A no-wash, multiplexable competitive immunoassay method

Abstract

This invention provides a wash-free, competitive immunoassay method capable of simultaneous multiplex detection. Specifically, this invention utilizes self-developed Raman microspheres (R-Sphere) with non-overlapping Raman signals to conjugate specific antibodies against the target molecule onto the Raman microspheres. The target molecule is then detected through an immunoassay. Raman microsphere-antibody conjugates that do not bind to the target molecule are adsorbed onto the container wall via carboxyl magnetic beads, thereby eliminating background signal interference. This method offers advantages such as wash-free operation, separation-free operation, ease of use, high accuracy, and the ability to simultaneously detect multiple signals.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and more specifically, this invention relates to a wash-free competitive immunoassay method that can perform multiplex detection simultaneously. Background Technology

[0002] Small molecule compounds refer to compounds with a molecular weight typically below 1000 Daltons and a simple structure. In the field of medical diagnostics, the range of small molecule compounds that need to be tested clinically is very wide, such as antihypertensive hormones, psychotropic drugs, sex hormones, small molecule therapeutic drugs, vitamins, etc.

[0003] High-performance liquid chromatography-mass spectrometry (HPLC-MS) has long been the gold standard for the detection of small molecule compounds. Although it has high accuracy and specificity, the key steps of the analytical process, such as pretreatment, are time-consuming and cumbersome, especially for biological samples such as blood and urine. Furthermore, HPLC-MS instruments are expensive and have high requirements for the working environment and the technical skills of operators. As a result, it cannot meet the needs of medical institutions at all levels for the rapid detection of various small molecule compounds in large quantities of blood, urine, saliva and other samples, which greatly limits its widespread application in resource-scarce areas.

[0004] Traditional enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA) have the advantages of requiring no sample pretreatment, being simple to operate, and having inexpensive instruments. However, in order to reduce background signals, repeated separation and cleaning steps are required, which makes the analysis process quite time-consuming and increases the workload. Furthermore, it is impossible to obtain multiple detection signals in a single detection within a single reaction unit.

[0005] Therefore, there is an urgent need to develop a rapid analysis technique for small molecule compounds that is easy to operate, highly sensitive, versatile, and capable of multiplex detection. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid analysis technique for small molecule compounds that is easy to operate, highly sensitive, versatile, and capable of multiplex detection.

[0007] In a first aspect of the invention, a wash-free, multiplex immunoassay method is provided, the method comprising the following steps:

[0008] (A) Preparation of Raman microsphere-antibody conjugate (R-Sphere-mAb), wherein the antibody is a specific antibody against the target molecule;

[0009] (B) Preparation of antigen-carboxylated magnetic bead conjugates, wherein the antigen is a macromolecular antigen formed by the target molecule and the protein carrier;

[0010] (C) The test sample, R-Sphere-mAb, and antigen-carboxyl magnetic bead conjugate are co-incubated under an external magnetic field.

[0011] Among them, R-Spheres without target molecules are adsorbed onto the container wall via carboxyl magnetic beads, while R-Spheres with target molecules are present in the solution; and

[0012] (D) Detect the Raman characteristic signal in the supernatant.

[0013] In another preferred embodiment, the Raman microspheres are selected from the group consisting of:

[0014] R-Sphere 041 : at Raman displacement 539cm -1 It has a characteristic signal peak;

[0015] R-Sphere 026 : at Raman displacement 735cm -1 It has a characteristic signal peak;

[0016] R-Sphere 067 : at Raman displacement 1075cm -1 It has a characteristic signal peak;

[0017] R-Sphere 013 : at Raman displacement 1629cm -1 It has a characteristic signal peak; and / or

[0018] R-Sphere 006 : at Raman displacement 2071cm -1 It has a characteristic signal peak.

[0019] In another preferred embodiment, a Raman microsphere is labeled with a specific antibody against a target molecule.

[0020] In another preferred embodiment, the target molecule is selected from the group consisting of: morphine (MOP), cocaine (COC), ketamine (KET), methamphetamine (MET), and / or amphetamine (AMP).

[0021] In another preferred embodiment, five types of Raman microspheres were used simultaneously to detect five target molecules, with no signal overlap or interference.

[0022] In another preferred embodiment, the protein carrier is selected from the group consisting of BSA (bovine serum albumin), RSA (rabbit serum albumin), HSA (human serum albumin), OVA (ovalbumin), or KLH (keyhole hemocyanin).

[0023] In another preferred embodiment, the positive reaction in step C is that the target molecule in the test sample binds to R-Sphere-mAb to form R-Sphere-mAb-target molecule.

[0024] In another preferred embodiment, the negative reaction in step C is that the carboxyl magnetic bead-antigen binds to R-Sphere-mAb to form R-Sphere-mAb-antigen-carboxyl magnetic beads, wherein the antigen is a macromolecular antigen formed by the target molecule and the protein carrier.

