Metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA, kit and preparation method and application of metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure

Abstract

The invention discloses a metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA, a kit and a preparation method and application of the metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure. A PSA capture magnetic bead, a detection sample and a metal nanoparticle detection probe form a sandwich structure, the PSA capture magnetic bead is obtained by biologically coupling a PSA capture antibody and a magnetic bead, an antigen is PSA, and the metal nanoparticle detection probe is 20-200nm gold / silver nanoparticles. The kit has high sensitivity and high specificity when being used for detecting the PSA, is simple to operate and rapid in detection, can detect the marker PSA of the prostatic cancer within about 10 minutes, and provides an important basis for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of detection reagents, and in particular to a metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA, a kit, and a preparation method and use thereof. Background Art

[0002] Cancer is a very important social public health security and economic issue in the 21st century. Prostate cancer is one of the common malignant tumors in men and one of the main causes of cancer death. Early diagnosis and accurate screening of prostate cancer are crucial to improving patient survival. Prostate-specific antigen (PSA) is a kallikrein-like serine protease that has been approved by the U.S. Food and Drug Administration (FDA) as a biomarker associated with the diagnosis and prognosis of prostate cancer. The detection of PSA levels in serum is the most commonly used detection method in hospitals. So far, various detection methods have been used for direct detection of PSA, such as fluorescent immunoassay, photoelectrochemical immunosensor, electrochemiluminescence, mass spectrometry, amperometric immunoassay, etc., but traditional detection methods have problems with insufficient sensitivity and specificity.

[0003] Surface-enhanced Raman scattering (SERS) is becoming increasingly popular in disease detection technology due to its non-destructive, ultra-sensitive, reliable and rapid detection capabilities. It is a surface-sensitive technology that can enhance the Raman scattering efficiency of molecules adsorbed on rough nanostructures, usually nanomaterials such as silver or gold. It can increase the sensitivity of analytical methods by several orders of magnitude (ranging from 10 4 to 10 11 ), and shows great potential in single-molecule assays. Immunoassay relies on the specific interaction between antibodies and complementary antigens and is a highly specific method for directly or indirectly detecting antigens. It has become a powerful analytical tool for clinical diagnosis, biochemical analysis, and environmental monitoring. By combining the advantages of SERS, such as high sensitivity and fast detection speed, with the advantages of high affinity and strong specificity between antigen and antibody. The signal is amplified by the enrichment of magnetic beads to improve the sensitivity of detection. Although a variety of Raman-active metal nanoparticles have been widely developed, such as gold and silver nanorods, gold and silver nanocubes, and gold and silver interstitial nanoflowers, etc., it is still an urgent need for research to find and design ideal SERS tags that can have simple preparation, high stability, and repeatability. As a highly sensitive spectroscopic technique, SERS combined with the antigen-antibody magnetic bead sandwich method can significantly improve the detection sensitivity and specificity of prostate cancer markers. It will be expected to achieve trace determination of PSA by combining highly sensitive SERS tags with specific immunoassays. Summary of the invention

[0004] The object of the present invention is to provide a metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA.

[0005] To achieve the above-mentioned purpose, the present invention provides a metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA, characterized in that the sandwich structure is formed by PSA capture magnetic beads, detection samples, and metal nanoparticle detection probes, wherein the PSA capture magnetic beads are obtained by bio-coupling of PSA capture antibodies and magnetic beads, the antigen is PSA, and the metal nanoparticle detection probes are 20-200nm gold / silver nanoparticles.

[0006] Further, the preparation method comprises the following steps:

[0007] S1. Preparation of PSA capture beads:

[0008] The PSA capture antibody is mixed with the carboxyl-activated magnetic beads, and the PSA capture antibody is bound to the carboxyl magnetic beads through biocoupling, and finally the excess unbound active sites are blocked to form PSA capture magnetic beads;

[0009] S2. Preparation of metal nanoparticle detection probes:

[0010] (1) Preparing metal particle sol: taking out the metal sol with uniform morphology and washing it once with pure water by centrifugation, thus obtaining the metal particle sol with surface plasmon resonance effect;

[0011] (2) adding the signal molecule to the sol obtained in step (1) to react, allowing the two to combine, and then adding the PSA detection antibody for incubation reaction to obtain the immunogold or silver signal nanoparticles, i.e., metal nanoparticle detection probes;

[0012] The S1 and S2 are not sequential and can be performed simultaneously;

[0013] S3. Prepare the antibody gold probe-antigen-antibody magnetic bead sandwich structure: mix the PSA capture magnetic beads, patient test serum samples, and metal nanoparticle detection probes to obtain a sandwich structure.

