Method for rapidly detecting PTH through SERS (Surface Enhanced Raman Scattering) based on aptamer-antibody sandwich structure

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a method for rapidly detecting PTH through SERS (Surface Enhanced Raman Scattering) based on an aptamer-antibody sandwich structure. A method for rapidly detecting PTH by SERS (Surface Enhanced Raman Scattering) based on an aptamer-antibody sandwich structure is characterized by comprising the following steps: preparing immunomagnetic beads to capture nanoparticles; preparing metal nano sol; preparing immune metal signal nano particles; preparing a sandwich type sandwich structure; mixing the immune magnetic bead capture nano particles, the immune metal signal nano particles and a sample to be detected to obtain the sandwich type sandwich structure; and detecting the PTH. According to the detection method, the antibody and the aptamer serve as specific recognition elements and are specifically combined with PTH, on one hand, the dispersed state of the nanometer material in a detection system can be stabilized, on the other hand, the target protein can be recognized with high specificity and high affinity, the stability is good, the preparation cost is low, the detection accuracy is improved to a great extent, and the detection method is suitable for popularization and application. And the detection speed is higher.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for rapid detection of PTH by SERS based on an aptamer-antibody sandwich structure. Background Art

[0002] In recent years, the incidence and mortality of thyroid cancer have continued to rise. Surgery is the main treatment for patients with differentiated thyroid cancer. Total / near-total thyroidectomy can significantly reduce the risk of local recurrence and prolong survival, and can guide the postoperative disease staging and risk stratification of patients. Although surgery can maximally remove potential lesions in tumors and normal tissues, due to the close anatomical relationship between the parathyroid glands and the thyroid gland, thyroid surgery will significantly increase the risk of parathyroid damage, causing hypoparathyroidism (HP) and hypocalcemia. There are two pairs of parathyroid glands, located on the back of the left and right lobes of the thyroid gland in the neck, which are light brown and the size of soybeans. The incidence of temporary and permanent HP after total thyroidectomy is 10% to 60% and 1% to 4%, respectively, which seriously affects the quality of life of patients after surgery. How to accurately, economically, conveniently, and dynamically identify the parathyroid glands and preserve them intact is the key to protecting their function and preventing postoperative complications. The risk of parathyroid injury is so high because the color and shape of the parathyroid glands are similar to those of the surrounding fat particles and lymph nodes, so the method of empirical identification by naked eyes is not reliable. This method also has high requirements for the surgeon, so auxiliary methods such as autologous transplantation technology, contrast agents, and lateral flow biosensors (LFB) have been widely developed. Although autologous transplantation technology can avoid permanent HP, it can still cause temporary HP after surgery, and there is a possibility of misdiagnosis and missed detection. Methylene blue positive development can cause adverse reactions such as neurotoxicity, gastrointestinal reactions, and hypoxic cyanosis, and the false positive rate is high. Nanocarbon negative development technology is prone to miss the inferior parathyroid glands and easily contaminate the surgical field, making it difficult to identify the parathyroid glands. Although the lateral flow biosensor (LFB) is simple and efficient for rapid determination of parathyroid hormone (PTH) in tissues, the sampling methods include needle aspiration, clamping and tissue homogenization. Among them, needle aspiration is the least harmful to the parathyroid gland, but the sensitivity of LFB is slightly insufficient; tissue homogenization and clamping sampling are more harmful to the parathyroid gland. Therefore, based on the current situation, it is urgent to develop a new technology to provide a new platform for intraoperative protection of the parathyroid gland.

