A rapid method for detecting PTH based on SERS using an aptamer-antibody sandwich structure

Abstract

This invention belongs to the field of biodetection technology, specifically disclosing a method for rapid detection of PTH using SERS based on an aptamer-antibody sandwich structure. The method for rapid detection of PTH using SERS based on an aptamer-antibody sandwich structure is characterized by the following steps: preparing immunomagnetic bead-capturing nanoparticles; preparing metal nanosol; preparing immunometal signal nanoparticles; preparing a sandwich structure: mixing the immunomagnetic bead-capturing nanoparticles and immunometal signal nanoparticles with the sample to be tested to obtain a sandwich structure; and detecting PTH. The detection method of this invention uses antibodies and aptamers as specific recognition elements, specifically binding to PTH. On the one hand, it can stabilize the dispersion state of nanomaterials in the detection system; on the other hand, it can recognize the target protein with high specificity and high affinity, and has good stability and low preparation cost, greatly improving the accuracy of detection and accelerating the detection speed.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for rapid detection of PTH based on an aptamer-antibody sandwich structure using SERS. Background Technology

[0002] In recent years, the incidence and mortality rates of thyroid cancer have been on a continuous upward trend. Surgery is the primary 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 postoperative disease staging and risk stratification. Although surgery can remove the tumor and potential lesions within normal tissue to the greatest extent possible, due to the close anatomical relationship between the parathyroid glands and the thyroid gland, thyroid surgery significantly increases the risk of parathyroid gland damage, leading to hypoparathyroidism (HP) and hypocalcemia. There are two pairs of parathyroid glands, located on the posterior surface of the left and right lobes of the thyroid gland in the neck, and they are light brownish-yellow in color and about the size of a soybean. The incidence of temporary and permanent HP after total thyroidectomy is 10%–60% and 1%–4%, respectively, seriously affecting the postoperative quality of life of patients. Accurate, economical, convenient, and real-time dynamic identification and complete preservation of the parathyroid glands are key to protecting their function and preventing postoperative complications. The risk of parathyroid gland damage is so high because the color and shape of the parathyroid glands are similar to those of surrounding fat granules and lymph nodes, making visual identification based on experience unreliable and requiring a high level of skill from the surgeon. Therefore, auxiliary methods such as autologous transplantation, contrast agents, and lateral flow biosensors (LFB) have been widely developed. While autologous transplantation can avoid permanent parathyroid hormone (HP), it can still cause temporary HP postoperatively and carries the risk of false positives and false negatives. Methylene blue positive contrast imaging can cause adverse reactions such as neurotoxicity, gastrointestinal reactions, and hypoxic cyanosis, and has a high false positive rate. Nanocarbon negative contrast imaging is prone to missing inferior parathyroid glands and can easily contaminate the surgical field, making parathyroid gland identification difficult. While lateral flow biosensors (LFBs) are simple and efficient for rapid measurement of parathyroid hormone (PTH) in tissues, sampling methods include needle aspiration, clamp sampling, and tissue homogenization. Needle aspiration causes the least damage to the parathyroid glands, but LFB sensitivity is slightly insufficient. Tissue homogenization and clamp sampling methods, on the other hand, cause greater damage to the parathyroid glands. Therefore, given the current situation, there is an urgent need to develop a new technology to provide a new platform for intraoperative protection of the parathyroid glands.

[0003] Surface-enhanced Raman spectroscopy (SERS) offers advantages such as non-destructive testing, resistance to water interference, high resolution and sensitivity, and simple and rapid analysis. Utilizing the coupling enhancement effect of gold nanoparticles, it can amplify the Raman signal of nearby molecules by millions of times. Combined with Raman resonance molecules, it can even achieve single-molecule detection sensitivity. It has been widely applied in surface science, materials science, biomedicine, drug analysis, food safety, and environmental monitoring, making it a highly promising trace analysis technique. Current research utilizes SERS to detect PTH. For example, Juang et al. prepared AgNPs@mrGO active substrates for rapid and sensitive label-free PTH detection, achieving a sensitivity of 0.2 ng / mL. Alejandro et al. prepared an immunogold colloidal test strip using a sandwich structure principle, employing a double-antibody sandwich method to achieve specific recognition, with a detection limit of 3.45 ng / mL. While the sensitivity is still relatively poor and falls far short of the detection limit required for clinical serum samples, it demonstrates the feasibility of the sandwich method for PTH detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for rapid detection of PTH based on an aptamer-antibody sandwich structure in SERS. It utilizes the principle of specific recognition between antibody aptamers and PTH antigens, and incubates PTH-capturing antibody-based immunomagnetic beads with PTH-modified immunometal signal nanoparticles to form a sandwich structure.