[0014] Furthermore, in step S1, the particle size of the magnetic beads after carboxyl activation is 1 micron.

[0015] Furthermore, in step S1, the carboxyl-activated magnetic beads are activated before being mixed with the PSA capture antibody. The activation is performed by resuspending the carboxyl-activated magnetic beads with PBS, and then adding them to a mixed solution of 10 mg / mL EDC and NHS prepared in a 1:1 ratio of the amounts of the substances for activation for 30 minutes.

[0016] Furthermore, in step S2 (1), the particle size of the metal particle sol is 20-200 nm.

[0017] Furthermore, in step S2 (1), the method for preparing the metal sol is to heat the solution containing metal ions to boiling under stirring; then add a reducing solution (such as sodium citrate or ascorbic acid, but not limited to these two) to the boiling solution containing metal ions for reaction, and cool to room temperature after the reaction to prepare the metal sol.

[0018] Furthermore, the molar concentration ratio of the signal molecule to the metal particles is (20-100):1;

[0019] Optionally, the reaction conditions in step (2) are incubation at room temperature with stirring for 1-15 min, centrifugation, discarding the supernatant and redispersing in a PBS solution.

[0020] The present invention also provides a kit for detecting PSA, characterized in that it contains the metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure.

[0021] The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure of the present invention is a prostate cancer detection technology based on SERS and the antigen-antibody magnetic bead sandwich method. The purpose of this technology is to overcome the defects of the prior art and provide a method for rapid and ultra-sensitive detection of PSA based on SERS.

[0022] Signal molecules with large Raman cross-sections and resonant with Raman lasers are selected to provide positive signals. Magnetic beads and gold nanoparticles are used to capture antibody support materials and detect antibody-coupled sensor probes, respectively. The SERS substrate uses magnetic beads with surface-modified carboxyl groups that are easy to collect and concentrate. The surface-modified carboxyl groups can be coupled with antibodies through activation. The magnetic attraction of the magnetic beads can achieve the purpose of concentrating and amplifying signals. In addition, as three-dimensional materials, magnetic beads can take advantage of their large surface area to increase the loading rate of magnetic beads, improve incubation efficiency, and reduce incubation time. Gold nanoparticles provide the role of enhancement and loading antibodies, provide enhanced SERS signals for the system, and improve sensitivity.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The method of the present invention uses antibodies as specific recognition elements and specifically binds to PSA. 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 speeds up detection.

[0025] 2. The present invention studies the effects of incubation solution, particle modification and actual sample detection 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 ECL results used in the hospital.

[0026] Beneficial effects: The method of the present invention has high sensitivity and high specificity, is simple to operate and can detect quickly. The prostate cancer marker PSA can be detected in about 10 minutes, providing an important basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Flowchart of SERS detection of prostate cancer markers.

[0028] Figure 2 : Figure 1 of the immunogold signal nanoparticles of the present invention.

[0029] Figure 3 : SEM image of the magnetic bead sandwich structure of the present invention.

[0030] Figure 4 : Raman signal screening diagram of fourteen antibody pairings of the present invention.

[0031] Figure 5 : The present invention preferably further pairs the four antibodies for Raman signal screening.

[0032] Figure 6 : Screening diagram of Raman signals of six magnetic beads of different models and manufacturers in the present invention.

[0033] Figure 7 : The pH of the buffer solution of the present invention explores the Raman signal diagram.

[0034] Figure 8 : The Raman signal diagram of the buffer PB concentration of the present invention.

[0035] Fig. 9 : Surface enhanced Raman spectra of different PSA concentrations of the present invention.

[0036] Fig.10 : Raman signal diagram of different antigen specificity verification of the present invention.

[0037] Fig.11 : Comparison chart of 75 actual serum samples and hospital ECL results in Example 6. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0039] Figure 1 FIG. 1 is a schematic diagram of the detection principle of the present invention. The detection method of the present invention utilizes the specific recognition principle between the antigen and antibody of PSA. After the immunomagnetic beads of the capture antibody of PSA specifically recognize PSA, they are incubated with the immunometallic signal nanoparticles of the detection antibody modified with PSA to form a sandwich structure. Figure 3 .