[0003] Surface-enhanced Raman spectroscopy (SERS) has the advantages of non-destructive detection, resistance to water interference, high resolution and sensitivity, simple and fast analysis, etc. It uses the coupling enhancement effect of gold nanosol to enhance the Raman signal of nearby molecules by a million times. Combined with Raman resonance molecules, it can even achieve single molecule detection sensitivity. It has been widely used in surface science, material science, biomedicine, drug analysis, food safety, environmental testing and other fields. It is a trace analysis technology with great potential. At present, there are studies using SERS technology to detect PTH. Among them, Juang et al. prepared AgNPs@mrGO active substrates for rapid and sensitive label-free detection of PTH, with a sensitivity of up to 0.2ng / Ml. Alejandro et al. prepared immunocolloidal gold test strips based on the sandwich structure principle, and used the double antibody sandwich method to achieve specific recognition. The minimum detection limit was 3.45ng / Ml, and the sensitivity was still relatively poor. Although there is a big gap for the detection limit required in clinical serum samples, it proves the feasibility of the sandwich method for PTH detection. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a method for rapid detection of PTH by SERS based on an aptamer-antibody sandwich structure. The method utilizes the specific recognition principle between an antibody aptamer and a PTH antigen, and incubates the immunomagnetic beads of the PTH capture antibody with the immunometallic signal nanoparticles of the PTH and the specific aptamer modified with PTH to form a sandwich structure.

[0005] Compared with the large steric hindrance of the traditional double antibody module, the aptamer-antibody sandwich with low Gibbs free energy and low dissociation constant has a higher recognition efficiency; nucleic acid aptamers have the great advantages of high affinity, small steric hindrance and stability between batches, which can reduce the false positives and false negatives caused by the double antibody module. The present invention constructs an aptamer-antibody sandwich mode PTH detection platform based on SERS technology, using highly sensitive nucleic acid aptamers and antibodies to specifically recognize PTH protein, and using the Raman signal molecule IR808 that resonates with the incident excitation light wavelength of 785nm as a probe. The gold-end modified aptamer, which is a SERRS tag constructed by aptamer No. 14 invented by Yanghoon Kim and Yoonshin Park, gold nanosol (AuNPs) and IR808, achieves the purpose of improving sensitivity. Magnetic beads as a substrate that can be collected and concentrated also make it possible to achieve ultra-sensitive detection. It specifically recognizes PTH protein and forms a "sandwich" structure to capture, separate and enrich trace PTH protein in the specimen eluate. By setting the detection threshold, a new method for intraoperative parathyroid identification that is accurate, economical, convenient, and real-time is constructed.

[0006] In order to achieve the above objectives, the technical solution of the present invention is: a method for rapid detection of PTH by SERS based on an aptamer-antibody sandwich structure, comprising the following steps:

[0007] S1: Preparation of immunomagnetic capture nanoparticles: The PTH capture antibody combination is modified on carboxyl magnetic beads through coupling to form immunomagnetic capture nanoparticles;

[0008] S2: preparation of metal nanosol;

[0009] S3: preparing immune metal signal nanoparticles: mixing the metal nanosol prepared in step S2 with the Raman reporter molecule, and then mixing with an aptamer having a thiol group at one end to obtain immune metal signal nanoparticles;

[0010] S4: preparing a sandwich structure: mixing the immunomagnetic capture nanoparticles and the immune metal signal nanoparticles with the sample to be tested to obtain a sandwich structure;

[0011] S5: Detection of PTH: The precipitate of the sandwich structure is taken out and dropped onto a silicon wafer coated with a gold film for Raman detection to obtain the Raman spectrum result of PTH.

[0012] In a preferred embodiment of the present invention, the capture antibody immunomagnetic beads in step S1 have magnetic properties and are modified with an aptamer that can specifically recognize PTH.

[0013] In a preferred embodiment of the present invention, step S1 comprises taking 100-1000 μl of 1-10 μg / ul carboxyl-modified magnetic beads, adding B&W washing buffer, vortexing to fully suspend the magnetic beads, and magnetically removing the supernatant; resuspending the magnetic beads in B&W washing buffer, adding 8-12 μL of 8-12 mg / mL EDC and NHS in a mixed solution prepared in a molar ratio of 1:1 to activate for 20-40 min, then adding 10-200 μl of 100 μg capture antibody, incubating at 3-5°C for 20-28 h, magnetically removing the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, and then adding 100-5000 μl 0.5-1.5wt% blocking solution was reacted at room temperature for 1-5h to block the remaining binding sites on the surface of the magnetic beads, magnetically separated, washed with B&W washing buffer, and finally redispersed in B&W washing buffer to obtain immunomagnetic beads to capture nanoparticles.