[0025] In another preferred embodiment, the carboxylated magnetic bead-antigen is a carboxylated magnetic bead-BSA-target molecule.

[0026] In another preferred embodiment, the target molecule in the "carboxylated magnetic bead-BSA-target molecule" is a molecule with the same structure as the target molecule in the sample to be tested.

[0027] In another preferred embodiment, prior to co-incubation, the target molecule in the “carboxy magnetic bead-BSA-target molecule” is pre-coupled with the carboxy magnetic bead and BSA.

[0028] In another preferred embodiment, the target molecule in the "sample to be tested" is more likely to bind to the Raman microsphere-antibody than the target molecule in the "carboxylated magnetic beads-BSA-target molecule".

[0029] In another preferred embodiment, the carboxyl magnetic bead is a magnetic bead with carboxyl groups attached to its surface.

[0030] In another preferred embodiment, the carboxyl magnetic beads are 1-10 μm magnetic beads, preferably 2-4 μm.

[0031] In another preferred embodiment, carboxyl magnetic beads are aggregated on the wall of the reaction vessel by an external magnetic field, thereby separating the negative signal (background signal) carried by the carboxyl magnetic beads from the solution (supernatant).

[0032] In another preferred embodiment, Raman magnetic beads that bind to the target molecule in the sample to be tested carry a positive signal in the solution (supernatant).

[0033] In another preferred embodiment, the solution (supernatant) is a homogeneous solution.

[0034] In another preferred embodiment, the concentration of the target molecule in the sample to be tested in the solution is positively correlated with the intensity of the Raman signal.

[0035] In another preferred embodiment, the sample to be tested is selected from the group consisting of saliva samples, blood samples, and urine samples.

[0036] In another preferred embodiment, the detection is performed by detecting Raman characteristic signals using a handheld Raman spectrometer.

[0037] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0038] In another preferred embodiment, the method is a non-therapeutic and non-diagnostic method.

[0039] In a second aspect of the invention, an immunoassay-based small molecule detection device that is wash-free and capable of simultaneous multiplex detection is provided, the detection device comprising the following modules:

[0040] (a) A reaction reagent module, wherein the reaction reagent comprises:

[0041] (a1) Raman microspheres, said Raman microspheres being conjugated to a specific antibody for detecting target molecules.

[0042] (a2) Carboxyl magnetic beads, wherein the carboxyl magnetic beads are coupled to a macromolecular antigen containing a target molecule for coupling to Raman microspheres unbound to the target molecule; and

[0043] (b) Detection module, the detection module comprising: a reaction container, an external magnetic field and a Raman spectrometer, the reaction container being used to hold the sample to be tested and the reaction reagent, the external magnetic field being used to aggregate unbound Raman microspheres of target molecules attached to carboxyl magnetic beads to the container wall, and the Raman spectrometer being used to detect Raman characteristic signals in the supernatant.

[0044] In another preferred embodiment, the reaction vessel is an immune micropore.

[0045] In another preferred embodiment, the content of the immune micropores is 100-1000 μL.

[0046] In another preferred embodiment, the applied magnetic field is a magnet.

[0047] In another preferred embodiment, a magnet is attached to the outer wall of the immunomicropore, and magnetic beads are visible to accumulate on the inner wall.

[0048] In another preferred embodiment, a Raman spectrometer is used to detect the Raman signal in the supernatant of the reaction vessel.

[0049] In another preferred embodiment, the Raman spectrometer is a handheld Raman spectrometer.

[0050] In a third aspect of the invention, an Au@Ag core-shell structured Raman microsphere is provided, said Raman microsphere being selected from the group consisting of:

[0051] Raman displacement 539cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0052] At Raman displacement 735cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0053] Raman displacement 1075cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0054] Raman displacement 1629cm -1 Raman microspheres with characteristic signal peaks; or

[0055] Raman displacement 2071cm -1 Raman microspheres with characteristic signal peaks.

[0056] In another preferred embodiment, the ratio of Au:Ag in the Raman microspheres is 1:3-1:6, preferably 1:4-1:5.

[0057] 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. Attached Figure Description

[0058] Figure 1 The following are displayed: (a) Characteristic Raman spectra of five R-Spheres;

[0059] (b) The detection principle of the analytical method of the present invention (taking MOP, COC and KET as examples);

[0060] (c) Raman spectrometer.

[0061] Figure 2 The standard curve for combined quantitative detection of MOP and KET is shown.

[0062] Figure 3 The standard curve for combined quantitative detection of MOP, COC, and KET is shown.

[0063] Figure 4 The standard curve for combined quantitative detection of MOP, COC, MET and KET is shown.