[0040] Example 1 Antibody Screening Experiment

[0041] Antibody screening is a key step in the sandwich structure experiment. In addition to being able to specifically and efficiently bind to PSA, the capture antibody modified on the magnetic beads and the detection antibody modified on the probe should also target different epitopes of PSA to avoid competitive inhibition between the two. Antibody specificity selection and optimization are key difficulties in the work. Although the principles of the sandwich structure are similar, the antigens targeted have unique structural and epitope characteristics. Antigen-antibody binding is highly specific, and the structural and epitope characteristics of different antigens vary significantly. For PSA detection, there are many types of antibodies available on the market, but it is not easy to accurately screen a pair of capture antibodies and detection antibodies that can strongly bind to PSA and work well together in the sandwich structure. It is necessary to characterize and evaluate antibodies against the same epitope from different companies or antibodies against different epitopes of PSA from the same company, including studies on antibody affinity, specific binding, cross-reactivity, and other aspects. For example, many antibodies that seem to have the ability to bind to PSA cannot produce accurate and reliable detection signals when actually constructing a sandwich structure due to steric hindrance or interference with the interaction with magnetic beads and markers. The inventors simulated the interaction between antibodies and PSA in a sandwich structure system, and after a large number of experimental screening and optimization combinations, they finally determined the appropriate antibody pair. This process consumed a lot of manpower, material resources and time, and was far from being covered by simple selection and replacement. The combination of different antibodies has a great influence on the strength of antigen binding (represented by Raman intensity here).

[0042] Antibodies: For different PSA epitopes, the inventors selected 14 different antibodies: A2052 (KLK3 Rabbit pAb from Abclonal), A4355 (PSA / KLK3 Rabbit mAb from Abclonal), AG1127 (PSA / KLK3 Mouse Monoclonal Antibody from Biotech), AG1131 (PSA / KLK3 Rabbit Polyclonal Antibody from Biotech), HA114-1M (mouse anti-human PSA monoclonal antibody from Shenzhen Heavy Chain Biotech), HA114-2M (mouse anti-human PSA monoclonal antibody from Shenzhen Heavy Chain Biotech), AG8656 (BeyoIHC from Biotech), TM PSA Rabbit Monoclonal Antibody), MS20032UD (PSA Mouse mAb, unconjugated, detector from Chengdu Zhengneng Biotechnology), MS20032UC (PSA Mouse mAb, capture from Chengdu Zhengneng Biotechnology), RP01540 (PSA / KLK3(D6B1)Xp@Rabbit mAb from Cell Signaling), 14119(from Cell Signaling), 75723(PSA / KLK3(D2A8)Rabbit mAb(BSA and Azide Free) from Cell Signaling), 35198(PSA / KLK3(D6B1)XP@RabbitmAb(BSA and Azide Free) from Cell Signaling), 76325(Total PSA / KLK3 MatchedAntibody Pair from Cell Signaling); there are differences in the antibodies used by different companies. The antibody was diluted according to the optimal usage method recommended in the instructions, such as 100-fold dilution, 500-fold dilution, 1000-fold dilution, and 2000-fold dilution.

[0043] Preparation steps:

[0044] S1. Preparation of PSA capture beads:

[0045] The PSA capture antibody (i.e., the diluted antibody) was mixed with the carboxyl-activated magnetic beads (1 μm carboxyl magnetic beads purchased from Thermo Fisher Scientific), and the PSA capture antibody was bound to the carboxyl magnetic beads by biocoupling. Finally, the excess unbound active sites were blocked with a BSA solution to form PSA capture magnetic beads; these were sequentially recorded as 1-14 (i.e., 14 antibodies were modified with magnetic beads);

[0046] S2. Preparation of metal nanoparticle detection probes:

[0047] (1) Preparation of metal particle sol: Take out the gold or silver sol with uniform morphology, centrifuge it and wash it with pure water, then you will get the metal particle sol with surface plasmon resonance effect. The morphology of gold particle sol is shown in Figure 2 ;