[0014] Further preferably, the particle size of the magnetic beads is 0.5-5 μm, more preferably 1-2 μm.

[0015] Further preferably, the B&W washing buffer comprises 1-200mM pH 5.5-8 buffer solution, 1-10wt% surfactant 1 and 15-100mM inorganic chloride salt; the PBS solution comprises 0.1-0.3M disodium hydrogen phosphate, 0.1-0.3M sodium dihydrogen phosphate, 140-160mM PB; the blocking solution comprises 1-20wt% BSA, 1-200mM pH 5.5-8 PB solution, 15-100mM inorganic chloride salt and 1-10wt% surfactant 2.

[0016] Further preferably, the buffer solution of pH 5.5-8 includes but is not limited to one of phosphate buffer (PB), citric acid-sodium citrate buffer (CPBS), boric acid-borax buffer (BB), and citric acid-borax buffer (CBS); the BSA includes but is not limited to one of casein, glycine, and skim milk; the surfactant 1 includes but is not limited to one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100; the surfactant 2 includes but is not limited to one of Triton-X400, TW-60, TW-80, CHAPS, and TRITON-X100; and the inorganic chloride salt includes but is not limited to one of NaCl, KCl, and MgCl2.

[0017] In a preferred embodiment of the present invention, the metal nanosol in step S3 comprises gold nanosol or silver nanoparticles.

[0018] Further preferably, the gold nanosol is prepared by the following method: taking 200 mL of a 0.01% chloroauric acid solution and heating it to boiling under magnetic stirring; then taking 2-4 mL of a 1% sodium citrate solution and adding it to the boiling chloroauric acid solution, boiling for 20-30 minutes until the reaction is completed, cooling to room temperature, and preparing a nano gold sol; the particle size of the gold nanosol is preferably 30-55 nm, and the ultraviolet absorption wavelength is 526 nm.

[0019] In a preferred embodiment of the present invention, the aptamer in step S3 consists of 40 bases and is modified with thiol-SH, SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'.

[0020] In a preferred embodiment of the present invention, the volume ratio of the metal nanosol to the Raman reporter molecule in step S3 is (50-500):(1-30), the concentration of the Raman reporter molecule is 25-35uM, and the Raman reporter molecule includes but is not limited to one of NBA, IR808, IR795, DTNB, 4-MBA, and 4-NTP.

[0021] In a preferred embodiment of the present invention, the step S3 specifically includes mixing the metal nanosol and the Raman reporter molecule in a volume ratio, incubating at room temperature for 1-30 minutes, centrifuging, discarding the supernatant, re-dispersing in a BB solution, and then mixing and modifying with a 1-10 μM aptamer with a thiol group at one end, and then incubating at -6--2°C for 20-28 hours, and washing with a BB solution to obtain immune metal signal nanoparticles; wherein the concentration of the BB solution is 1.5-2.5 mM.

[0022] In a preferred embodiment of the present invention, step S4 specifically comprises mixing the immunomagnetic bead capture nanoparticles, PTH and immunogold particles, incubating at room temperature for 5-60 minutes, and washing with a PBS-T solution after magnetic separation to obtain a precipitate with a sandwich structure, wherein the PBS-T solution is a mixed solution of PBS solution and TW-20, and the mass fraction of TW-20 in the PBS-T solution is 1-10wt%.

[0023] In a preferred embodiment of the present invention, in step S5, the precipitate is taken out and dropped onto a silicon wafer coated with a gold film or directly onto an aluminum foil for SERS detection.

[0024] In a preferred embodiment of the present invention, the Raman spectrum test in step S5 is performed by a portable Raman spectrometer or other types of Raman spectrometers, and the excitation wavelength is preferably 785 nm.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The detection method of the present invention uses antibodies and aptamers as specific recognition elements, which specifically bind to PTH. On the one hand, it can stabilize the dispersed state of the nanomaterial in the detection system, and on the other hand, it can recognize the target protein with high specificity and high affinity, and has good stability and low preparation cost, which greatly improves the accuracy of detection and makes the detection speed faster.

