A method and apparatus for spectral analysis

Abstract

The present application relates to a kind of spectral analysis method and device. Specifically, the present application provides a kind of spectral analysis method, the method includes the following steps: (a) providing a sample to be measured, the sample to be measured contains test object to be detected;(b) in a container, the sample to be measured is mixed with capture particle and detection particle, to form the first mixture containing " capture particle-test object-detection particle " complex;(c) after washing " capture particle-test object-detection particle " complex, " capture particle-test object-detection particle " complex is dispersed into suspension, and the determination of spectral signal is carried out under the condition that suspension is in continuous motion.The spectral analysis method of the present application can improve the repeatability and stability of analysis, so as to improve the accuracy of determination.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, and specifically relates to a spectral analysis method and apparatus. Background Technology

[0002] In the field of immunoassay, qualitative or quantitative detection of various antigens or antibodies is often required. Existing technologies, based on the principles of competitive inhibition and double-antibody sandwich assays, have led to the development of various immunoassay methods, such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, time-resolved fluorescence immunoassay, fluorescence immunoassay, and surface-enhanced Raman spectroscopy (SERS). These methods can be used to identify pathogenic microorganisms, quantify specific proteins in the human body, and thus provide auxiliary diagnosis or monitoring for diseases, demonstrating a wide range of applications. Among these, the SERS method has attracted considerable attention from researchers due to its extremely high sensitivity, simultaneous detection of multiple indicators, and stable optical signal.

[0003] The basic principle of SERS immunoassay is as follows: A capture antibody (Ab1) targeting the analyte is immobilized on a solid-phase Raman-enhancing substrate (typically a gold or silver film, gold or silver nanoparticles, or a composite nanoparticle composed of gold, silver, and inorganic materials). A signaling antibody (Ab2) targeting the analyte is also immobilized on nanoparticles of a similar material. Simultaneously, Raman reporter molecules (RR) are immobilized on the surface of these nanoparticles. When a positive sample forms a "ternary sandwich complex" with Ab1 and Ab2, the near-surface area of ​​the nanoparticles (typically less than 5 nm) and the narrow gaps between the closely clustered nanoparticles create "hotspot structures," greatly enhancing the RR signal. The more analytes present, the more "ternary sandwich complexes" are formed, resulting in more "hotspot structures" and RR, and consequently, a stronger Raman signal, thus enabling quantitative and qualitative analysis of the target analyte.

[0004] Because the Raman signal (RR) on the surface of nanoparticles is easily affected by "hot spot structures," and the number and state of "hot spot structures" formed in each immune reaction are difficult to be consistent, the Raman signal measured in the end fluctuates greatly, which seriously affects the quantitative detection performance of SERS.

[0005] Therefore, continuous improvement of SERS magnetic immunoassay technology is needed in this field to reduce Raman signal variability and enhance the repeatability and stability of SERS magnetic immunoassay. Summary of the Invention

[0006] The purpose of this invention is to provide a SERS magnetic immunoassay method that improves the repeatability and stability of SERS magnetic immunoassay, thereby enabling accurate determination of analytes in serum, plasma, whole blood, or buffer samples.

[0007] Another object of the present invention is to provide a spectral analysis system for detecting analytes.

[0008] A first aspect of the present invention provides a spectral analysis method, comprising the steps of:

[0009] (a) Provide a sample to be tested, said sample containing the test substance to be detected;

[0010] (b) In a container, the sample to be tested is mixed with capture particles and detection particles to form a first mixture containing a complex of “capture particles-test sample-detection particles”;

[0011] The capturing particles are magnetic particles loaded with a first capturing agent; and the detection particles are solid-phase carriers loaded with a second capturing agent and labeled with signal molecules.

[0012] The first and second trapping agents are both specifically targeted at the test substance and combine with the test substance to form a ternary complex of "first trapping agent-test substance-second trapping agent";

[0013] (c) After cleaning the complex of “captured particles-test substance-detection particles”, the complex of “captured particles-test substance-detection particles” is dispersed into a suspension, and the spectral signal is measured while the suspension is in a continuous motion state.

[0014] In another preferred embodiment, the determination of the spectral signal includes:

[0015] Excitation light is shone into the bottom of the container, and the spectral signal generated by the "capture particle-test material-detection particle" complex in continuous motion after irradiation is measured.

[0016] In another preferred embodiment, the test substance to be detected is selected from the group consisting of procalcitonin, interleukin-6, or combinations thereof.

[0017] In another preferred embodiment, the spectral analysis method is surface-enhanced Raman spectroscopy.

[0018] In another preferred embodiment, the detection particles are Raman detection particles.

[0019] In another preferred embodiment, the spectral signal is a Raman spectral signal.

[0020] In another preferred embodiment, the signal molecule is a Raman signal molecule.

[0021] In another preferred embodiment, the concentration of procalcitonin in the suspension is 0.001-1000 ng / ml, more preferably 0.01-1000 ng / ml, even more preferably 0.01-500 ng / ml, even more preferably 0.05-100 ng / ml, even more preferably 0.05-50 ng / ml, even more preferably 0.1-30 ng / ml, even more preferably 0.1-10 ng / ml, and most preferably 0.5-1 ng / mL.