[0048] (2) adding the signal molecule to the sol obtained in step (1), allowing the two to combine, and then adding the PSA detection antibody (diluted antibody) for incubation reaction to obtain immunogold or silver signal nanoparticles, i.e., metal nanoparticle detection probes; denoted as AN (i.e., metal nanoparticles modified with 14 antibodies);

[0049] For example, gold and silver sol with uniform morphology: take 200mL of 0.01% chloroauric acid solution and heat it to boiling under magnetic stirring; then take 4.5mL of 1% sodium citrate solution and add it to the boiling chloroauric acid solution, wait for the reaction to end after boiling for 30 minutes, and cool it to room temperature to make nano-gold sol. Other metal sols are prepared in the same way;

[0050] The PSA capture magnetic beads No. 1-14 obtained in S1 and the metal nanoparticle detection probe obtained in S2 were mixed and incubated with 2 ng / mL PSA, and then Figure 1 The detection process shown in the figure obtains the Raman signal intensity, and the Raman signal intensity is processed to obtain the heat map as shown in the attached figure. Figure 4 The colors in the figure change from white to black, indicating that the intensity of the Raman signal is getting stronger. Figure 4 It can be seen that the preliminary experimental results show that antibodies No. 4, 9, 10, and 12 have strong detection signals, while the signals of other antibodies are weak and almost invisible. Subsequent work will be mainly carried out on this basis. These four antibodies are PSA MousemAb, unconjugated, detector (recorded as antibody No. 1) purchased from Chengdu Zhengneng Bio; PSA Mouse mAb, capture (recorded as antibody No. 2); mouse anti-human PSA monoclonal antibody (recorded as antibody No. 3) from Shenzhen Heavy Chain Bio; PSA / KLK3 Rabbit mAb (recorded as antibody No. 4) from Abclonal. It can be seen that these four antibodies have strong binding ability.

[0051] The four screened antibodies were modified with magnetic beads and labeled as Mag1-Mag4, which means that carboxyl magnetic beads (1 micron carboxyl magnetic beads purchased from Thermo Fisher Scientific) were modified with different antibodies No. 1-4 targeting different PSA antigen epitopes. The four antibodies were modified with metal nanoparticles and labeled as Au1-Au4.

[0052] Further screening experiments: Four antibodies with strong binding ability after initial screening were used as examples for further screening experiments, as follows:

[0053] Antibody treatment: The four antibodies are diluted by a certain multiple according to the instructions, such as 100 times, 500 times, 1000 times, and 2000 times, and then modified and bound to metal nanoparticles and carboxyl magnetic beads, respectively. This is the above steps S1 and S2.

[0054] S3. Preparation of antibody gold probe-antigen-antibody magnetic bead sandwich structure: PSA capture magnetic beads, PSA antigen diluted with serum without PSA, and metal nanoparticle detection probe are mixed to obtain a sandwich structure. There are 16 different combinations: Mag1&Au1, Mag1&Au2, Mag1&Au3, Mag1&Au4; Mag2&Au1, Mag2&Au2, Mag2&Au3, Mag2&Au4; Mag3&Au1, Mag3&Au2, Mag3&Au3, Mag3&Au4; Mag4&Au1, Mag4&Au2, Mag4&Au3, Mag4&Au3.

[0055] After step S3, Figure 1 The Raman signal intensity after detection is obtained by the process of Figure 5 . During use, the Raman intensity of different combinations varies greatly. Some combinations such as Mag2&Au3, Mag2&Au4, Mag2&Au4, Mag3&Au2, Mag3&Au4, Mag4&Au2, and Mag4&Au3 have very high Raman intensities, while some combinations such as Mag1&Au1, Mag2&Au1, Mag3&Au4, and Mag4&Au1 have very low Raman intensities. Since the signal intensities of different combinations vary greatly, in order to ensure the reliability of the results, the experiment needs to be repeated and verified many times. The inventor repeated and verified many times and obtained the same result. Taking into account the intensity of the detection signal and the signal difference between positive and negative samples, the combination with the strongest signal was selected while ensuring that there was no false positive. That is, the Mag2@Au4 combination was selected. The No. 2 antibody modified on the magnetic beads was PSA Mouse mAb, capture (denoted as No. 2 antibody); the No. 4 antibody modified on the metal nanoparticle probe was PSA / KLK3Rabbit mAb from Abclonal (denoted as No. 4 antibody). Subsequent work was carried out on this basis.