[0027] 2. The present invention studies the influence of incubation solution, particle modification and actual sample detection process during the detection process, including incubation, washing, magnetic adsorption, detection, etc., and realizes ultra-fast and sensitive detection with results within 10 minutes. The results are highly consistent with the CMIA results used in hospitals, which can provide protection for medical staff performing emergency surgery.

[0028] 3. The detection method of the present invention helps to solve the practical clinical problems of "difficulty in locating the parathyroid glands during surgery, insufficient accuracy of existing detection technologies, high damage, high time and economic costs, and poor repeatability", and provides new ideas and methods for the refined development of thyroid cancer surgery, the improvement of patients' postoperative safety and quality of life, and the promotion of new models of tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Figure 1 Schematic diagram of the detection principle of the present invention, (a) is a schematic diagram of the positional relationship between the thyroid gland and the parathyroid gland; (b) is a schematic diagram of the detection process;

[0031] Figure 2 Schematic diagram of the particle modification process of the present invention and SEM images of the sandwich structures of Example 1 and Example 2, (a) is a schematic diagram of the particle modification process, (b) is a SEM image of the sandwich structure of Example 1, and (c) is a SEM image of the sandwich structure of Example 2;

[0032] Figure 3 Surface enhanced Raman spectra of different PTH concentrations in Example 1 of the present invention, (a) is a surface enhanced Raman spectra of different PTH concentrations, and (b) is a linear fitting diagram of the characteristic peak intensity;

[0033] Figure 4: (a) is a signal distribution diagram of a large number of actual samples detected in Example 1 of the present invention, (b) is a distribution diagram of the results of three large numbers of actual samples detected in this embodiment, (c) is a schematic diagram of the accuracy compared with the CMIA results, (d) is a detailed distribution diagram of the results of a large number of thyroid samples, (e) is a detailed distribution diagram of the results of a large number of peripheral tissue samples, and (f) is a detailed distribution diagram of the results of a large number of parathyroid samples. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0035] A method for rapid detection of PTH by SERS based on an aptamer-antibody sandwich structure comprises the following steps:

[0036] S1: Preparation of immunomagnetic capture nanoparticles: The PTH capture antibody combination is modified on carboxyl magnetic beads through coupling to form immunomagnetic capture nanoparticles;

[0037] S2: preparation of metal nanosol;

[0038] S3: preparing immune metal signal nanoparticles: mixing the metal nanosol prepared in step S2 with the Raman reporter molecule, and then mixing with an aptamer having a thiol group at one end to obtain immune metal signal nanoparticles;

[0039] S4: preparing a sandwich structure: mixing the immunomagnetic capture nanoparticles and the immune metal signal nanoparticles with the sample to be tested to obtain a sandwich structure;

[0040] S5: Detection of PTH: The precipitate of the sandwich structure is taken out and dropped onto a silicon wafer coated with a gold film for Raman detection to obtain the Raman spectrum result of PTH.

[0041] In step S1, the capture antibody immunomagnetic beads capture nanoparticles with magnetism and are modified with aptamers that can specifically recognize PTH.

[0042] The step S1 comprises taking 100-1000 μl of 1-10 μg / ul carboxyl-modified magnetic beads, adding B&W washing buffer, vortexing to fully suspend the magnetic beads, and magnetically aspirating to discard the supernatant; resuspending the magnetic beads in B&W washing buffer, adding 8-12 μL of 8-12 mg / mL EDC and NHS in a mixed solution prepared in a molar ratio of 1:1 to activate for 20-40 min, then adding 10-200 μl of 100 μg capture antibody, incubating at 3-5°C for 20-28 h, magnetically aspirating to discard the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, and then adding 100-5000 μl 0.5-1.5wt% blocking solution was reacted at room temperature for 1-5h to block the remaining binding sites on the surface of the magnetic beads, magnetically separated, washed with B&W washing buffer, and finally redispersed in B&W washing buffer to obtain immunomagnetic beads to capture nanoparticles.