[0005] Compared to the large steric hindrance of traditional dual-antibody modules, aptamer-antibody sandwiches, with their low Gibbs free energy and low dissociation constant, exhibit higher recognition efficiency. Nucleic acid aptamers offer significant advantages such as high affinity, low steric hindrance, and batch-to-batch stability, reducing false positives and false negatives caused by dual-antibody modules. This invention constructs a PTH detection platform based on SERS technology using an aptamer-antibody sandwich model. It utilizes highly sensitive nucleic acid aptamers and antibodies to specifically recognize PTH proteins, while employing the Raman signal molecule IR808, which resonates with the incident excitation wavelength of 785 nm, as a probe. A gold-terminated aptamer, specifically aptamer number 14 invented by Yanghoon Kim and Yoonshin Park, is combined with gold nanoparticles (AuNPs) and IR808 to construct a SERRS tag, thereby enhancing sensitivity. Magnetic beads, as a collectable and concentrated substrate, also enable ultrasensitive detection. This method specifically recognizes PTH protein and forms a "sandwich" structure to capture, separate, and enrich trace amounts of PTH protein in the sample eluent. By setting a detection threshold, a new, accurate, economical, convenient, and real-time dynamic method for intraoperative parathyroid gland identification is constructed.

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

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

[0008] S2: Preparation of metal nanosols;

[0009] S3: Preparation of immune metal signaling nanoparticles: The metal nanosol prepared in step S2 is mixed with Raman reporter molecules, and then mixed with an aptamer with a thiol group at one end to obtain immune metal signaling nanoparticles.

[0010] S4: Preparation of sandwich structure: Immunomagnetic beads and immunometal signal nanoparticles are mixed with the sample to be tested to obtain a sandwich structure.

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

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

[0013] In a preferred embodiment of the present invention, step S1 includes taking 100-1000 μl of carboxyl-modified magnetic beads with a concentration of 1-10 μg / μl, adding B&W washing buffer, vortexing to fully suspend the magnetic beads, and magnetically discarding the supernatant; resuspending the magnetic beads in B&W washing buffer, adding 8-12 μL of a mixed solution of 8-12 mg / mL EDC and NHS 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 discarding the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, and then adding 100-5000 μl of [the solution is missing here]. The remaining binding sites on the surface of the magnetic beads were blocked by reacting with 0.5-1.5 wt% blocking solution at room temperature for 1-5 h. The beads were then magnetically separated, washed with B&W washing buffer, and finally redispersed in B&W washing buffer to obtain immunomagnetic bead capture nanoparticles.

[0014] More preferably, the magnetic beads have a particle size of 0.5-5 μm, and more preferably 1-2 μm.

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

[0016] More preferably, the buffer solution with pH 5.5-8 includes, but is not limited to, one of phosphate buffer (PB), citrate-sodium citrate buffer (CPBS), borate-borax buffer (BB), and citrate-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 includes gold nanosol or silver nanoparticles.

[0018] More preferably, the gold nanosol is prepared by the following method: 200 mL of 0.01% chloroauric acid solution is heated to boiling under magnetic stirring; then 2-4 mL of 1% sodium citrate solution is added to the boiling chloroauric acid solution, and the reaction is stopped after boiling for 20-30 minutes. The solution is then cooled to room temperature to prepare the gold nanosol; 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 a thiol group -SH,SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'.

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

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

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

[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 dropped onto an aluminum foil for SERS detection.

[0024] In a preferred embodiment of the present invention, the Raman spectroscopy test in step S5 is performed using a portable Raman spectrometer or other type of Raman spectrometer, 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 dispersion state of nanomaterials in the detection system, and on the other hand, it can recognize target proteins with high specificity and high affinity. It also has good stability, low preparation cost, and greatly improves the accuracy of detection and speeds up the detection process.