[0056] Example 2 Magnetic bead screening experiment

[0057] Surface modification of magnetic beads is also a challenge. Complex modification processes are required to ensure antibody activity, directional fixation, and avoid steric hindrance affecting detection sensitivity, in order to achieve stable and efficient antibody loading and high specificity and sensitivity of detection. The modification processes of magnetic beads from different companies are different. After selecting antibody pairs that can bind specifically and strongly to the target antigen while ensuring that other conditions remain unchanged and that fit the spatial conformation and reaction kinetics of the magnetic bead sandwich detection system, in order to improve the sensitivity of detection under the premise that the detection signal can be stably repeated, this embodiment conducts screening experiments on carboxyl magnetic beads of different sizes from different companies.

[0058] Magnetic beads: 1 μm (denoted as No. 1 magnetic beads) and 2.8 μm carboxyl magnetic beads (denoted as No. 2 magnetic beads) purchased from Thermo Fisher Scientific (Dynabeads TM MyOnerx TM Carboxylic Acid Qty: 10 mL (10 mg / mL); 200 nm (denoted as No. 3 magnetic beads), 500 nm (denoted as No. 4 magnetic beads) and 1 μm (denoted as No. 5 magnetic beads) carboxyl magnetic beads were purchased from Bio-Tech; 1 μm carboxylic magnetic beads (denoted as No. 6 magnetic beads) were purchased from Suzhou Beaver Biotechnology.

[0059] The above 6 kinds of magnetic beads and the screened antibody PSA Mouse mAb were captured and subjected to step S1 to prepare PSA capture magnetic beads, which were respectively denoted as MagA-MagF.

[0060] Metal nanoparticle detection probe: Au4 in Example 1.

[0061] Follow the steps in S3. Figure 1 The Raman signal intensity after detection is obtained by the process of Figure 6 The results showed that the signal of magnetic beads No. 1, i.e., the 1-micron carboxyl magnetic beads purchased by Thermo Fisher Scientific, was exceptionally good compared with the signal of other magnetic beads, which were very weak, so subsequent work was carried out using these magnetic beads.

[0062] Example 3 Optimization experiment of buffer formulation conditions of detection system

[0063] After the introduction of the antibody, in order to avoid possible problems such as nonspecific adsorption and signal interference, the reaction buffer system needs to be finely optimized, and the adaptation conditions of the buffer formula need to be screened based on the ionic strength, pH value and other conditions designed for the PSA antibody.

[0064] pH value: The inventors adjusted the pH value of the phosphate buffer solution PB by adjusting the phosphate buffer (H2PO4 - and HPO4 2- ) ratio to adjust the pH range of the solution to 6-8. The experimental results are as follows Figure 7 As shown, it can be seen that when the pH is in the range of 6-7.5, the detection effect is better as the pH value increases, but when it exceeds 7.5, the detection effect decreases. Therefore, when the pH is 7-7.5, especially when the pH is 7.5, the detection effect is best.

[0065] Buffer concentration: The inventors conducted experiments on different PB concentrations, with the conventional concentration being 20 mM. The experimental results are as follows Figure 8 As shown, it can be seen that when the concentration of PB buffer increases from 0 to 5 times the normal concentration, the Raman intensity also increases almost linearly. When the concentration is 5 times, the Raman intensity reaches the highest and then gradually weakens. Therefore, when the solution is 2.5-5.0 times, especially 5.0 times, the Raman intensity is good, which is the best condition under this detection system.

[0066] Embodiment 4:

[0067] S1. Preparation of immunomagnetic bead capture nanoparticles: Take 200 μL of 1 micron carboxyl-modified magnetic beads (purchased from Thermo Fisher Scientific) with a concentration of 10 μg / μL, add 1 mL of PBS buffer, vortex to mix to fully suspend the magnetic beads, magnetically discard the supernatant, and then wash twice with 200 μL of PBS buffer; resuspend the above magnetic beads in PBS buffer, add 10 μL of 10 mg / mL EDC and NHS in a 1:1 mixed solution for activation for 30 minutes, and wash with PBS buffer: resuspend in PBS buffer and add 80 μg capture antibody (PSA Mouse mAb, capture from Chengdu Zhengneng Biotechnology), incubate at 4°C overnight, then wash with PBS buffer, resuspend in PBS buffer, add blocking solution, react at room temperature, block the remaining binding sites on the surface of the magnetic beads, magnetically separate, and wash with PBS buffer, and finally redisperse in PBS buffer to obtain the immunomagnetic bead capture nanoparticles.