[0022] In another preferred embodiment, the concentration of interleukin-6 in the suspension is 0.001-1000 ng / ml, more preferably 0.01-500 ng / ml, more preferably 0.01-100 ng / ml, more preferably 0.01-50 ng / ml, more preferably 0.01-30 ng / ml, more preferably 0.01-10 ng / ml, and most preferably 0.01-1 ng / mL.

[0023] In another preferred embodiment, the sample to be tested is serum, plasma, whole blood, or a buffer solution.

[0024] In another preferred embodiment, in step (c), the washing solution used for washing is PBS buffer.

[0025] In another preferred embodiment, the motion includes flow.

[0026] In another preferred embodiment, the flow includes eddies.

[0027] In another example, the motion includes eddies.

[0028] In another preferred embodiment, the cleaning includes the following steps:

[0029] The complex of "captured particles-test material-detected particles" is aggregated on the side wall of the container, and then the complex of "captured particles-test material-detected particles" is cleaned with a cleaning solution.

[0030] In another preferred embodiment, the “capture particle-test material-detection particle” complex is aggregated on the side wall of the container under the influence of a magnetic field.

[0031] In another preferred embodiment, the magnetic field is generated by a magnetic material.

[0032] In another preferred embodiment, the magnetic material is a magnet.

[0033] In another preferred embodiment, the washing solution is PBS buffer.

[0034] In another preferred embodiment, the number of cleaning cycles is 1-6.

[0035] In another preferred embodiment, the cleaning is performed 1, 2, 3, 4, 5, or 6 times.

[0036] In another preferred embodiment, the cleaning solution is removed from the container after cleaning is completed.

[0037] In another preferred embodiment, after the cleaning is completed, the magnetic material is removed.

[0038] In another preferred embodiment, after the cleaning is completed, the magnetic material is removed, so that the cleaned "captured particle-test material-detected particle" complex is located at the bottom of the container.

[0039] In another preferred embodiment, the "capture particle-test substance-detection particle" complex is dispersed into a suspension using a buffer solution (such as PBS buffer).

[0040] In another preferred embodiment, the buffer solution comprises PBS buffer.

[0041] In another preferred embodiment, keeping the suspension in a state of continuous motion includes the following steps:

[0042] The suspension is agitated, thus keeping it in a state of continuous motion.

[0043] In another preferred embodiment, keeping the suspension in a state of continuous motion includes the following steps:

[0044] After aspirating an appropriate amount of suspension from the container, return the aspirated amount of suspension to the container. Repeat this process, aspirating an appropriate amount of suspension from the container and returning the aspirated amount of suspension to the container, to keep the suspension in a continuous state of motion.

[0045] In another preferred embodiment, the appropriate amount of suspension is 5-50% of the suspension volume (e.g., volume), more preferably 5-50%, even more preferably 5-50%, even more preferably 5-40%, even more preferably 10-30%, even more preferably 15-25%.

[0046] In another preferred embodiment, aspiration is performed using a pipette.

[0047] In another preferred embodiment, the material of the container is selected from the group consisting of: plastic, glass, ceramic, or combinations thereof.

[0048] In another preferred embodiment, the bottom material of the container is selected from the group consisting of: plastic, glass, ceramic, or combinations thereof.

[0049] In another preferred embodiment, the bottom material is selected from the group consisting of polymers, resins, or combinations thereof.

[0050] In another preferred embodiment, the polymer comprises a homopolymer or a copolymer.

[0051] In another preferred embodiment, the bottom material is selected from the group consisting of polystyrene, polyvinyl chloride, polycarbonate, polyethylene terephthalate, polyethylene, polypropylene, melamine, or combinations thereof.

[0052] In another preferred embodiment, the container is transparent or translucent.

[0053] In another preferred embodiment, the bottom of the container is transparent or translucent.

[0054] In another preferred embodiment, the thickness of the bottom of the container is 0.2-1.0 mm.

[0055] In another preferred embodiment, the excitation light is emitted from below the bottom of the container.

[0056] In another preferred embodiment, the excitation light is emitted from directly below the center of the bottom of the container.

[0057] In another preferred embodiment, the incident angle α of the excitation light relative to the normal of the bottom plane of the container is 0-70 degrees, more preferably 0-60 degrees, and even more preferably 0-45 degrees. (That is, when the incident angle α is 0°, the excitation light is incident perpendicularly on the bottom of the container).

[0058] In another preferred embodiment, α is 0 degrees.

[0059] In another preferred embodiment, the excitation light is a laser.

[0060] In another preferred embodiment, the wavelength of the excitation light is 300-600 nm, more preferably 600-900 nm, and even more preferably 900-1200 nm.

[0061] In another preferred embodiment, the Raman spectral signal is at 1074±5 cm⁻¹ (preferably 1074±2 cm⁻¹) in the Raman spectrum. -1 The peak value of the characteristic peak at point 10.