[0064] Figure 5 The standard curves for combined quantitative detection of MOP, COC, MET, AMP, and KET are displayed. Detailed Implementation

[0065] Through extensive and in-depth research, the inventors have invented a wash-free, multiplex competitive immunoassay method. Specifically, this invention utilizes self-developed Raman microspheres (R-Spheres) with non-overlapping Raman signals. Specific antibodies targeting the target molecule are conjugated to the Raman microspheres, and the target molecule is detected through an immunoassay. Raman microsphere-antibody conjugates that do not bind to the target molecule are adsorbed onto the container wall by carboxyl magnetic beads, thus eliminating background signal interference. The characteristic Raman signal is detected using a handheld Raman spectrometer under an applied magnetic field. This invention is based on this principle.

[0066] The method of the present invention has the advantages of being wash-free, separation-free, easy to operate, and highly accurate, and can simultaneously detect multiple signals.

[0067] the term

[0068] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0069] 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”.

[0070] In this invention, the amount (molar amount) of carboxyl magnetic beads-BSA-target molecules is greater than the amount (molar amount) of Raman microspheres. This results in all Raman microsphere signals being aggregated on the reaction vessel wall by the carboxyl magnetic beads when there are no target molecules in the sample to be tested, which are negative signals or background signals. At this time, no Raman signal (positive signal) can be detected in the solution.

[0071] When the target molecule is present in the test sample, the target molecule in the test sample has a strong ability to bind with the Raman microsphere-antibody. Even if the "Raman microsphere-antibody" has already been coupled with the "carboxy magnetic bead-BSA-target molecule", the target molecule in the test sample can still displace the "carboxy magnetic bead-BSA-target molecule" and thus bind to the "Raman microsphere-antibody". At this time, a positive signal appears in the solution.

[0072] Then, by attracting the carboxyl magnetic beads with an external magnetic field, the negative signal is separated from the solution and enriched on the wall of the reaction vessel. At this point, only the positive signal exists in the solution.

[0073] In this invention, the target molecule described in "carboxylated magnetic beads-BSA-target molecule" is a molecule with the same structure as the target molecule to be detected in the sample.

[0074] In this invention, "solution" and "supernatant" can be used interchangeably.

[0075] In this invention, "negative signal" and "background signal" can be used interchangeably.

[0076] In this invention, "container wall" and "reaction vessel wall" can be used interchangeably.

[0077] In this invention, "Raman microsphere-antibody" and "R-Sphere-mAb" can be used interchangeably.

[0078] Surface-enhanced Raman spectroscopy

[0079] Raman spectroscopy, a type of molecular vibrational spectroscopy, can reflect the characteristic structure of molecules. However, Raman scattering is a very weak process. Surface-enhanced Raman spectroscopy (SERS) involves adsorbing the analyte onto rough surfaces such as silver, gold, or copper, significantly increasing the intensity of its Raman signal. The corresponding spectrum is called surface-enhanced Raman spectroscopy. SERS has demonstrated significant application potential over existing technologies in molecular imaging and rapid qualitative and quantitative detection of multiple targets with high precision, and has been widely used in the analysis of proteins, bacteria, pathogens, and small molecules.

[0080] Raman microspheres

[0081] In this invention, five nanoscale Raman microspheres (R-Spheres) were independently developed based on the SERS principle. These are Au@Ag core-shell structured nanomaterials that not only have high luminescence intensity but also feature non-overlapping Raman spectra, which is the basis for achieving high-throughput detection.

[0082] The five types of Raman microspheres used in this invention are:

[0083] Raman displacement 539cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0084] At Raman displacement 735cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0085] Raman displacement 1075cm -1 Raman microspheres with characteristic signal peaks at the point of contact;

[0086] Raman displacement 1629cm -1 Raman microspheres with characteristic signal peaks; and

[0087] Raman displacement 2071cm -1 Raman microspheres with characteristic signal peaks are used. Five types of Raman microspheres have non-overlapping characteristic signal peaks, enabling the simultaneous detection of multiple targets.

[0088] Carboxyl magnetic beads

[0089] Carboxyl magnetic beads (CMBs) are magnetic microspheres with carboxyl (-COOH) functional groups on their surface, making them a multifunctional magnetic material. These carboxyl groups endow the beads with excellent biocompatibility and chemical stability, while also providing the possibility of covalently binding with various biomolecules (such as proteins, antibodies, and nucleic acids). Due to their unique and highly efficient adsorption properties, CMBs exhibit efficient adsorption performance for various biomolecules and heavy metal ions, making them suitable for rapid separation and purification. They can be used not only as carriers for immunoassays but also in various applications in environmental remediation and materials science.

[0090] In addition to the carboxyl magnetic beads used in this experiment, there are also amino magnetic beads and SA magnetic beads. However, amino magnetic beads cannot be coupled to the protein molecules used in this experiment because their own groups cannot be used. SA magnetic beads require biotin modification, which is more complex. Carboxyl magnetic beads, on the other hand, can be directly coupled to the amino groups on protein molecules, are easy to operate, have high utilization, and are convenient for immune responses.