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

[0028] 3. The detection method of the present invention helps to solve the practical clinical problems of "difficulty in intraoperative parathyroid gland localization, insufficient accuracy of existing detection technology, high invasiveness, 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 postoperative safety and quality of life of patients, and the promotion of new models of tumor diagnosis and treatment. Attached Figure Description

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

[0030] Figure 1 The following is a 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 The images show schematic diagrams of the particle modification process of the present invention and SEM images of the sandwich structure of Examples 1 and 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 The following are surface-enhanced Raman spectra of different PTH concentrations in Example 1 of the present invention: (a) is a surface-enhanced Raman spectrum of different PTH concentrations, and (b) is a linear fitting diagram of the characteristic peak intensities.

[0033] Figure 4The following are signal distribution diagrams for a large number of actual samples detected in Embodiment 1 of the present invention: (a) is a schematic diagram of actual sampling; (b) is a distribution diagram of the results of detecting a large number of actual samples in this embodiment; (c) is a schematic diagram of the accuracy comparison with CMIA results; (d) is a detailed distribution diagram of a large number of thyroid samples; (e) is a detailed distribution diagram of a large number of peripheral tissue samples; and (f) is a detailed distribution diagram of a large number of parathyroid gland samples. Detailed Implementation

[0034] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

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

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

[0037] S2: Preparation of metal nanosols;

[0038] S3: Preparation of immune metal signaling nanoparticles: The metal nanosol prepared in step S2 is mixed with Raman reporter molecules, and then mixed with an aptamer with a thiol group at one end to obtain immune metal signaling nanoparticles.

[0039] S4: Preparation of sandwich structure: Immunomagnetic beads and immunometal signal nanoparticles are mixed with the sample to be tested to obtain a sandwich structure.

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

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

[0042] Step S1 includes taking 100-1000 μl of carboxyl-modified magnetic beads with a concentration of 1-10 μg / μl, adding B&W washing buffer, vortexing to fully suspend the magnetic beads, and magnetically discarding the supernatant; resuspending the magnetic beads in B&W washing buffer, adding 8-12 μL of a mixed solution of 8-12 mg / mL EDC and NHS at 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℃ for 20-28 h, magnetically discarding the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, and then adding 100-5000 μl of... The remaining binding sites on the surface of the magnetic beads were blocked by reacting with 0.5-1.5 wt% blocking solution at room temperature for 1-5 h. The beads were then magnetically separated, washed with B&W washing buffer, and finally redispersed in B&W washing buffer to obtain immunomagnetic bead capture nanoparticles.

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

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

[0045] The buffer solution with pH 5.5-8 includes, but is not limited to, one of phosphate buffer (PB), citrate-sodium citrate buffer (CPBS), borate-borax buffer (BB), and citrate-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. 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 nanosol is prepared by the following method: 200 mL of 0.01% chloroauric acid solution is heated to boiling under magnetic stirring; then 2-4 mL of 1% sodium citrate solution is added to the boiling chloroauric acid solution. After boiling for 20-30 minutes, the reaction is completed and cooled to room temperature to prepare the gold nanosol. The preferred particle size of the gold nanosol is 30-55 nm, and the ultraviolet absorption wavelength is 526 nm.

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

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

[0050] Step S3 specifically includes mixing metal nanosol with Raman reporter molecules at a volume ratio, incubating at room temperature for 1-30 min, centrifuging, discarding the supernatant, redispersing in BB solution, then mixing and modifying with 1-10 μM of aptamers with a thiol group at one end, and then incubating at -6 to -2℃ for 20-28 h. After washing with BB solution, immune metal signal nanoparticles are obtained, wherein the concentration of BB solution is 1.5-2.5 mM.

[0051] Step S4 specifically includes mixing immunomagnetic beads to capture nanoparticles, PTH and immunogold particles, incubating at room temperature for 5-60 min, and washing with PBS-T solution after magnetic separation to obtain a sandwich-structured precipitate. The PBS-T solution is a mixture of PBS solution and TW-20, and the mass fraction of TW-20 in the PBS-T solution is 1-10 wt%.

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

[0053] In step S5, the Raman spectroscopy test is performed using a portable Raman spectrometer or other types of Raman spectrometers, with the excitation wavelength preferably being 785 nm.