[0062] In another preferred embodiment, the trapping particles are magnetic particles loaded with a trapping agent and having the structure of Formula I:

[0063] Z1-Z2-Z3(I)

[0064] In the formula,

[0065] Z1 is a magnetic particle;

[0066] Z2 is the first connecting element; and

[0067] Z3 is the primary capture agent.

[0068] In another preferred embodiment, the first linker is selected from the group consisting of linkers containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linkers, or combinations thereof.

[0069] In another preferred embodiment, the polypeptide linker includes an avidin-biotin linker arm.

[0070] In another preferred embodiment, the avidin is streptavidin.

[0071] In another preferred embodiment, the first capturing agent is a first antibody.

[0072] In another preferred embodiment, the detected particle is a microparticle having the structure of Formula II:

[0073] Y1-Y2-Y3-Y4-Y5(II)

[0074] In the formula,

[0075] Y1 represents precious metal particles;

[0076] Y2 is a Raman signal molecule; and

[0077] Y3 is the coating layer;

[0078] Y4 is the second connecting element;

[0079] Y5 is the second trapping agent.

[0080] In another preferred embodiment, the noble metal particles are selected from the group consisting of Au particles, Ag particles, or combinations thereof.

[0081] In another preferred embodiment, the precious metal particles are selected from the group consisting of colloidal gold, colloidal silver, or combinations thereof.

[0082] In another preferred embodiment, the Raman signaling molecule is selected from the group consisting of: 4-mercaptobenzoic acid, 4-mercaptothiophenol, nitrothiophenol, aminothiophenol, benzimidazole, benzothiazole, mercaptopyridine, isothiocyanate, 5,5-dimercaptobis(nitrobenzoic acid), 4-acetaminophensulfuric acid, 2-thiouracil, or combinations thereof.

[0083] In another preferred embodiment, the coating is an Ag coating, an Au coating, or a combination thereof.

[0084] In another preferred embodiment, the second linker is selected from the group consisting of linkers containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linkers, or combinations thereof.

[0085] In another preferred embodiment, the polypeptide linker includes an avidin-biotin linker arm.

[0086] In another preferred embodiment, the second capturing agent is a second antibody.

[0087] In another preferred embodiment, the Raman spectral signal is the peak value of the characteristic peak in the Raman spectrum.

[0088] In another preferred embodiment, the Raman signal molecule is 4-mercaptobenzoic acid.

[0089] In another preferred embodiment, the bottom material of the container is polystyrene.

[0090] In another preferred embodiment, the method is an ex vivo method.

[0091] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0092] In a second aspect, the present invention provides a spectral analysis system, the system comprising:

[0093] (i) An excitation light emission source emits excitation light, which enters from below the bottom of a container containing a continuously moving "capture particle-test material-detection particle" complex.

[0094] (ii) A spectral signal acquisition unit, wherein the spectral signal acquisition unit is used to acquire the spectral signal generated after the complex of “capture particle-test substance-detection particle” is irradiated;

[0095] (iii) Output unit.

[0096] In another preferred embodiment, the spectral analysis system further includes:

[0097] (iv) A dispersion unit for continuously moving the “capture particle-test material-detection particle” complex in the container.

[0098] In another preferred embodiment, the dispersing unit includes a stirrer, an oscillator, or a combination thereof.

[0099] In another preferred embodiment, the stirrer includes a stirring head (such as a stirring blade).

[0100] In another preferred embodiment, the stirring head (such as a stirring blade) stirs the container, causing the complex of "captured particles-test material-detected particles" contained in the container to move continuously.

[0101] In another preferred embodiment, the oscillator includes a shaking table.

[0102] In another preferred embodiment, the shaker oscillates the container, causing the complex of "captured particles-test material-detected particles" contained in the container to move continuously.

[0103] In another preferred embodiment, the spectral analysis system further includes a detection container for holding the sample to be tested and / or the “capture particle-test substance-detection particle” complex.

[0104] In another preferred embodiment, the test sample and / or the complex of the “captured particle-test substance-detected particle” is in a liquid state.

[0105] In another preferred embodiment, the spectral analysis system is a spectral analysis method used in the method described in the first aspect of the present invention.

[0106] In another preferred embodiment, the spectral analysis system is a surface-enhanced Raman spectroscopy system.

[0107] In another preferred embodiment, the detection particles are Raman detection particles.

[0108] In another preferred embodiment, the spectral signal is a Raman spectral signal.

[0109] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0110] Attached Figure

[0111] Figure 1 The present invention illustrates a preferred embodiment of a spectral analysis system. Detailed Implementation

[0112] Through extensive experimental and comparative studies, the inventors have established a SERS magnetic immunoassay method. Unexpectedly, it was discovered that when the SA-M complex of the analyte is in a continuous motion state, the measured Raman signal intensity fluctuation is minimal, with a CV% of less than 5%. This indicates that the SERS measurement of the SA-M complex under continuous motion exhibits the strongest repeatability and stability, thus enabling accurate determination of the analyte content and meeting the requirements of quantitative immunoassay.