[0091] The immunoassay method of the present invention

[0092] This invention establishes an innovative analytical technique for small molecule compounds based on R-Sphere signal tagging, combined with competitive immunoassay, using small molecule compounds such as morphine (MOP), cocaine (COC), ketamine (KET), methamphetamine (MET), and amphetamine (AMP) as template analytes. A small Raman spectrometer is used to detect the characteristic peaks of R-Sphere in homogeneous solutions, enabling efficient and highly sensitive simultaneous quantitative detection of MOP, COC, KET, MET, and AMP. This technique offers numerous advantages, including simple operation, speed, and multiplexing capabilities.

[0093] Preferably, the specific steps of the present invention include:

[0094] (1) The specific antibody (mAb) of the small molecule compound is labeled on R-Sphere to form R-Sphere-mAb. The small molecule is coupled with BSA to form antigen (small molecule-BSA). The small molecule-BSA is then coupled to carboxyl magnetic beads to form small molecule-BSA-carboxyl magnetic beads.

[0095] (2) The test sample, R-Sphere-mAb, and small molecule-BSA-carboxylic magnetic beads were co-incubated in an immunoassay microwell (container content approximately 400 μl). During the immunoassay reaction, the target molecule (target small molecule) of the test sample and the small molecule-BSA-carboxylic magnetic beads simultaneously competed to bind to R-Sphere-mAb.

[0096] When there is no target molecule in the sample to be tested: R-Sphere-mAb reacts and combines with small molecule-BSA-carboxyl magnetic beads to generate R-Sphere-mAb-small molecule-BSA-carboxyl magnetic beads, which are adsorbed onto the container wall through the carboxyl magnetic beads, so no Raman signal can be detected in the supernatant.

[0097] When the target molecule is present in the sample: Since the small molecules in the sample have a dominant reaction with R-Sphere-mAb, the target molecule reacts and binds with R-Sphere-mAb and exists in the supernatant, while the R-Sphere-mAb that does not bind with the target molecule is adsorbed onto the container wall by carboxyl magnetic beads.

[0098] After incubation, an external magnetic field is applied. The lower the sample concentration, the more R-Sphere-mAb binds to the small molecule -BSA-carboxylic magnetic beads, the fewer R-Spheres are present in the solution, and the weaker the Raman signal; conversely, the higher the sample concentration, the fewer R-Sphere-mAb binds to the small molecule -BSA-carboxylic magnetic beads, the more R-Spheres bind to the target molecule in the solution, and the stronger the Raman signal.

[0099] Compared with the prior art, the advantages of the present invention are as follows:

[0100] 1. No washing or separation required: Raman magnetic beads bound to the target molecule are present in the supernatant, while Raman microspheres not bound to the target molecule are adsorbed onto the container wall by carboxyl magnetic beads. No washing or separation steps are required, thus eliminating the interference of background signals.

[0101] 2. High accuracy: The target molecule binds to the Raman magnetic bead-antibody to form a homogeneous solution, reducing errors caused by signal inhomogeneity. Furthermore, the signal intensity is positively correlated with the concentration of the target molecule, thus improving the accuracy of the signal.

[0102] 3. Multiple detection: The Raman microspheres of this invention have non-overlapping Raman characteristic signals. Each signal detects one target molecule without interfering with each other. Therefore, multiple target molecules can be detected simultaneously, reducing manpower and material costs.

[0103] 4. Easy to operate: The handheld Raman spectrometer is easy to carry and can realize real-time on-site detection.

[0104] 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.

[0105] 1. Experimental apparatus

[0106] Electronic balance (BSM-220.4, Shanghai Zhuojing Electronic Technology Co., Ltd.);

[0107] Benchtop high-speed refrigerated centrifuge (CT14RD, Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.);

[0108] Fixed mixer (MX-F, Beijing Dalongxingchuang Experimental Instrument Co., Ltd.);

[0109] Track shaking table for decolorization (TS-1, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.);

[0110] Intelligent constant temperature oscillation instrument (GB-3A, Shanghai Leichuang Instrument Co., Ltd.);

[0111] Ultraviolet-Vis absorption spectrometer (L6S, Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.);

[0112] Handheld Raman spectrometer (BLADE-785B-OEM, Shanghai Liqiong Optoelectronic Technology Co., Ltd.)

