Nanoplasmon resonance biochip based on sandwich ELISA and method for rapid qualitative and quantitative detection of target

Abstract

The invention discloses a nanoplasma resonance biochip based on sandwich ELISA and a method for rapid qualitative and quantitative detection of a target, belonging to the field of plasma resonance quantitative detection technology of antigens; including a substrate and a coated antibody of a target modified on the substrate; the substrate includes a substrate and a 1-20nm titanium film layer, a 2-80nm silver film layer and a 2-70nm gold film layer plated on the surface of the substrate, or a 1-20nm titanium film layer, a 2-70nm silver film layer. The target that can be detected by this method includes tumor markers (such as alpha-fetoprotein, prostate-specific antigen), animal diseases (such as African swine fever virus), food small molecules (such as sulfonamide), etc. All chemical substances that can be qualitatively and quantitatively detected using enzyme-linked immunosorbent assay technology have a very wide range of applications; the chip microplate prepared by integrating the above-mentioned SPR chip is used for target quantitative detection, which can significantly improve its detection sensitivity and reduce the detection limit.

Description

Technical Field

[0001] The invention belongs to the technical field of quantitative detection of antigens by plasma resonance, in particular to rapid qualitative and quantitative detection by nanoplasmon resonance based on sandwich ELISA. Background Art

[0002] As a traditional sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA) is fast and inexpensive, and is widely used in serum marker detection and analysis. However, this detection method has limited detection limits, a relatively cumbersome and time-consuming detection process, enzyme-labeled antibodies are not resistant to storage, and enzyme-catalyzed reaction conditions are relatively harsh. Therefore, it is of great significance to develop a biomarker detection strategy with stable signals, high sensitivity, universality and ease of implementation in clinical diagnosis and treatment monitoring.

[0003] Nano surface plasmon resonance technology (NanoSPR) is a new qualitative and quantitative detection technology that is completely different from plasma resonance chip technology (SPR) and local surface plasmon resonance technology (LSPR). Surface plasmon resonance (SPR) is mainly based on the total reflection mode, using the attenuated total reflection prism coupling method to achieve laser excitation of surface plasmon waves. By detecting the change in the total reflection angle, the extinction spectrum is shifted, and the information of biochemical reactions is obtained. Local surface plasmon resonance technology (LSPR) is when light is incident on nanoparticles composed of precious metals, and the frequency of the incident photons matches the overall vibration frequency of the precious metal nanoparticles or metal conduction electrons, the nanoparticles or metals will have a strong absorption effect on the photon energy, and a strong resonance absorption peak will appear in the spectrum.

[0004] However, NanoSPR detects biochemical reactions by coupling the incident light to the metal nanostructure and using the sensitivity of the wavelength of the surface plasmon resonance to the dielectric environment around the nanostructure. When there is a difference between the refractive index of the adsorbed molecule and the surrounding environment, the reaction between the biomolecules adsorbed on the substrate surface and the target molecules will change the refractive index of the substrate surface, causing changes in the resonance peak and achieving detection of the target substance. Therefore, the resonance analysis of the NanoSPR sensor does not require the use of a complex optical system like the traditional SPR technology, and there are also obvious differences in technical principles from the LSPR.

[0005] In the prior art, for example, Chinese patent application CN110779905A provides a gold nano-labeled test strip based on surface enhanced Raman scattering, a preparation method and a method of use, in which gold nanorods are coated on a conjugate pad, and Raman molecules and bovine serum albumin are modified on the surface of the gold nanorods. Raman molecules are used as Raman reporter molecules to feedback the signal of maximum surface enhanced Raman scattering under excitation light. Bovine serum albumin (BSA) can enhance the stability of gold nanorods and prevent gold nanorods from aggregating with each other and affecting performance. The above-mentioned test strip is coated with tumor marker detection antibodies such as mouse anti-human alpha-fetoprotein detection antibodies, thereby achieving the interception of antigen tumor markers.

[0006] For another example, Chinese patent application CN101617229A provides a method for enzyme determination of LSPR, which utilizes an immobilized enzyme and a substrate to generate an insoluble precipitate, which aggregates on the surface of the LSPR, causing a change in the surface reflected light or transmitted light; specifically, a first capture antibody for detecting the target is fixed on the gold-coated surface, and then the first capture antibody and the second capture antibody are used to bind to the target, and then enzyme substrates nitro blue tetrazolium chloride (NBT), 5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt (BCIP), 3,3′,5,5′-tetramethylbenzidine (TMB), 4-chloro-1-naphthol (4-CN) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) are added to form an insoluble precipitate of the conjugate, which causes the extinction spectrum of the gold particles to shift toward the long wavelength direction during detection.