[0113] In the experiments of this invention, streptavidin (SA)-labeled magnetic microparticles (SA-M) were used as a solid-phase carrier. The Ab2 of this invention is labeled on Au-RR@Ag core-shell nanomaterials, i.e., “Au-RR@Ag-Ab2”. After the sample, biotin-labeled capture antibody biotin-Ab1, and “Au-RR@Ag-Ab2” were added to the reaction vessel in an appropriate ratio and reacted for a certain period, an appropriate amount of SA-M was added to the reaction vessel. A magnetic field was applied to the sidewall outside the reaction vessel to cause all SA-M to aggregate, including the SA-M complex generated by the immune reaction. After repeatedly washing the aggregated SA-M and SA-M complex, the magnetic field was removed, and an appropriate amount of buffer solution was added to the reaction vessel. The SA-M and SA-M complex were dispersed in an appropriate manner to form a suspension. While maintaining the suspension in the reaction vessel in a continuous state of motion, the signal intensity was measured using a Raman spectrometer.

[0114] The experiments of this invention show that the dispersion state of the "SA-M complex" during measurement has a significant impact on the repeatability and stability of the measured values. The Raman signal of the SA-M complex suspension when it is stationary and the SA-M complex that is repeatedly aggregated fluctuates greatly, and the repeatability and stability are poor, which cannot meet the requirements of quantitative immunoassay. However, when the "SA-M complex" suspension is in continuous motion, the Raman signal fluctuation is minimal, and the repeatability and stability are the strongest, which can fully meet the requirements of quantitative immunoassay.

[0115] the term

[0116] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0117] As used in this article, “Raman”, “Surface Enhanced Raman” and “SERS” are used interchangeably.

[0118] Spectral analysis methods

[0119] This invention provides a spectroscopic analysis method, which exhibits the strongest repeatability and stability in SERS determination of magnetic microparticle complexes of analytes in a continuously moving state.

[0120] Typically, the spectral analysis method of the present invention includes the following steps:

[0121] (a) Provide a sample to be tested, said sample containing the test substance to be detected;

[0122] (b) In a container, the sample to be tested is mixed with capture particles and detection particles to form a first mixture containing a complex of “capture particles-test sample-detection particles”;

[0123] The capturing particles are magnetic particles loaded with a first capturing agent; and the detection particles are solid-phase carriers loaded with a second capturing agent and labeled with signal molecules.

[0124] The first and second trapping agents are both specifically targeted at the test substance and combine with the test substance to form a ternary complex of "first trapping agent-test substance-second trapping agent";

[0125] (c) After cleaning the complex of “captured particles-test substance-detection particles”, the complex of “captured particles-test substance-detection particles” is dispersed into a suspension, and the spectral signal is measured while the suspension is in a continuous motion state.

[0126] In a preferred embodiment of the present invention, the method is an in vitro method.

[0127] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0128] In a preferred embodiment of the present invention, the spectral analysis method is a surface-enhanced Raman spectroscopy method.

[0129] In another preferred embodiment, the detection particles are Raman detection particles.

[0130] In another preferred embodiment, the spectral signal is a Raman spectral signal.

[0131] In another preferred embodiment, the signal molecule is a Raman signal molecule.

[0132] As stated in this article, "the suspension is in a state of continuous motion" means that the suspension is non-static and in a state of fluid motion, which is a fluid state. Fluid motion can include the flow of eddies.

[0133] Typically, the movement described includes flow.

[0134] Typically, the flow includes (but is not limited to) eddies.

[0135] The spectral analysis method described in this invention can be used for the quantitative determination of a test substance to be detected. Preferably, the test substance to be detected includes (but is not limited to): procalcitonin, interleukin-6, or a combination thereof.

[0136] In another preferred embodiment, the sample to be tested includes (but is not limited to) serum, plasma, whole blood, or buffer solution.

[0137] In another preferred embodiment of the invention, the cleaning includes the following steps:

[0138] The complex of "captured particles-test material-detected particles" is aggregated on the side wall of the container, and then the complex of "captured particles-test material-detected particles" is cleaned with a cleaning solution.

[0139] In this invention, there is no particular limitation on the method of keeping the suspension in a continuous state of motion, as long as it meets the purpose of this invention.

[0140] In a preferred embodiment, keeping the suspension in a continuous state of motion includes the following steps:

[0141] The suspension is agitated, thus keeping it in a state of continuous motion.

[0142] In another preferred embodiment, keeping the suspension in a state of continuous motion includes the following steps:

[0143] After aspirating an appropriate amount of suspension from the container, return the aspirated amount of suspension to the container. Repeat this process, aspirating an appropriate amount of suspension from the container and returning the aspirated amount of suspension to the container, to keep the suspension in a continuous state of motion.

[0144] In a preferred embodiment of the invention, the trapping particles are magnetic particles loaded with a trapping agent and having the structure of Formula I:

[0145] Z1-Z2-Z3(I)

[0146] In the formula,

[0147] Z1 is a magnetic particle;

[0148] Z2 is the first connecting element; and

[0149] Z3 is the primary capture agent.

[0150] In another preferred embodiment, the first linker is selected from the group consisting of linkers containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linkers, or combinations thereof.