[0113] 2. Experimental reagents

[0114] R-Sphere041, R-Sphere026, R-Sphere067, R-Sphere013, R-Sphere006 Raman microspheres (Shanghai Xinpu Biotechnology Co., Ltd.);

[0115] Sucrose, sodium bicarbonate, menthol, aspirin, calcium chloride dihydrate, sodium chloride, magnesium chloride hexahydrate, potassium chloride, dipotassium hydrogen phosphate trihydrate, potassium carbonate, sodium carboxymethyl cellulose, tris(hydroxymethyl)aminomethane (Tris), dilute hydrochloric acid, ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC·HCl) (China National Pharmaceutical Group Chemical Reagent Co., Ltd.);

[0116] 2.8μm carboxyl magnetic beads (Shanghai Taoyu International Trade Co., Ltd.);

[0117] Hydroxysuccinimide (NHS) (Shanghai Yuanye Biotechnology Co., Ltd.);

[0118] Morpholine ethanesulfonic acid (MES) (Shanghai Aladdin Biotechnology Co., Ltd.);

[0119] Bovine serum albumin (Shanghai Anbe Medical Equipment Trading Co., Ltd.);

[0120] MET national standard, mAbMET, MET-BSA, AMP national standard, mAbAMP, AMP-BSA (Hangzhou LONGi Biotechnology Co., Ltd.);

[0121] MOP National Standard Product, mAbMOP, MOP-BSA, KET National Standard Product, mAbKET, KET-BSA, COC National Standard Product, mAbCOC, COC-BSA (Shanghai Yuansi Standard Technology Co., Ltd.)

[0122] 3. Solution preparation

[0123] 3.1 Preparation of artificial saliva

[0124] Weigh out 0.02g magnesium chloride hexahydrate, 0.02g calcium chloride dihydrate, 0.03g sodium chloride, 0.08g potassium chloride, 0.08g dipotassium hydrogen phosphate trihydrate, 0.05g potassium carbonate, and 0.02g sodium carboxymethyl cellulose, respectively, and dissolve them in ultrapure water to 100mL. Store at 4-8℃ for later use.

[0125] 3.2 Preparation of R-Sphere-mAb

[0126] Take 1 mL of the prepared R-Sphere solution, add 7.5 μg mAb, mix and react at room temperature for 30 minutes, centrifuge (8000 rpm, 4℃, 10 min), discard the supernatant, add 20 μL of pure water and 10% BSA solution, react for 30 min, centrifuge (8000 rpm, 4℃, 10 min) to remove unreacted excess substances, redissolve in 100 mL of pure water to obtain R-Sphere-mAb, store at 4℃ for later use.

[0127] 3.3 Preparation of small molecule-BSA-carboxylated magnetic beads

[0128] Take 60 μl of 2.8 μm carboxyl magnetic beads, aggregate for 1 min, remove the supernatant, add 100 μl of 0.1 mol / L MES aqueous solution (pH 5.0), shake thoroughly to mix, then add 10 μl of 10 mg / mL EDC-HCl aqueous solution and NHS aqueous solution (prepare fresh), shake thoroughly to mix for 30 min, then add 10 μg of 1 mg / mL small molecule-BSA, shake thoroughly to mix for 3 hours, aggregate for 1 min, remove the supernatant, wash the magnetic beads three times with 100 μl of TBS-T aqueous solution, and finally add 100 μl of TBS-T aqueous solution to obtain small molecule-BSA-carboxyl magnetic beads, store at 4 °C for later use.

[0129] 3.4 Detection Methods

[0130] A certain amount of the prepared mixed series of standard solutions / test solutions, R-Sphere-mAb, and small molecule-BSA-carboxyl magnetic beads were placed in a reaction vessel and subjected to a constant-temperature oscillation reaction at 37°C for 45 minutes. An external magnetic field was applied to concentrate the magnetic beads on the side wall of the reaction vessel, and the Raman signal at the characteristic peak of R-Sphere in the supernatant was detected using a handheld Raman spectrometer.

[0131] Example 1. Quantitative detection of morphine and ketamine

[0132] Raman microspheres were prepared by mixing Au and Ag in a certain proportion. Five types of Raman microspheres exhibited non-overlapping Raman signal characteristic peaks, such as... Figure 1 As shown in a.

[0133] A schematic diagram of the analysis method of this invention is shown below. Figure 1 As shown in b, the actual detection and shooting image is as follows. Figure 1 As shown in c.

[0134] 1.1 Preparation of MOP and KET series standard solutions

[0135] Using artificial saliva as the matrix solution, MOP and KET national standards were diluted to prepare certain concentrations. Finally, the two samples were mixed in equal volumes to prepare a series of mixed standard solutions of 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, which were stored at 4℃ for later use.

[0136] 1.2 Establishment of the standard curve

[0137] The results obtained after testing using the above immunoassay method are shown in Table 1 below. A standard curve was plotted with the concentrations of the MOP and KET mixed standard solutions on the x-axis and the corresponding Raman signal values ​​on the y-axis. (See Table 1 for details.) Figure 2 As shown, the regression equation for MOP is y = -0.4261x. 2+134.6x+1313.9, the correlation coefficient is r 2 =0.9972; the regression equation for KET is y = -0.3023x 2 +118.98x+770.47, the correlation coefficient is r. 2 =0.9960.