[0007] However, both of the above methods have shortcomings such as low detection sensitivity, complex detection optical path system, and low detection flux. Summary of the invention

[0008] In view of the deficiencies of the above existing technologies, the present invention provides a nanoplasmonic resonance biochip based on sandwich ELISA, and a method for rapid qualitative and quantitative detection of a target object using the chip. The method selects a nanoplasmonic resonance biochip with a specific size structure (hereinafter referred to as the chip), and uses the TMB ions generated by the reaction of the enzyme-labeled secondary antibody with the TMB solution to etch the precious metals (Ag, Au) on the surface of the biochip, thereby causing a significant decrease in the spectral peak OD value of the full spectrum absorption, thereby achieving rapid qualitative and quantitative detection of the target object; the technical principle of the detection method of the present invention is completely different from the above existing technologies. It is specifically achieved through the following technologies.

[0009] A nanoplasmonic resonance biochip based on sandwich ELISA comprises a substrate and a coated antibody modified on the substrate; the substrate comprises a base and a titanium film layer, a silver film layer, and a gold film layer with a thickness of 1-20 nm, which are sequentially plated on the surface of the base from bottom to top, or comprises a base and a titanium film layer, a silver film layer, and a silver film layer with a thickness of 1-20 nm, which are sequentially plated on the surface of the base from bottom to top, and a silver film layer with a thickness of 2-70 nm, which are sequentially plated on the surface of the base from bottom to top, and the coated antibody is used to specifically bind to the target; the surface of the base is imprinted with nanopores arranged in a matrix. .

[0010] The substrate of the NanoSPR biochip is prepared by a currently known preparation method, that is, firstly, a nanocup array is made on a silicon wafer nanocolumn mold by laser interference lithography, and then a UV-curable polymer solution is coated on the clean silicon wafer nanocolumn mold, and a glue spreader is used to evenly spread the glue solution on the surface, and then a polyethylene terephthalate (PET) sheet is slowly attached to the nanocolumn mold covered with the UV-curable polymer so that it is completely attached to the mold and placed under ultraviolet light for curing treatment; then the PET sheet together with the UV-curable polymer with the nanocup array is peeled off from the mold to obtain a base; finally, a titanium film, a silver film, and a gold film are sequentially evaporated on the surface of the nanocup array to obtain the substrate of the NanoSPR biochip.

[0011] As described above, the substrate of the NanoSPR biochip provided by the present invention has two structural forms, namely, one structure is substrate + titanium film layer (1-20nm) + silver film layer (2-80nm) + gold film layer (2-70nm), and the other structure is substrate + titanium film layer (1-20nm) + silver film layer (2-70nm). The SPR chips prepared using the substrates of these two structural forms can obtain relatively accurate quantitative detection results with low detection limits. All kinds of target standards and their coated antibodies can be purchased on the market.

[0012] The above-mentioned nanoplasmon resonance biochip (NanoSPR biochip) can be used to detect all antigens or chemical substances (i.e., targets) with specific antibodies and secondary antibodies. For example, it can be widely used in the rapid qualitative and quantitative detection of tumor markers (alpha-fetoprotein AFP, prostate-specific antigen PSA, carcinoembryonic antigen CEA, etc.), animal diseases (African swine fever virus, avian influenza virus, swine fever virus antibody, swine avian influenza, canine parvovirus, canine distemper, feline distemper antibody, etc.), and small molecules in food (antibiotics, toxins, fipronil, aflatoxin, additional additives, etc.).

[0013] Preferably, the substrate includes a base and a titanium film layer with a thickness of 2-9nm, a silver film layer with a thickness of 5-30nm, and a gold film layer with a thickness of 2-10nm, which are sequentially plated on the surface of the base from bottom to top; further preferably, the substrate includes a base and a titanium film layer with a thickness of 2-5nm, a silver film layer with a thickness of 5-10nm, and a gold film layer with a thickness of 2-5nm, which are sequentially plated on the surface of the base from bottom to top.

[0014] Preferably, the substrate comprises a base and a titanium film layer with a thickness of 2-9 nm and a silver film layer with a thickness of 5-30 nm, which are sequentially plated on the surface of the base from bottom to top. Further preferably, the substrate comprises a base and a titanium film layer with a thickness of 2-5 nm and a silver film layer with a thickness of 5-10 nm, which are sequentially plated on the surface of the base from bottom to top.

[0015] A method for preparing a nanoplasmonic resonance biochip based on sandwich ELISA comprises the following steps:

[0016] S1, preparing and cleaning the substrate, diluting the coating antibody with CBS buffer to obtain a coating antibody working solution, coating the substrate with the coating antibody working solution, and incubating at 4-37° C. for 2-24 h;

[0017] S2, wash the plate with PBST buffer, tap the plate, add 150 μl blocking agent, and incubate at 25-37°C for 30-120 min;

[0018] S3. Then, 150 μl of a protective agent is added, and the mixture is incubated at 25-37° C. for 1-60 min. The protective agent is removed by drying to prepare the nanoplasmon resonance biochip.