[0151] In another preferred embodiment, the polypeptide linker includes an avidin-biotin linker arm.

[0152] In another preferred embodiment, the avidin is streptavidin.

[0153] In another preferred embodiment, the first capturing agent is a first antibody.

[0154] In another preferred embodiment of the invention, the detection particle is a microparticle having the structure of Formula II:

[0155] Y1-Y2-Y3-Y4-Y5(II)

[0156] In the formula,

[0157] Y1 represents precious metal particles;

[0158] Y2 is a Raman signal molecule; and

[0159] Y3 is the coating layer;

[0160] Y4 is the second connecting element;

[0161] Y5 is the second trapping agent.

[0162] In another preferred embodiment, the noble metal particles are selected from the group consisting of Au particles, Ag particles, or combinations thereof.

[0163] In another preferred embodiment, the precious metal particles are selected from the group consisting of colloidal gold, colloidal silver, or combinations thereof.

[0164] In another preferred embodiment, the Raman signaling molecule is selected from the group consisting of: 4-mercaptobenzoic acid, 4-mercaptothiophenol, nitrothiophenol, aminothiophenol, benzimidazole, benzothiazole, mercaptopyridine, isothiocyanate, 5,5-dimercaptobis(nitrobenzoic acid), 4-acetaminophensulfuric acid, 2-thiouracil, or combinations thereof.

[0165] In another preferred embodiment, the coating is an Ag coating, an Au coating, or a combination thereof.

[0166] In another preferred embodiment, the second linker is selected from the group consisting of linkers containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linkers, or combinations thereof.

[0167] In another preferred embodiment, the polypeptide linker includes an avidin-biotin linker arm.

[0168] In another preferred embodiment, the second capturing agent is a second antibody.

[0169] In another preferred embodiment, the Raman spectral signal is the peak value of the characteristic peak in the Raman spectrum.

[0170] In another preferred embodiment, the Raman signal molecule is 4-mercaptobenzoic acid.

[0171] In another preferred embodiment, the bottom material of the container is polystyrene.

[0172] Spectral Analysis System

[0173] The present invention also provides a spectral analysis system, which can be used in the spectral analysis method described in the present invention.

[0174] For ease of explanation, the following is combined with Figure 1 Further description of the spectral analysis system of the present invention should be made, and it should be understood that the accompanying drawings do not limit the scope of the spectral analysis system.

[0175] Typically, the spectral analysis system includes:

[0176] (i) An excitation light emission source emits excitation light, which enters from below the bottom of a container containing a continuously moving "capture particle-test material-detection particle" complex.

[0177] (ii) A spectral signal acquisition unit, wherein the spectral signal acquisition unit is used to acquire the spectral signal generated after the complex of “capture particle-test substance-detection particle” is irradiated;

[0178] (iii) Output unit.

[0179] In a preferred embodiment of the present invention, the spectral analysis system further includes:

[0180] (iv) A dispersion unit for continuously moving the “capture particle-test material-detection particle” complex in the container.

[0181] In another preferred embodiment, the spectral analysis system is a surface-enhanced Raman spectroscopy system.

[0182] In another preferred embodiment, the detection particles are Raman detection particles.

[0183] In another preferred embodiment, the spectral signal is a Raman spectral signal.

[0184] In another preferred embodiment, the dispersing unit includes a stirrer, an oscillator, or a combination thereof.

[0185] In another preferred embodiment, the stirrer includes a stirring head (such as a stirring blade).

[0186] Preferably, the stirring head (such as a stirring blade) stirs the container, causing the complex of "captured particles-test material-detected particles" contained in the container to move continuously.

[0187] In another preferred embodiment, the oscillator includes a shaking table.

[0188] Preferably, the shaker oscillates the container, causing the complex of "captured particles-test material-detected particles" contained in the container to move continuously.

[0189] In another preferred embodiment, the spectral analysis system further includes a detection container for holding the sample to be tested and / or the “capture particle-test substance-detection particle” complex.

[0190] In another preferred embodiment, the test sample and / or the complex of the “captured particle-test substance-detected particle” is in a liquid state.

[0191] The main advantages of this invention include:

[0192] This invention provides a spectroscopic analysis method. In the method, it was unexpectedly discovered that the Raman signal intensity fluctuation was minimal when the magnetic microparticle complex of the analyte was in continuous motion. This indicates that the repeatability and stability of the measurement of the magnetic microparticle complex of the analyte were strongest when it was in continuous motion, thus enabling accurate determination of the content of the analyte and meeting the requirements of quantitative immunoassay.