[0043] The particle size of the magnetic beads is 0.5-5 μm, more preferably 1-2 μm.

[0044] The B&W washing buffer includes 1-200mM pH 5.5-8 buffer solution, 1-10wt% surfactant 1 and 15-100mM inorganic chloride salt; the PBS solution includes 0.1-0.3M disodium hydrogen phosphate, 0.1-0.3M sodium dihydrogen phosphate, 140-160mM PB; the blocking solution includes 1-20wt% BSA, 1-200mM pH 5.5-8 PB solution, 15-100mM inorganic chloride salt and 1-10wt% surfactant 2.

[0045] The buffer solution of pH 5.5-8 includes but is not limited to one of phosphate buffer (PB), citric acid-sodium citrate buffer (CPBS), boric acid-borax buffer (BB), and citric acid-borax buffer (CBS); the BSA includes but is not limited to one of casein, glycine, and skim milk; the surfactant 1 includes but is not limited to one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100; the surfactant 2 includes but is not limited to one of Triton-X400, TW-60, TW-80, CHAPS, and TRITON-X100; and the inorganic chloride salt includes but is not limited to one of NaCl, KCl, and MgCl2.

[0046] The metal nanosol in step S3 includes gold nanosol or silver nanosol.

[0047] The gold nano-sol is prepared by the following method: taking 200 mL of a 0.01% chloroauric acid solution and heating it to boiling under magnetic stirring; then taking 2-4 mL of a 1% sodium citrate solution and adding it to the boiling chloroauric acid solution, boiling for 20-30 minutes until the reaction is finished, cooling to room temperature, and preparing the nano-gold sol; the particle size of the gold nano-sol is preferably 30-55 nm, and the ultraviolet absorption wavelength is 526 nm.

[0048] In the step S3, the aptamer consists of 40 bases and is modified with thiol-SH, SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'.

[0049] In step S3, the volume ratio of the metal nanosol to the Raman reporter molecule is (50-500):(1-30), the concentration of the Raman reporter molecule is 25-35uM, and the Raman reporter molecule includes but is not limited to one of NBA, IR808, IR795, DTNB, 4-MBA, and 4-NTP.

[0050] The step S3 specifically includes mixing the metal nanosol and the Raman reporter molecule according to a volume ratio, incubating at room temperature for 1-30 minutes, centrifuging, discarding the supernatant, re-dispersing in a BB solution, and then mixing and modifying with a 1-10 μM aptamer with a thiol group at one end, and then incubating at -6--2°C for 20-28 hours, and washing with a BB solution to obtain immune metal signal nanoparticles, wherein the concentration of the BB solution is 1.5-2.5 mM.

[0051] The step S4 specifically comprises mixing the immunomagnetic bead capture nanoparticles, PTH and immunogold particles, incubating at room temperature for 5-60 minutes, and washing with a PBS-T solution after magnetic separation to obtain a precipitate with a sandwich structure, wherein the PBS-T solution is a mixed solution of PBS solution and TW-20, and the mass fraction of TW-20 in the PBS-T solution is 1-10wt%.

[0052] In step S5, the precipitate is taken out and dropped onto a silicon wafer coated with a gold film or directly dropped onto an aluminum foil for SERS detection.

[0053] The Raman spectrum test in step S5 is performed by a portable Raman spectrometer or other types of Raman spectrometers, and the excitation wavelength is preferably 785 nm.

[0054] Figure 1This is a schematic diagram of the detection principle of the present invention. Immunomagnetic capture nanoparticles, PTH and immunogold signal nanoparticles are mixed and incubated at room temperature for 0.5-5 hours. After magnetic separation, they are washed with PBS-T solution to obtain a sandwich-like structure precipitate, and then a sandwich-like structure is constructed. Then rapid magnetic separation is performed, and a portable Raman spectrometer is used to achieve rapid detection of the new coronavirus.