[0138] Table 1. Combined quantitative detection data of MOP and KET

[0139]

[0140] 1.3 Spike Recovery Experiment

[0141] A mixed sample solution containing MOP and KET national standards diluted to a final concentration of 10 ng / mL was designated as sample a; a mixed sample solution containing MOP and KET national standards diluted to a final concentration of 25 ng / mL was designated as sample b; and a mixed sample solution containing MOP and KET national standards diluted to a final concentration of 100 ng / mL was designated as sample c. 1 mL of sample a solution was added to 100 μL of sample b solution to obtain concentration 1; 1 mL of sample a solution was added to 100 μL of sample c solution to obtain concentration 2; and 1 mL of sample b solution was added to 100 μL of sample c solution to obtain concentration 3. Following the above experimental steps, the Raman signal response values ​​were obtained using a handheld Raman spectrometer. The corresponding concentration values ​​were obtained by substituting these values ​​into the above standard curve formula, and the recovery rate was calculated (Table 2).

[0142] Table 2. Spiked Recovery Experiment Data

[0143]

[0144]

[0145] The experimental results in the examples show that the concentration range of MOP and KET (0-200.0 ng / mL) exhibits a good correlation with the Raman signal, and this range can be considered the linear range of the standard system. It can be seen that the Raman signal increases with increasing sample concentration, which is consistent with the principle of competitive immunoassay. Furthermore, the recoveries at all three concentration points are between 90% and 110%, indicating that the detection method is accurate and can be used for the simultaneous detection of MOP and KET.

[0146] Any two of the five types of Raman microspheres can be selected to achieve simultaneous detection of MOP and KET.

[0147] Example 2. Combined quantitative detection of morphine, cocaine and ketamine

[0148] 2.1 Preparation of MOP, COC and KET series standard solutions

[0149] Using artificial saliva as the matrix solution, MOP, COC and KET national standards were diluted to prepare certain concentrations. Finally, the three samples were mixed in equal volumes to prepare a series of standard solutions of 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL and 100 ng / mL, which were stored at 4℃ for later use.

[0150] 2.2 Establishment of the Standard Curve

[0151] The results obtained after testing using the above immunoassay method are shown in Table 3 below. A standard curve was plotted with the concentrations of the mixed series of MOP, COC, and KET standard solutions on the x-axis and the corresponding Raman signal values ​​on the y-axis. (See Table 3 for details.) Figure 3 As shown, the regression equation for MOP is y = -1.2655x. 2 +246.01x+1084.2, the correlation coefficient is r 2 =0.9935; the regression equation for COC is y = -1.0601x 2 +206.03x+701.82, correlation coefficient r 2 =0.9925; the regression equation for KET is y = -1.3298x 2 +229.4x+814.03, the correlation coefficient is r. 2 =0.9933.

[0152] Table 3. Combined quantitative detection data of MOP, COC and KET

[0153]

[0154] 2.3 Spike Recovery Experiment

[0155] A mixed sample solution containing MOP, COC, and KET national standards diluted to a final concentration of 10 ng / mL was prepared as sample a; a mixed sample solution containing MOP, COC, and KET national standards diluted to a final concentration of 25 ng / mL was prepared as sample b; and a mixed sample solution containing MOP, COC, and KET national standards diluted to a final concentration of 50 ng / mL was prepared as sample c. 1 mL of sample a solution was added to 100 μL of sample b solution to obtain concentration 1; 1 mL of sample a solution was added to 100 μL of sample c solution to obtain concentration 2; and 1 mL of sample b solution was added to 100 μL of sample c solution to obtain concentration 3. Following the above experimental steps, the Raman signal response values ​​were obtained using a handheld Raman spectrometer. The corresponding concentration values ​​were obtained by substituting these values ​​into the above standard curve formula, and the recovery rate was calculated (Table 4).

[0156] Table 4. Spiked Recovery Experiment Data

[0157]

[0158] The experimental results in the examples show that the concentration ranges of MOP, COC, and KET (0-100.0 ng / mL) exhibit good correlation with Raman signals, and this range can be considered the linear range of the standard system. It can be seen that the Raman signal increases with increasing sample concentration, which is consistent with the principle of competitive immunoassay. Furthermore, the recoveries at all three concentration points are between 90% and 110%, indicating that the detection method is accurate and can be used for the simultaneous detection of MOP, COC, and KET.

[0159] Any three of the five types of Raman microspheres can be selected to achieve simultaneous detection of MOP, COC, and KET.

[0160] Example 3. Combined quantitative detection of morphine, cocaine, methamphetamine and ketamine

[0161] 3.1 Preparation of MOP, COC, MET and KET series standard solutions

[0162] Using artificial saliva as the matrix solution, national standards MOP, COC, MET, and KET were diluted to prepare certain concentrations. Finally, the four samples were mixed in equal volumes to prepare a series of standard solutions of 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, which were stored at 4℃ for later use.