[0193] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0194] Example

[0195] Reagents:

[0196] Procalcitonin (PCT) and interleukin-6 (IL-6) paired monoclonal antibodies (the capture antibody is replaced by Ab1, and the detection antibody is replaced by Ab2, commercially available);

[0197] Bovine serum albumin (68KD, batch number: Y161201, Genview Biotech);

[0198] Chloroauric acid (analytical grade, batch number: 20180423, Sinopharm Chemical Reagent Co., Ltd.);

[0199] Disodium hydrogen phosphate (analytical grade, batch number: 20141015, Sinopharm Chemical Reagent Co., Ltd.);

[0200] Sodium dihydrogen phosphate (analytical grade, batch number: 20140922, Sinopharm Chemical Reagent Co., Ltd.);

[0201] Sodium chloride (analytical grade, batch number: 20180223, Sinopharm Chemical Reagent Co., Ltd.);

[0202] SH-PEG-COOH (analytical grade, batch number: D06112, Pengsheng Biotechnology); 4-mercaptobenzoic acid (analytical grade, batch number: 20160617, Sinopharm Chemical Reagent Co., Ltd.);

[0203] Ethanol (analytical grade, batch number: 20170918, Sinopharm Chemical Reagent Co., Ltd.)

[0204] Silver nitrate (analytical grade, batch number: 20161001, Shanghai Reagent Factory No. 1);

[0205] Trisodium citrate (analytical grade, batch number: 20161209, Sinopharm Chemical Reagent Co., Ltd.)

[0206] EDC (analytical grade, batch number: 20160817, Sinopharm Chemical Reagent Co., Ltd.).

[0207] All the water used in the above experiments was deionized water.

[0208] Streptavidin (SA)-labeled magnetic beads (SA-M), Shanghai Yingruicheng Biotechnology Co., Ltd.

[0209] 1μm streptavidin magnetic beads (SA-M).

[0210] equipment:

[0211] L6S UV-Vis spectrophotometer (Shanghai Yidian Analytical Instrument Co., Ltd.);

[0212] CT14RD Benchtop High-Speed ​​Refrigerated Centrifuge (Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.);

[0213] FA1004 electronic balance (Shanghai Sunny Hengping Scientific Instruments Co., Ltd.);

[0214] Leici GB-3A Thermostatic Timer Stirrer (Shanghai Leici Chuangyi Instrument Co., Ltd.);

[0215] Seed3000 Raman spectrometer (Shanghai Ruhai Company);

[0216] Orbital Shaker TS-1 (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.)

[0217] Transparent polystyrene micropores.

[0218] Example 1: Comparative test on the stability of decreased procalcitonin (PCT) and interleukin-6 (IL-6) measurement data of SA-M complex in different dispersion states

[0219] 1. Preparation of Au-4MBA@Ag-Ab2

[0220] 1.1 Preparation of colloidal gold: After boiling 100 mL of 0.01% chloroauric acid aqueous solution, 1.75 mL of 1% trisodium citrate solution was quickly added, and boiling was continued for 5 min. Its UV-Vis value was approximately 518 nm.

[0221] 1.2 Preparation of Au-4MBA@Ag: Take 1 mL of colloidal gold solution, add 1 mM of 4-mercaptobenzoic acid (4-MBA) solution, react for a period of time, centrifuge and redissolve in 27 μM trisodium citrate solution, heat to boiling, add 60 μL of 20 mM silver nitrate solution and 60 μL of 20 mM trisodium citrate solution dropwise, continue boiling for ten minutes, cool to room temperature, and store at 4 °C for later use.

[0222] 1.3 Preparation of Au-4MBA@Ag-Ab2: Centrifuge 1 mL of the synthesized Au-4MBA@Ag solution, discard the supernatant, and reconstitute in ultrapure water. Add 2 μL of 0.1 mM SH-PEG-COOH and store at 4℃ overnight. After centrifuging to remove excess SH-PEG-COOH, add 5 μg of anti-PCT detection antibody Ab2 for conjugation for 30 min. Add an appropriate amount of 1 μM EDC for fixation three times, once every 30 min. Finally, add 20 μL of 10% BSA solution for blocking for 30 min, and then add EDC for fixation once more. Finally, centrifuge, discard the supernatant, and dissolve in 100 μL of ultrapure water for later use at 4℃ to obtain Au-4MBA@Ag-Ab2 solution.

[0223] Preparation of 2Biotin-Ab1

[0224] 2.1 Biotin-labeled Ab1: 100 μL of 1.5 mg / mL anti-PCT capture antibody (Ab1) was added to 10 μL of Biotin, and the mixture was shaken at 4°C for 2 h. Then, 100 μL of 10% glycine solution was added and the mixture was reacted for 30 min. The mixture was then placed in a dialysis bag and dialyzed at 4°C in the dark for three days. The PBS dialysate was changed every 8 h to obtain Biotin-Ab1 solution.

[0225] 3. Stability comparison test of the test

[0226] 3.1 Sample preparation:

[0227] Procalcitonin (PCT) samples at concentrations of 0.5 ng / mL and 1 ng / mL were prepared using calf serum as a matrix.

[0228] A 0.1 ng / mL interleukin-6 (IL-6) sample was prepared using fetal bovine serum as a matrix.

[0229] 3.2 Raman Detection Methods under Different Dispersion States

[0230] The excitation light of the Seed3000 Raman spectrometer enters from below the center of the bottom of the polystyrene micropores, and the excitation light is incident perpendicularly on the bottom of the polystyrene micropores.

[0231] The Raman signal intensity of procalcitonin (PCT) is 1074 cm⁻¹ in the Raman spectrum. -1 The intensity of the peak value of the characteristic peak.