[0054] Figure 1The diagram illustrates the detection principle of this invention. Immunomagnetic beads, PTH, and immunogold signaling nanoparticles are mixed and incubated at room temperature for 0.5-5 hours. After magnetic separation, the mixture is washed with PBS-T solution to obtain a sandwich-structured precipitate. Rapid magnetic separation is then performed, and PTH is detected quickly using a portable Raman spectrometer.

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

[0056] In the following examples, the capture antibody was provided by Shenzhen Feipeng or Jiangsu Dongkang Company. The capture antibody immunomagnetic beads capture nanoparticles that are magnetic and 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 of 7.4, 0.4wt% TW-20, and 100mM sodium chloride. The molar ratio of sodium dihydrogen phosphate to disodium hydrogen phosphate in the PB solution is 1:1. The PBS buffer contains 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 mixture of PBS and TW-20, wherein the mass fraction of TW-20 is 4wt%.

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

[0059] Example 1

[0060] (1) Preparation of immunomagnetic bead capture nanoparticles: Take 200 μL of carboxyl-modified magnetic beads (1 μm in diameter) with a concentration of 10 μg / μL and add 1 mL of B&W washing buffer. Vortex mix to fully suspend the magnetic beads. Magnetically remove the supernatant and wash twice with 200 μL of B&W washing buffer. Resuspend the magnetic beads in B&W washing buffer and add 10 μL of a mixed solution of 10 mg / mL EDC and NHS prepared in a 1:1 molar ratio for activation for 30 min. Then add 100 μg of capture antibody and incubate at 4 °C for 24 h. Magnetically remove the supernatant and wash with B&W washing buffer. Resuspend in B&W washing buffer and add 200 μL of blocking solution. React at room temperature for 1 h to block the remaining binding sites on the surface of the magnetic beads. Magnetic separation and washing with B&W washing buffer are performed. Finally, redisperse in B&W washing buffer to obtain the immunomagnetic bead capture nanoparticles.

[0061] (2) Preparation of gold nanoparticle sol: Take 200 mL of 0.01% chloroauric acid solution and heat it to boiling under magnetic stirring; then take 4.5 mL of 1% sodium citrate solution and add it to the boiling chloroauric acid solution. After boiling for 30 min, the reaction is completed and cooled to room temperature to prepare gold nanoparticle sol with a particle size of 55 nm and an ultraviolet absorption wavelength of 526 nm.

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

[0063] (4) Preparation of sandwich structure: 20 μL of the prepared immunomagnetic bead capture nanoparticles and 40 μL of immunogold signaling nanoparticles were mixed and then mixed with 200 μL of PTH (100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 10 pg / ml). After incubation at room temperature for 5 min, magnetic separation was performed, followed by washing once with 200 μL of PBS-T solution to construct a sandwich structure. Figure 2 (b) is a SEM image of the sandwich structure.

[0064] (5) Detection of PTH: The precipitate that forms the Meiji sandwich structure is taken out and dropped onto aluminum foil. After drying, Raman detection is performed directly using a portable Raman spectrometer (laser wavelength 785nm). The Raman spectrum results of HBsAg are quickly detected within 10 minutes.

[0065] Figure 3 The images show surface-enhanced Raman spectra at different PTH concentrations in this embodiment. (a) shows the surface-enhanced Raman spectra at different PTH concentrations, and (b) shows the linear fitting graph of the characteristic peak intensities. As can be seen from the figures, the linearity is good, and the sensitivity reaches 9.5 pg / mL.

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

[0067] Example 2

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

[0069] (2) Preparation of silver nanosol: 200 ml of 1 mM AgNO3 aqueous solution was heated to boiling, and 6 ml of 1% sodium citrate aqueous solution was added. The solution color gradually changed from colorless and transparent to milky white with a slight green tinge. The mixture was kept at a gentle boil for 3 hours, then the reaction was stopped and cooled in a water bath. This yielded Ag nanosol with a particle size of approximately 80 nm, which was stored away from light. To synthesize silver nanosol of different sizes, simply change the amount of sodium citrate added.