[0163] 3.2 Establishment of the standard curve

[0164] The results obtained after testing using the above immunoassay method are shown in Table 5 below. A standard curve was plotted with the concentrations of the mixed series of MOP, COC, MET, and KET standard solutions on the x-axis and the corresponding Raman signal values ​​on the y-axis. (See Table 5 for details.) Figure 4 As shown, the regression equation for MOP is y = -0.3149x. 2 +112.48x+1022.9, the correlation coefficient is r. 2 =0.9986; the regression equation for COC is y = -0.2813x 2 +94.463x +985.08, correlation coefficient r 2 =0.9927; the regression equation for MET is y = -0.2879x 2 +93.517x+837.88, the correlation coefficient is r. 2 =0.9941; the regression equation for KET is y = -0.3615x 2 +110.35x+1248.9, the correlation coefficient is r. 2 =0.9976.

[0165] Table 5. Combined quantitative detection data of MOP, COC, MET and KET

[0166]

[0167]

[0168] 3.3 Spike Recovery Experiment

[0169] A mixed sample solution containing MOP, COC, MET, and KET national standards diluted to a final concentration of 10 ng / mL was designated as sample a; a mixed sample solution containing MOP, COC, MET, and KET national standards diluted to a final concentration of 25 ng / mL was designated as sample b; and a mixed sample solution containing MOP, COC, MET, and KET national standards diluted to a final concentration of 50 ng / mL was designated as sample c. 1 mL of sample a solution was added to 100 μL of sample b solution to obtain concentration 1; 1 mL of sample a solution was added to 100 μL of sample c solution to obtain concentration 2; and 1 mL of sample b solution was added to 100 μL of sample c solution to obtain concentration 3. Following the above experimental steps, Raman signal response values ​​were obtained using a handheld Raman spectrometer. The corresponding concentration values ​​were obtained by substituting these values ​​into the above standard curve formula, and the recovery rate was calculated (Table 6).

[0170] Table 6. Spiked Recovery Experiment Data

[0171]

[0172] The experimental results in the examples show that the concentration ranges of MOP, COC, MET, and KET (0-200.0 ng / mL) exhibit good correlation with Raman signals, and this range can be considered the linear range of the standard system. It can be seen that the Raman signal increases with increasing sample concentration, which is consistent with the principle of competitive immunoassay. Furthermore, the recoveries at all three concentration points are between 90% and 110%, indicating that the detection method is accurate and can be used for the simultaneous detection of MOP, COC, MET, and KET.

[0173] Any four of the five types of Raman microspheres can be selected to achieve simultaneous detection of MOP, COC, KET, and MET.

[0174] Example 4. Combined quantitative detection of morphine, cocaine, methamphetamine, amphetamine, and ketamine.

[0175] 4.1 Preparation of MOP, COC, MET, AMP and KET series standard solutions

[0176] Using artificial saliva as the matrix solution, national standards for MOP, COC, MET, AMP, and KET were diluted to prepare certain concentrations. Finally, the five samples were mixed in equal volumes to prepare a series of standard solutions of 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, which were stored at 4℃ for later use.

[0177] 4.2 Establishment of the Standard Curve

[0178] The results obtained after testing using the above immunoassay method are shown in Table 7 below. A standard curve was plotted with the concentrations of the mixed series of standard solutions (MOP, COC, MET, AMP, and KET) on the x-axis and the corresponding Raman signal values ​​on the y-axis. (See Table 7 for details.) Figure 5 As shown, the regression equation for MOP is y = -0.2921x. 2 +90.844x+1220.3, the correlation coefficient is r. 2 =0.9961; the regression equation for COC is y = -0.279x 2 +91.989x+1129.6, correlation coefficient r 2 =0.9923; the regression equation for MET is y = -0.3051x 2 +97.988x+1388.3, ​​the correlation coefficient is r. 2 =0.9937; the regression equation for AMP is y = -0.3147x 2 +93.798x+1252, the correlation coefficient is r. 2 =0.9913; the regression equation for KET is y = -0.1706x 2 +64.931x+710.03, correlation coefficient is r 2 =0.9956.

[0179] Table 7. Combined quantitative detection data of MOP, COC, MET, AMP and KET

[0180]

[0181]

[0182] 4.3 Spike Recovery Experiment

[0183] A mixed sample solution containing MOP, COC, MET, AMP, and KET national standards diluted to a final concentration of 10 ng / mL was prepared as sample a; a mixed sample solution containing MOP, COC, MET, AMP, and KET national standards diluted to a final concentration of 25 ng / mL was prepared as sample b; and a mixed sample solution containing MOP, COC, MET, AMP, and KET national standards diluted to a final concentration of 50 ng / mL was prepared as sample c. 1 mL of sample a solution was added to 100 μL of sample b solution to obtain concentration 1; 1 mL of sample a solution was added to 100 μL of sample c solution to obtain concentration 2; and 1 mL of sample b solution was added to 100 μL of sample c solution to obtain concentration 3. Following the above experimental steps, the Raman signal response values ​​were obtained using a handheld Raman spectrometer. The corresponding concentration values ​​were obtained by substituting these values ​​into the above standard curve formula, and the recovery rate was calculated (Table 8).