[0232] The Raman signal intensity of interleukin-6 (IL-6) is 1074 cm⁻¹ in the Raman spectrum. -1 The intensity of the peak value of the characteristic peak.

[0233] 3.2.1 Raman detection of suspensions under continuous flow

[0234] Add 50 μL of the test sample (LPCT) to a transparent polystyrene microwell. Add 2 μL of Au-4MBA@Ag-Ab2 and 3 μL of Biotin-Ab1, and react at room temperature for 10 min. Then add 50 μL of SA-M and continue reacting for 5 min. Use a magnet to aggregate SA-M and its complex onto the sidewall of the polystyrene microwell. Remove the liquid from the polystyrene microwell, wash with 200 μL of PBS buffer, and repeat this process three times. Finally, remove the magnet and add 50 μL of [unspecified ingredient] to the polystyrene microwell. PBS buffer was used to disperse SA-M and the SA-M complex into a suspension. 10 μL of the suspension was aspirated from the polystyrene micropores using a pipette and immediately returned to the polystyrene micropores. The "absorption and return" operation was repeated continuously to keep the suspension in a continuous vortex flow state. During the continuous vortex flow state, the Raman signal intensity of the SA-M complex in the continuous vortex flow state was measured 10 times in parallel.

[0235] 3.2.2 Raman Detection of Suspension under Static Condition

[0236] Take 50 μL of the test sample and add it to a transparent polystyrene microwell. Add 2 μL of Au-4MBA@Ag-Ab2 and 3 μL of Biotin-Ab1 respectively and react at room temperature for 10 min. Then add 50 μL of SA-M and continue to react for 5 min. Then use the magnetic field of a magnet to gather SA-M and SA-M complex on the sidewall of the polystyrene microwell. Remove the liquid in the polystyrene microwell of the reaction container, add 200 μL of PBS buffer to wash, and then remove the PBS buffer. Repeat this process 3 times. Finally, remove the magnet and add 50 μL of PBS buffer to the polystyrene microwell to disperse SA-M and SA-M complex into a suspension. Measure the Raman signal intensity of the suspension in a static state 10 times in parallel.

[0237] 3.2.3 Raman detection under repeated aggregation conditions

[0238] Add 50 μL of the test sample (LPCT) to a transparent polystyrene microwell. Add 2 μL of Au-4MBA@Ag-Ab2 and 3 μL of Biotin-Ab1, and react at room temperature for 10 min. Then add 50 μL of SA-M and continue reacting for 5 min. Use a magnet to aggregate SA-M and its complex onto the sidewall of the polystyrene microwell. Remove the liquid from the polystyrene microwell, wash with 200 μL of PBS buffer, and then remove the PBS buffer. Repeat this process three times. Finally, remove the magnet and add 50 μL of [unspecified substance] to the polystyrene microwell. PBS buffer was used to disperse SA-M and the SA-M complex into a suspension. Then, a magnet was used to gather SA-M and the SA-M complex at the bottom detection area of ​​the polystyrene micropores. The pull signal intensity of the first gathered SA-M complex was measured. After the first measurement, the magnet was removed, and the liquid in the polystyrene micropores was blown away with a pipette to allow SA-M and the SA-M complex to re-form a suspension in the polystyrene micropores. Then, the magnet was used again to gather SA-M and the SA-M complex at the bottom detection area of ​​the polystyrene micropores, and the pull signal intensity of the second gathered SA-M complex was measured. This process was repeated 10 times, and the Raman signal intensity of the SA-M complex gathered in 10 repeated measurements was measured.

[0239] 3.3 Raman detection results under different conditions

[0240] The Raman signal intensity detection results of the SA-M complex of the test samples containing 0.5 ng / mL procalcitonin (PCT) and 0.1 ng / mL interleukin-6 (IL-6) under different dispersion conditions (continuous flow, static and repeated aggregation) are shown in Tables 1, 2 and 3:

[0241] Table 1. Raman signal intensity of SA-M complex in 0.5 ng / mL PCT test samples under different dispersion states.

[0242]

[0243] Table 2. Raman signal intensity of SA-M complex in 1 ng / mL PCT test samples under different dispersion states.

[0244]

[0245]

[0246] Table 3. Raman signal intensity of SA-M complex in 0.1 ng / mL IL-6 test samples under different dispersion states.

[0247]

[0248] As shown in Tables 1, 2, and 3, the Raman signal intensity of the SA-M complex of procalcitonin (PCT) and interleukin-6 (IL-6) fluctuated greatly under both static and repeatedly aggregated conditions, failing to meet the requirements of quantitative immunoassay. However, the Raman signal intensity of the suspension of the SA-M complex of PCT and IL-6 under continuous flow conditions showed the smallest fluctuation, with CV% less than 5%. This indicates that the SA-M complex exhibits the strongest repeatability and stability in SERS measurements under continuous flow conditions, thus enabling accurate determination of the analyte content and meeting the requirements of quantitative immunoassay.