[0068] S2. Preparation of nano-gold sol: Take 200mL of 0.01% chloroauric acid solution and heat it to boiling under magnetic stirring; then take 4.5mL of 1% sodium citrate solution and add it to the boiling chloroauric acid solution. After boiling for 30 minutes, the reaction is completed and cooled to room temperature to prepare nano-gold sol. Take out the gold sol with uniform morphology and wash it with pure water by centrifugation, which is the metal particle sol with surface plasmon resonance effect;

[0069] Preparation of immunogold signal nanoparticles: Take 1000 μL of the metal particle sol obtained above and mix it with 10 μL of 30 μM Raman signal molecules (4-ATP / IR808 / MB-methylene blue / R6G-rhodamine 6G / MG-malachite green), incubate at room temperature for 15 minutes, centrifuge, discard the supernatant, add 20 μg of detection antibody (PSA / KLK3 RabbitmAb from Abclonal) at -4°C and incubate overnight, wash with PBS buffer, add blocking solution to block the remaining active sites, incubate at room temperature, centrifuge, wash with PBS solution, and finally discard the supernatant, and redisperse in 1000 μL of blocking solution to obtain immunogold signal nanoparticles, i.e., metal nanoparticle detection probes.

[0070] S3. Preparation of sandwich structure: Mix 20 μL of the prepared immunomagnetic capture nanoparticles (concentration 0.5 mg / mL), 40 μL of immunogold signal nanoparticles, and 200 μL of PSA (0 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL), incubate at room temperature for 10 min, and wash once with 200 μL of PBS solution after magnetic separation to construct a sandwich structure.

[0071] Detection of PSA: Take out the precipitate that forms a sandwich structure and drop it on a silicon wafer coated with a gold film or directly on an aluminum foil for SERS detection. After it dries, use a portable Raman spectrometer to directly perform Raman detection (laser wavelength 785nm) to achieve rapid detection of PSA Raman spectrum results within 10 minutes.

[0072] Results Fig. 9 It can be seen that PSA concentrations of 0.5 ng / mL and above can be well detected, and the detection signal shows a linear relationship with the PSA concentration.

[0073] Example 5

[0074] S1. Preparation of immunomagnetic beads to capture nanoparticles: Take 200 μL of 10 μg / μL carboxyl-modified magnetic beads, add 1 mL of washing buffer, vortex to mix to fully suspend the magnetic beads, magnetically discard the supernatant, and then wash twice with 200 μL of washing buffer; resuspend the above magnetic beads in PBS buffer, add 10 μL of a mixed solution of 10 mg / mL EDC and NHS prepared in a 1:1 ratio of the amount of substance to activate for 30 minutes, wash with washing buffer, resuspend in PBS buffer, add 150 μg of capture antibody, incubate at 4°C overnight, wash with washing buffer, resuspend in PBS buffer, add blocking solution BSA, react at room temperature, block the remaining binding sites on the surface of the magnetic beads, magnetically separate, and wash with PBS buffer, and finally redisperse in PBS buffer to obtain the immunomagnetic beads to capture nanoparticles.

[0075] S2. Preparation of nano silver sol: Heat 200mL of 1mM AgNO3 aqueous solution to boiling, add 6mL of 1% sodium citrate aqueous solution, the solution color gradually changes from colorless and transparent to milky white with a slight green tint, continue to keep slightly boiling for 3h, stop the reaction, and cool in a water bath. Ag nanoparticles with a particle size of about 80nm are obtained, and are stored away from light. If you need to synthesize silver nanoparticles of different sizes, just change the amount of sodium citrate added.

[0076] Preparation of immunogold signal nanoparticles: 1000 μL of silver nanoparticles were mixed with 10 μL of 30 μM Raman signal molecules, incubated at room temperature for 15 min, centrifuged, the supernatant was discarded, and the mixture was re-dispersed in PBS solution for activation. 20 μg of detection antibody was added and incubated overnight at -4°C. After washing with PBS buffer, a blocking solution was added to block the remaining active sites. The mixture was incubated at room temperature, centrifuged, and washed with PBS buffer solution. Finally, the supernatant was discarded and the mixture was re-dispersed in 1000 μL of blocking solution to obtain immunogold signal nanoparticles.