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

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

[0072] (5) Detection of 3μM aptamer incubation overnight: The precipitate that formed the Meiji sandwich structure was taken out and dropped directly onto aluminum foil. After drying, Raman detection was performed directly using a portable Raman spectrometer (laser wavelength 785nm). The Raman spectrum results of HBsAg were quickly detected within 10 minutes.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid detection of PTH based on SERS using an aptamer-antibody sandwich structure, characterized in that, Includes the following steps: S1: Preparation of immunomagnetic bead capture nanoparticles: PTH capture antibody combination is modified onto carboxyl magnetic beads through coupling to form immunomagnetic bead capture nanoparticles; S2: Preparation of metal nanosols; S3: Preparation of immune metal signaling nanoparticles: The metal nanosol prepared in step S2 is mixed with Raman reporter molecules, and then mixed with an aptamer with a thiol group at one end to obtain immune metal signaling nanoparticles; the aptamer consists of 40 bases and is modified with thiol-SH, SH-C6-5'-CACGAAAGATCAATTACATGCTTATCATTTTATTCATTGG-3'; S4: Preparation of sandwich structure: Immunomagnetic bead capture nanoparticles and immunometal signal nanoparticles are mixed with the sample to be tested to obtain sandwich structure. S5: Detection of PTH: The sandwich-structured precipitate is taken out and dropped onto a silicon wafer coated with a gold film for Raman detection to obtain the Raman spectrum of PTH.

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

3. The method for rapid detection of PTH based on an aptamer-antibody sandwich structure according to claim 1, characterized in that, Step S1 includes taking 100-1000 µl of carboxyl-modified magnetic beads with a concentration of 1-10 µg / µl, 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 a mixed solution of 8-12 mg / mL EDC and NHS in a 1:1 molar ratio to activate for 20-40 min, then adding 10-200 μl of 100 μg capture antibody, incubating at 3-5℃ for 20-28 h, magnetically removing the supernatant, washing with B&W washing buffer, resuspending in B&W washing buffer, then adding 100-5000 μl of blocking solution, reacting at room temperature for 1-5 h to block the remaining binding sites on the surface of the magnetic beads, magnetic separation, washing with B&W washing buffer, and finally redispersing in B&W washing buffer to obtain immunomagnetic bead capture nanoparticles.

4. The method for rapid detection of PTH based on an aptamer-antibody sandwich structure using SERS as described in claim 3, characterized in that, The B&W washing buffer comprises 1-200 mM pH 5.5-8 buffer solution, 1-10 wt% surfactant 1, and 15-100 mM inorganic chloride salt; the PBS solution comprises 0.1-0.3 M disodium hydrogen phosphate, 0.1-0.3 M... Sodium dihydrogen phosphate, 140-160 mM PB; the blocking solution comprises 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 based on an 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 based on an aptamer-antibody sandwich structure according to claim 1, characterized in that, In step S3, the volume ratio of metal nanosol to Raman reporter molecules is (50-500):(1-30), the concentration of Raman reporter molecules is 25-35 μM, and the Raman reporter molecules include, but are not limited to, one of NBA, IR808, IR795, DTNB, 4-MBA, and 4-NTP.

7. The method for rapid detection of PTH based on an aptamer-antibody sandwich structure using SERS as described in claim 1, characterized in that, Step S3 specifically includes mixing metal nanosol with Raman reporter molecules at a volume ratio, incubating at room temperature for 1-30 min, centrifuging, discarding the supernatant, redispersing in BB solution, then mixing and modifying with 1-10 µM aptamers with a thiol group at one end, incubating at -6 to -2℃ for 20-28 h, and washing with BB solution to obtain immune metal signal nanoparticles, wherein the concentration of BB solution is 1.5-2.5 mM.

8. The method for rapid detection of PTH based on an aptamer-antibody sandwich structure using SERS as described in claim 1, characterized in that, Step S4 specifically includes mixing immunomagnetic beads to capture nanoparticles, PTH and immunogold particles, incubating at room temperature for 5-60 min, and washing with PBS-T solution after magnetic separation to obtain a sandwich-structured precipitate. The PBS-T solution is a mixture of PBS solution and TW-20, and the mass fraction of TW-20 in the PBS-T solution is 1-10 wt%.

9. The method for rapid detection of PTH based on an aptamer-antibody sandwich structure using SERS as described in claim 1, characterized in that, In step S5, the Raman spectroscopy test is performed using a portable Raman spectrometer or other types of Raman spectrometer, with an excitation wavelength of 785 nm.

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