[0249] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A surface-enhanced Raman spectroscopy method for use in a Raman spectroscopy analyzer, comprising the following steps: (a) Provide a sample to be tested, said sample containing the test substance to be detected; (b) In a container, the sample to be tested is mixed with capture particles and detection particles to form a first mixture containing a complex of "capture particles-test sample-detection particles"; in, The capturing particles are magnetic particles loaded with a first capturing agent; while the detection particles are solid-phase carriers loaded with a second capturing agent and labeled with signal molecules. The first and second trapping agents are both specifically targeted at the test substance and bind with the test substance to form a ternary complex of "first trapping agent-test substance-second trapping agent"; (c) After washing the complex of "captured particles-test material-detection particles", the complex of "captured particles-test material-detection particles" is dispersed into a suspension, and the spectral signal is measured while the suspension is in a state of continuous motion; the motion includes flow, and the flow includes eddies. The process of keeping the suspension in a continuous state of motion includes the following steps: After aspirating an appropriate amount of suspension from the container, return the aspirated amount of suspension to the container. Repeat this process, aspirating an appropriate amount of suspension from the container and returning the aspirated amount of suspension to the container, to keep the suspension in a continuous state of motion.

2. The method as described in claim 1, characterized in that, The test substance to be detected is selected from the following group: procalcitonin, interleukin-6, or a combination thereof.

3. The method as described in claim 1, characterized in that, The detection particles are Raman detection particles.

4. The method as described in claim 2, characterized in that, The concentration of procalcitonin in the suspension is 0.001-1000 ng / ml; The concentration of interleukin-6 in the suspension is 0.001-1000 ng / ml.

5. The method as described in claim 4, characterized in that, The concentration of procalcitonin in the suspension is 0.01-1000 ng / ml; The concentration of interleukin-6 in the suspension is 0.01-500 ng / ml.

6. The method as described in claim 5, characterized in that, The concentration of procalcitonin in the suspension is 0.01-500 ng / ml; The concentration of interleukin-6 in the suspension is 0.01-100 ng / ml.

7. The method as described in claim 1, characterized in that, The sample to be tested is serum, plasma, whole blood, or buffer solution.

8. The method as described in claim 1, characterized in that, The cleaning process includes the following steps: The complex of "captured particles-test material-detected particles" is aggregated on the side wall of the container, and then the complex of "captured particles-test material-detected particles" is cleaned with a cleaning solution. In this process, the complex of "captured particles-test material-detected particles" is aggregated on the side wall of the container under the action of a magnetic field.

9. The method as described in claim 1, characterized in that, The process of keeping the suspension in a continuous state of motion includes the following steps: The suspension is agitated, thus keeping it in a state of continuous motion.

10. The method as described in claim 1, characterized in that, The trapping particles are magnetic particles loaded with trapping agents and having a structure of Formula I: Z1-Z2-Z3(I) In the formula, Z1 is a magnetic particle; Z2 is the first connecting element; and Z3 is the primary capture agent.

11. The method as described in claim 10, characterized in that, The first linking element is selected from the group consisting of linking molecules containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linking elements, or combinations thereof. The first capturing agent is the first antibody.

12. The method as described in claim 11, characterized in that, The peptide linker includes an avidin-biotin linker arm.

13. The method as described in claim 1, characterized in that, The detected particles are microparticles having the structure of Formula II: Y1-Y2-Y3-Y4-Y5(II) In the formula, Y1 represents precious metal particles; Y2 is a Raman signal molecule; and Y3 is the coating layer; Y4 is the second connecting element; Y5 is the second trapping agent.

14. The method as described in claim 13, characterized in that, The detected particles include one or more features selected from the group consisting of: (1) The precious metal particles are selected from the group consisting of Au particles, Ag particles, or combinations thereof; (2) The Raman signaling molecules are selected from the group consisting of: 4-mercaptobenzoic acid, 4-mercaptothiophenol, nitrothiophenol, aminothiophenol, benzimidazole, benzothiazole, mercaptopyridine, isothiocyanate, 5,5-dimercaptobis(nitrobenzoic acid), 4-acetaminophensulfuric acid, 2-thiouracil, or combinations thereof. (3) The coating is an Ag coating, an Au coating, or a combination thereof; (4) The second linking element is selected from the group consisting of linking molecules containing thiol, hydroxyl, aldehyde and / or carboxyl groups, polypeptide linking elements, or combinations thereof; (5) The second capturing agent is a second antibody.

15. A spectral analysis system, characterized in that, The system includes: (i) An excitation light emission source emits excitation light, which enters from below the bottom of a container containing a continuously moving "capture particle-test material-detection particle" complex. (ii) A spectral signal acquisition unit, wherein the spectral signal acquisition unit is used to acquire the spectral signal generated after the complex of "captured particle-test substance-detected particle" is irradiated; (iii) Output unit.

16. The spectral analysis system as described in claim 15, characterized in that, The spectral analysis system also includes: (iv) A dispersion unit for continuously moving the "capture particle-test material-detection particle" complex in the container.

17. The spectral analysis system as described in claim 15, characterized in that, The dispersion unit includes a stirrer, an oscillator, or a combination thereof.

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