[0055] Figure 2 (a) Schematic diagram of the particle modification process.

[0056] In the following examples, the capture antibody is provided by Shenzhen Feipeng or Jiangsu Dongkang Company. The capture antibody immunomagnetic beads capture nanoparticles have magnetism and are modified with aptamers that can specifically recognize PTH.

[0057] In the following examples, the B&W washing buffer contains 0.2M PB solution with a pH value of 7.4, 0.4wt% TW-20, 100mM sodium chloride, and the molar ratio of sodium dihydrogen phosphate to sodium dihydrogen phosphate in the PB solution is 1:1; PBS buffer (containing 0.2M disodium hydrogen phosphate and 0.2M sodium dihydrogen phosphate); the blocking solution contains 5wt% BSA, 0.2M PB, 100mM sodium chloride and 1wt% TW-20; the PBS-T solution is a mixed solution of PBS and TW-20, in which the mass fraction of TW-20 is 4wt%.

[0058] In the following examples, the aptamer with a thiol group at one end was provided by Sangon. The aptamer consisted of 40 bases and was modified with a thiol group -SH, SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'.

[0059] Example 1

[0060] (1) Preparation of immunomagnetic capture nanoparticles: Take 200ul of 10ug / ul carboxyl-modified magnetic beads (diameter 1μm), add 1mL B&W washing buffer, vortex to mix to fully suspend the magnetic beads, magnetically remove the supernatant, and then wash twice with 200ul B&W washing buffer; resuspend the above magnetic beads in B&W washing buffer, add 10uL of 10mg / mL EDC and NHS mixed solution prepared in a 1:1 ratio of substance for activation for 30min, then add 100ug capture antibody, incubate at 4°C for 24h, magnetically remove the supernatant and wash with B&W washing buffer, resuspend in B&W washing buffer, then add 200uL blocking solution, react at room temperature for 1h, block the remaining binding sites on the surface of the magnetic beads, magnetically separate, and wash with B&W washing buffer, and finally redisperse in B&W washing buffer to obtain the immunomagnetic capture nanoparticles.

[0061] (2) Preparation of nano-gold sol: 200 mL of 0.01% chloroauric acid solution was heated to boiling under magnetic stirring; then 4.5 mL of 1% sodium citrate solution was added to the boiling chloroauric acid solution, and the reaction was allowed to boil for 30 min. The solution was cooled to room temperature to prepare a nano-gold sol with a particle size of 55 nm and an ultraviolet absorption wavelength of 526 nm.

[0062] (3) Preparation of immunogold signal nanoparticles: 1000ul gold nanosol was mixed with 10ul 30uM Raman reporter IR808, incubated at room temperature for 10min, centrifuged, the supernatant was discarded, and redispersed in BB solution. Then, 3μM aptamer was added and incubated overnight at -4°C. After washing with BB, immunogold signal nanoparticles were obtained. The concentration of BB solution was 1.5-2.5mM.

[0063] (4) Preparation of sandwich structure: 20ul of the prepared immunomagnetic capture nanoparticles, 40ul of the immunogold signal nanoparticles, and 200ul of PTH (100ng / ml, 10ng / ml, 1ng / ml, 0.1ng / ml, 10pg / ml) were mixed and incubated at room temperature for 5min. After magnetic separation, the mixture was washed once with 200uL PBS-T solution to construct a sandwich structure. Figure 2 (b) is the SEM image of the sandwich structure.

[0064] (5) Detection of PTH: The precipitate forming the Meiji sandwich structure is taken out and dropped onto aluminum foil. After it dries, Raman detection is directly performed using a portable Raman spectrometer (laser wavelength 785 nm). The Raman spectrum results for rapid detection of HBsAg can be achieved within 10 minutes.

[0065] Figure 3 The surface enhanced Raman spectra of different PTH concentrations in this example are shown in Figure 1, (a) is the surface enhanced Raman spectra of different PTH concentrations, and (b) is the linear fitting diagram of the characteristic peak intensity. As can be seen from the figure, the linearity is good and the sensitivity can reach 9.5 pg / mL.