[0184] Table 8. Spiked Recovery Experiment Data

[0185]

[0186]

[0187] The experimental results in the examples show that the concentration ranges of MOP, COC, MET, AMP, and KET (0-200.0 ng / mL) exhibit good correlation with Raman signals, and this range can be considered the linear range of the standard system. It can be seen that the Raman signal increases with increasing sample concentration, which is consistent with the principle of competitive immunoassay. Furthermore, the recoveries at all three concentration points are between 90% and 110%, indicating that the detection method is accurate and can be used for the simultaneous detection of MOP, COC, MET, AMP, and KET.

[0188] 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 wash-free, multiplex immunoassay method, characterized in that, The method includes the following steps: (A) Preparation of Raman microsphere-antibody conjugate (R-Sphere-mAb), wherein the antibody is a specific antibody against the target molecule; (B) Preparation of antigen-carboxylated magnetic bead conjugates, wherein the antigen is a macromolecular antigen formed by the target molecule and the protein carrier; (C) The test sample, R-Sphere-mAb, and antigen-carboxyl magnetic bead conjugate are co-incubated under an external magnetic field. Among them, R-Spheres without target molecules are adsorbed onto the reaction vessel wall via carboxyl magnetic beads, while R-Spheres with target molecules are present in the solution; and (D) Detect the Raman characteristic signal in the supernatant.

2. The method as described in claim 1, characterized in that, The Raman microspheres are selected from the following group: R-Sphere 041 : at Raman displacement 539cm -1 It has a characteristic signal peak; R-Sphere 026 : at Raman displacement 735cm -1 It has a characteristic signal peak; R-Sphere 067 : at Raman displacement 1075cm -1 It has a characteristic signal peak; R-Sphere 013 : at Raman displacement 1629cm -1 It has a characteristic signal peak; and / or R-Sphere 006 : at Raman displacement 2071cm -1 It has a characteristic signal peak.

3. The method as described in claim 1, characterized in that, The target molecules are selected from the following group: morphine (MOP), cocaine (COC), ketamine (KET), methamphetamine (MET), and / or amphetamine (AMP).

4. The method as described in claim 1, characterized in that, The positive reaction in step C is that the target molecule in the test sample binds to R-Sphere-mAb to form R-Sphere-mAb-target molecule.

5. The method as described in claim 1, characterized in that, The negative reaction in step C is that the antigen-carboxy magnetic beads bind to R-Sphere-mAb to form R-Sphere-mAb-antigen-carboxy magnetic beads, wherein the antigen is a macromolecular antigen formed by the target molecule and the protein carrier.

6. The method as described in claim 1, characterized in that, In step C, the solution is a homogeneous solution.

7. The method as described in claim 1, characterized in that, The concentration of the target molecule in the sample to be tested is positively correlated with the intensity of the Raman signal.

8. The method as described in claim 1, characterized in that, The samples to be tested are selected from the following group: saliva samples, blood samples, and urine samples.

9. A wash-free, multiplex-detection-based small molecule detection device, characterized in that, The detection equipment includes the following modules: (a) A reaction reagent module, wherein the reaction reagent comprises: (a1) Raman microspheres, said Raman microspheres being conjugated to a specific antibody for detecting target molecules. (a2) Carboxyl magnetic beads, wherein the carboxyl magnetic beads are coupled to a macromolecular antigen containing a target molecule for coupling to Raman microspheres unbound to the target molecule; and (b) Detection module, the detection module comprising: a reaction container, an external magnetic field and a Raman spectrometer, the reaction container being used to hold the sample to be tested and the reaction reagent, the external magnetic field being used to aggregate unbound Raman microspheres of target molecules attached to carboxyl magnetic beads to the container wall, and the Raman spectrometer being used to detect Raman characteristic signals in the supernatant.

10. A Raman microsphere with an Au@Ag core-shell structure, characterized in that, The Raman microspheres are selected from the following group: Raman displacement 539cm -1 Raman microspheres with characteristic signal peaks at the point of contact; At Raman displacement 735cm -1 Raman microspheres with characteristic signal peaks at the point of contact; Raman displacement 1075cm -1 Raman microspheres with characteristic signal peaks at the point of contact; Raman displacement 1629cm -1 Raman microspheres with characteristic signal peaks; or Raman displacement 2071cm -1 Raman microspheres with characteristic signal peaks.

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