[0077] S3. Preparation of sandwich structure: Mix 20 μL of the prepared immunomagnetic capture nanoparticles, 40 μL of the immunosilver signal nanoparticles, and 200 μL of PSA (100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL), incubate at room temperature for 10 min, and wash once with 200 μL of PBS solution after magnetic separation to construct a sandwich structure.

[0078] Detection of PSA: Take out the precipitate that forms the Meiji sandwich structure and drop it directly on the aluminum foil. After it dries, use a portable Raman spectrometer to directly perform Raman detection (laser wavelength 785nm), and achieve rapid detection of PSA within 10 minutes.

[0079] Example 6

[0080] The preparation and detection steps are the same as those in Example 4, except that PBS buffer, BSA, S PrO, NPrO, cTnT, CKMB, EpCAM, and CK19 are detected.

[0081] The results are as follows Fig.10 .from Fig.10 It can be seen that the sandwich structure prepared by the present invention has very high specificity.

[0082] Example 7

[0083] Samples: 75 patients' sera, each 200 mL.

[0084] The preparation and detection steps are the same as those in Example 4, except that in step S3, 200 mL of patient serum is used to replace 200 mL of PSA.

[0085] The results are as follows Fig.11 .from Fig.11 It can be seen that the results of the detected Raman signals after data processing are highly consistent with the test results of the hospital.

[0086] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA, characterized in that: A sandwich structure is formed by PSA capture magnetic beads, detection samples, and metal nanoparticle detection probes, wherein the PSA capture magnetic beads are obtained by bio-coupling of PSA capture antibodies and magnetic beads, the antigen is PSA, and the metal nanoparticle detection probes are 20-200nm gold / silver nanoparticles.

2. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Preparation of PSA capture beads: The PSA capture antibody is mixed with the carboxyl-activated magnetic beads, and the PSA capture antibody is bound to the carboxyl magnetic beads through biocoupling, and finally the excess unbound active sites are blocked to form PSA capture magnetic beads; S2. Preparation of metal nanoparticle detection probes: (1) Preparing metal particle sol: taking out the metal sol with uniform morphology and washing it once with pure water by centrifugation, thus obtaining the metal particle sol with surface plasmon resonance effect; (2) adding the signal molecule to the sol obtained in step (1) to react, allowing the two to combine, and then adding the PSA detection antibody for incubation reaction to obtain the immunogold or silver signal nanoparticles, i.e., metal nanoparticle detection probes; The S1 and S2 are not sequential and can be performed simultaneously; S3. Prepare the antibody gold probe-antigen-antibody magnetic bead sandwich structure: mix the PSA capture magnetic beads, patient test serum samples, and metal nanoparticle detection probes to obtain a sandwich structure.

3. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA as claimed in claim 2, characterized in that: In the step S1, the particle size of the magnetic beads after carboxyl activation is 1 micron.

4. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA as claimed in claim 2, characterized in that: In the step S1, the carboxyl-activated magnetic beads are activated before being mixed with the PSA capture antibody. The activation is performed by resuspending the carboxyl-activated magnetic beads with PBS, and then adding them to a mixed solution of 10 mg / mL EDC and NHS prepared in a 1:1 ratio of the amounts of the substances for activation for 30 minutes.

5. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA as claimed in claim 2, characterized in that: In step S2 (1), the particle size of the metal particle sol is 20-200 nm.

6. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA as claimed in claim 2, characterized in that: In step S2 (1), the method for preparing the metal sol is to heat the solution containing metal ions to boiling under stirring; then add a reducing solution to the boiling solution containing metal ions to react, and cool to room temperature after the reaction to prepare the metal sol.

7. The metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure for detecting PSA as claimed in claim 2, characterized in that: The molar concentration ratio of the signal molecule to the metal particle is (20-100):1; Optionally, the reaction conditions in step (2) are incubation at room temperature with stirring for 1-15 min, centrifugation, discarding the supernatant and redispersing in a PBS solution.

8. A kit for detecting PSA, characterized in that: Contains the metal nanoparticle detection probe-antigen-antibody magnetic bead sandwich structure as described in any one of claims 1-3.

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