate-bicarbonate buffer, pH 9.4
  • Blocking Buffer 50 mM TRIS buffer, pH 8.0 with 2% BSA (bovine serum albumin) with 0.05% Tween 20, pH 8.0
  • Wash Buffer 50 mM TRIS buffered saline with 0.05% Tween 20, pH 8.0
  • Procedure A An immunoassay of 4-aminophenyl phosphate is prepared as follows:
  • Procedure B Immunoassays of hydroquinone diphosphate, 4- hydroxynaphthyl phosphate, and 4-aminonaphthyl phosphate are prepared as follows:
  • Blocking Empty the plate. Add 200 ⁇ of blocking buffer and incubate for 1 hour at room temperature.
  • the plate can be stored at 4 ° C for
  • Washing Wash the plate with 300 pL of wash buffer per well 5 times. Blot the plate after the last wash on a stack of paper towels.
  • Antigen A per well (standards as well as samples to be tested). Incubate for 1 hour at room temperature on a plate shaker. Samples should be freshly diluted in the assay buffer before adding to the plate.
  • alkaline phosphatase conjugated antibody specific to Antigen A in assay buffer to approximately 1
  • EXAMPLE 16 COLORIMETRIC DETECTION OF ALKALINE PHOSPHATASE CONJUGATE WITH OXIDIZING AGENT
  • Alkaline phosphatase was analyzed via Raman spectroscopy using 4- aminophenylphosphate as the substrate, with oxidizing agent (sodium
  • “Positive” refers to samples whose mean Raman signal recorded at 3300 cm “1 was greater than the negative mean signal recorded at 3300 cm “1 (+ 2 SD).
  • Figure 28 A is a logarithmic plot of Raman spectral values at
  • Figure 29 A shows Raman spectra of 4-aminophenylphosphate as a function of alkaline phosphatase conjugate concentration ranging from 0-1000 ng/mL with the addition of oxidizing agent, while Figure 29 B shows the range 0-10 ng/mL. The limit of detection was approximately 0.25 ng/mL.
  • EXAMPLE 18 COLORIMETRIC DETECTION OF ALKALINE PHOSPHATASE WITHOUT OXIDIZING AGENT
  • Alkaline phosphatase was analyzed via colorimetry using p- nitrophenylphosphate as the substrate.
  • Alkaline phosphatase was analyzed via Raman spectroscopy using 4- aminophenylphosphate as the substrate, without oxidizing agent.
  • Figure 30 A is a logarithmic plot of Raman spectral values at 3300 cm "1 recorded for 4-aminophenylphosphate as a function of alkaline phosphatase concentration; Figure 30 B shows the linear plot.
  • Figure 31 A shows Raman spectra of 4-aminophenylphosphate as a function of alkaline phosphatase concentration ranging from 0-2500 mU/mL, while Figure 31 B shows the range 0-25 mU/mL. The limit of detection was approximately 1 mU/mL.
  • EXAMPLE 20 RAMAN DETECTION OF FREE PEROXIDASE USING A SINGLE LENS RAMAN OPTICS BASED DETECTOR AND A FLUORESCENCE-BASED DETECTOR
  • HRPO Horseradish peroxidase
  • Negative control reaction components with 0 pg/ml HRPO in 50 mM Imidazole, 50 mM phosphate, and 3 mM EDTA at pH 6.5 containing 0.1% BSA.
  • the plate was incubated at room temperature for 30 minutes. 6. The plate was read in the indicated detectors.
  • Tecan fluorescence detector set to read at increments of 10 nm from 580 nm to 800 nm ("Tecan").
  • the analytical limit of detection was estimated as the concentration read from the fitted 4PL curve corresponding to the mean signal of the negative control + 2 standard deviation (SD) units (associated with the negative measurement). This evaluation included: evaluation of the shape of the standard curve, estimation of the LOD, and graphical estimation of standard curve shift due to Raman detection.
  • the Raman signal was measured as: (i) the matnitude of the shifted backscatter signal at a single Raman shift number at 3,200 cm “1 (Raman SV); (ii) a sample to negative ratio of the measured values (S/N); or (iii) an "area under the curve” measurement designated as "single quantifiable result" or "SQR” value. The latter value quantitates the difference between the Raman spectra of a reactive sample and its respective background (negative sample) over a specified Raman shift range.
  • EXAMPLE 21 RAMAN DETECTION OF A PEROXIDASE-BASED ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) USING A SINGLE LENS RAMAN OPTICS BASED DETECTOR AND A FLUORESCENCE-BASED DETECTOR
  • M1000 set to read at increments of 10 nm from 580 nm to 800 nm ("Tecan").
  • TMB tetramethyl benzidnine
  • EXAMPLE 23 RAMAN DETECTION OF FREE ALKALINE PHOSPHATASE USING A SINGLE-LENS-RAMAN-OPTICS-BASED DETECTOR AND A
  • 4-aminoaphthylphosphate (4-ANP) synthesized according to the procedure in Masson et al., Talanta, 64:174-180 (2004).
  • M1000 set to read at increments of 10 nm from 580 nm to 800 nm ("Tecan").
  • Figure 40 shows the response of alkaline phosphatase on Tecan fluorescence detector at different wavelengths. The data indicated highest signal at 580 nm.
  • Table 24 shows the mean Tecan signal at 580 nm TABLE 24
  • Tables 25 and 26 show the results derived from the same microtiter plate on a Raman reader.
  • EXAMPLE 24 RAMAN DETECTION OF FREE ALKALINE PHOSPHATASE USING A SINGLE-LENS-RAMAN-OPTICS-BASED DETECTOR AND A

Abstract

La présente invention concerne des systèmes pour la détection rapide et sensible d'organismes et de molécules dans des échantillons. Des réactifs qui produisent des produits à activité Raman sont utilisés en combinaison avec la diffusion de lumière Raman et un système de détection qui utilise au moins une source de lumière non à base de laser, tel qu'un système de détection de fluorescence. De tels composés peuvent comprendre des phosphates permettant la détection de phosphatases et des essais d'enzyme à base de phosphate, tels que des immunoessais et des essais à base d'ADN. La présente description peut également être utilisée pour mesurer une cinétique enzymatique.
PCT/US2011/030166 2010-03-28 2011-03-28 Procédés pour détecter la diffusion raman en utilisant des composés aromatiques comprenant du phosphate et au moins une source de lumière non laser WO2011123386A1 (fr)

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EP11714460A EP2553117A1 (fr) 2010-03-28 2011-03-28 Procédés pour détecter la diffusion raman en utilisant des composés aromatiques comprenant du phosphate et au moins une source de lumière non laser

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