[0066] Figure 4 The following are the signal distribution diagrams of a large number of actual samples detected in this embodiment, (a) is a schematic diagram of actual sampling, (b) is a distribution diagram of the results of three large numbers of actual samples detected by this method, (c) is a schematic diagram of accuracy compared with CMIA results, (d) is a detailed distribution diagram of the results of a large number of thyroid samples, (e) is a detailed distribution diagram of the results of a large number of peripheral tissue samples, and (f) is a detailed distribution diagram of the results of a large number of parathyroid samples. It can be seen from the figure that the concentration of the parathyroid gland can be clearly distinguished from other tissues with an accuracy of up to 96%.

[0067] Example 2

[0068] (1) Preparation of immunomagnetic capture nanoparticles: Take 200ul of 10ug / ul carboxyl-modified magnetic beads (diameter 1μm), add 1mL B&W washing buffer, vortex to mix to fully suspend the magnetic beads, magnetically remove the supernatant, and then wash twice with 200ul B&W washing buffer; resuspend the above magnetic beads in B&W washing buffer, add 10uL of 10mg / mL EDC and NHS mixed solution prepared in a 1:1 ratio of substance to activate for 30min, then add 100ug capture antibody, incubate at 4°C for 24h, magnetically remove the supernatant and wash with B&W washing buffer, resuspend in B&W washing buffer, add 200uL blocking solution, react at room temperature for 1h, block the remaining binding sites on the surface of the magnetic beads, magnetically separate, and wash with B&W washing buffer, and finally redisperse in B&W washing buffer to obtain the immunomagnetic capture nanoparticles.

[0069] (2) Preparation of nano silver sol: 200 ml of 1 mM AgNO3 aqueous solution was heated to boiling, and 6 ml of 1% sodium citrate aqueous solution was added. The color of the solution gradually changed from colorless and transparent to milky white with a slight green tint. The solution was kept at a slight boil for 3 h, and the reaction was stopped. The solution was cooled in a water bath. Ag nano sol with a particle size of about 80 nm was obtained and stored away from light. If silver nano sols of different sizes need to be synthesized, the amount of sodium citrate added can be changed.

[0070] (3) Preparation of immunogold signal nanoparticles: 1000ul of silver nanosol was mixed with 10ul of 30uM Raman reporter molecule IR808, incubated at room temperature for 15min, centrifuged, the supernatant was discarded, and the solution was redispersed in BB. 3μM aptamer was added and incubated overnight at -4°C for 24h. After washing with BB, immunogold signal nanoparticles were obtained, wherein the concentration of BB solution was 2mM.

[0071] (4) Preparation of sandwich structure: 20ul of the prepared immunomagnetic capture nanoparticles, 40ul of the immunosilver signal nanoparticles, and 200ul of PTH (100ng / ml, 10ng / ml, 1ng / ml, 0.1ng / ml, 10pg / ml) were mixed and incubated at room temperature for 5min. After magnetic separation, the mixture was washed once with 200Ul of PBS-T solution to construct a sandwich structure. Figure 2 (c) is the SEM image of the sandwich structure.

[0072] (5) Detection of 3 μM aptamer incubated overnight: The precipitate forming the Meiji sandwich structure was taken out and dropped directly onto aluminum foil. After it dried, Raman detection was performed directly using a portable Raman spectrometer (laser wavelength 785 nm), and the Raman spectroscopy results for rapid detection of HBsAg were achieved within 10 minutes.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid detection of PTH by SERS based on an aptamer-antibody sandwich structure, characterized in that: The steps include: S1: Preparation of immunomagnetic capture nanoparticles: The PTH capture antibody combination is modified on carboxyl magnetic beads through coupling to form immunomagnetic capture nanoparticles; S2: preparation of metal nanosol; S3: preparing immune metal signal nanoparticles: mixing the metal nanosol prepared in step S2 with the Raman reporter molecule, and then mixing with an aptamer having a thiol group at one end to obtain immune metal signal nanoparticles; S4: preparing a sandwich structure: mixing the immunomagnetic capture nanoparticles and the immune metal signal nanoparticles with the sample to be tested to obtain a sandwich structure; S5: Detection of PTH: The precipitate of the sandwich structure is taken out and dropped onto a silicon wafer coated with a gold film for Raman detection to obtain the Raman spectrum result of PTH.

2. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: In the step S1, the capture antibody immunomagnetic beads capture nanoparticles with magnetism and are modified with aptamers that can specifically recognize PTH.

3. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: The step S1 includes taking 100-1000 μl of carboxyl-modified magnetic beads with a concentration of 1-10 μg / ul, adding B&W washing buffer, vortexing to fully suspend the magnetic beads, and magnetically removing the supernatant; resuspending the above magnetic beads in B&W washing buffer, adding 8-12 μL of 8-12 mg / mL EDC and NHS in a mixed solution prepared according to a molar ratio of 1:1 to activate for 20-40 minutes, then adding 10-200 μl 100 μg capture antibody, incubating at 3-5°C for 20-28 hours, magnetically removing the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, and then adding 100-5000 μl blocking solution, reacting at room temperature for 1-5 hours to block the remaining binding sites on the surface of the magnetic beads, magnetically separating, and washing with B&W washing buffer, and finally redispersing in B&W washing buffer to obtain immunomagnetic beads to capture nanoparticles.

4. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 3, characterized in that: The B&W washing buffer solution includes 1-200 mM pH 5.5-8 buffer solution, 1-10 wt % surfactant 1 and 15-100 mM inorganic chloride salt; the PBS solution includes 0.1-0.3 M disodium hydrogen phosphate, 0.1-0.3 M Sodium dihydrogen phosphate, 140-160 mM PB; the blocking solution includes 1-20 wt % BSA, 1-200 mM PB solution with pH 5.5-8, 15-100 mM inorganic chloride salt and 1-10 wt % surfactant 2.

5. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 4, characterized in that: The buffer solution includes but is not limited to one of PB, CPBS, BB, and CBS; the BSA includes but is not limited to one of casein, glycine, and skim milk; the surfactant 1 includes but is not limited to one of TW-20, TW-60, TW-80, CHAPS, and TRITON-X100; the surfactant 2 includes but is not limited to one of Triton-X400, TW-60, TW-80, CHAPS, and TRITON-X100; and the inorganic chloride salt includes but is not limited to one of NaCl, KCl, and MgCl2.

6. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: The aptamer in step S3 is composed of 40 bases and modified with thiol-SH, SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'.

7. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: In step S3, the volume ratio of the metal nanosol to the Raman reporter molecule is (50-500):(1-30), the concentration of the Raman reporter molecule is 25-35uM, and the Raman reporter molecule includes but is not limited to one of NBA, IR808, IR795, DTNB, 4-MBA, and 4-NTP.

8. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: The step S3 specifically includes mixing the metal nanosol and the Raman reporter molecule according to a volume ratio, incubating at room temperature for 1-30 minutes, centrifuging, discarding the supernatant, re-dispersing in a BB solution, and then mixing and modifying with a 1-10 μM aptamer with a thiol group at one end, and then incubating at -6--2°C for 20-28 hours, and washing with a BB solution to obtain immune metal signal nanoparticles, wherein the concentration of the BB solution is 1.5-2.5 mM.

9. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: The step S4 specifically includes mixing the immunomagnetic bead capture nanoparticles, PTH and immunogold particles, incubating at room temperature for 5-60 minutes, and washing with a PBS-T solution after magnetic separation to obtain a precipitate with a sandwich structure, wherein the PBS-T solution is a mixed solution of PBS solution and TW-20, and the mass fraction of TW-20 in the PBS-T solution is 1-10wt%.

10. The method for rapid detection of PTH by SERS based on aptamer-antibody sandwich structure according to claim 1, characterized in that: The Raman spectrum test in step S5 is performed by a portable Raman spectrometer or other types of Raman spectrometers, and the excitation wavelength is preferably 785 nm.

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