[0019] In the above preparation method, when the selected substrate structure is the above-mentioned substrate-titanium film layer-silver film layer-gold film layer, the coating antibody is generally diluted with 10mM CBS buffer to a concentration of 2μg / ml for AFP coating antibody, and then 50μl of coating antibody working solution is added; when the selected substrate structure is the above-mentioned substrate-titanium film layer-silver film layer, the coating antibody is generally diluted with 10mM CBS buffer to a concentration of 20μg / ml for AFP coating antibody, and then 1μl of coating antibody working solution is added.

[0020] The raw materials of CBS buffer are: 8-12g potassium carbonate, 15-20g sodium bicarbonate, 5-10ml preservative Proclin300, 500ml purified water. The raw materials of PBST buffer are: 20-30g disodium hydrogen phosphate dodecahydrate, 2-5g sodium dihydrogen phosphate, 1-3g potassium chloride, 60-80g sodium chloride, 5-10ml Tween-20, 5-10ml preservative Proclin300, 10L double distilled water. The blocking agent is a complex solution with a concentration of 10μg / ml prepared by dissolving bovine serum albumin in CBS solution. The protective agent is a complex solution with a concentration of 10-200μg / ml prepared by dissolving a sugar (one of dextran, glucose, sucrose, and trehalose) in a PBST solution.

[0021] A chip microplate integrates any one of the above-mentioned nanoplasmonic resonance biochips.

[0022] A detection kit for rapid qualitative and quantitative detection of a target object contains any one of the above-mentioned nanoplasmonic resonance biochips, or contains the above-mentioned chip microplate.

[0023] More preferably, the detection kit further comprises a washing solution, a target standard substance, and a detection antibody for the target, wherein the detection antibody is an enzyme-labeled secondary antibody, the enzyme used for the enzyme-labeled secondary antibody is horseradish peroxidase, and the enzyme substrate is at least one of 3,3',5,5'-tetramethylbenzidine, 4-chloro-l-naphthol, and 3,3'-diaminobenzidine tetrahydrochloride. For example, the detection antibody can be an HRP-labeled detection antibody, etc.

[0024] A method for rapid qualitative and quantitative detection of a target object not for the purpose of diagnosis and treatment, using any of the above-mentioned nanoplasmonic resonance biochips, or using the above-mentioned chip microplate, or using the above-mentioned detection kit for detection. Whether the above-mentioned NanoSPR biochip is used directly, or a chip microplate integrated with the NanoSPR biochip is used, or a kit equipped with the above-mentioned NanoSPR biochip or chip microplate, the detection method and principle are the same, which is to first clean and read the initial value, then add the target standard and TMB solution with a concentration gradient, and cause a metal (Ag, Au) etching reaction on the chip surface, detect the endpoint value, calculate the difference between the initial value and the endpoint value, and then draw a standard curve, and finally add the sample to be tested, record the endpoint value, and obtain the corresponding concentration according to the standard curve.

[0025] More preferably, the above method for rapid qualitative and quantitative detection of a target comprises the following steps:

[0026] P1. After washing the nanoplasmon resonance biochip with washing solution, add washing solution again, put it into the microplate reader to record the initial value, and discard the remaining liquid;

[0027] P2. Add 30-150 μl of target standard with different concentration gradients, or add the sample to be tested after being diluted by a specific multiple, and the enzyme-labeled detection antibody with appropriate concentration of the target to several nanoplasmonic resonance biochips, shake the plate to react, and discard the liquid in the microwells; then wash with washing solution, tap the plate and spin dry;

[0028] P3. Add 25-100 μl TMB solution to each nanoplasmon resonance biochip. After vibrating the plate for 10-20 minutes, place the chip in a microplate reader to record the endpoint value. Calculate the reaction value by subtracting the endpoint value from the initial value. Draw a standard curve of the reaction values ​​of target standards with different concentration gradients. Record the endpoint value of the diluted sample to be tested, and read the concentration of the sample to be tested against the standard curve.

[0029] In the above step P2, the TMB solution was purchased from Beijing Solebow Technology Co., Ltd. When the selected substrate structure is the above-mentioned substrate-titanium film layer-silver film layer-gold film layer, the general concentration of the detection antibody is 2pg / ml, and the dosage is 50μl. When the selected substrate structure is the above-mentioned substrate-titanium film layer-silver film layer, the general concentration of the detection antibody is 20pg / ml, and the dosage is 50μl; or the concentration is 1ng / ml, and the dosage is 1μl; or the concentration is 0.1ng / ml, and the dosage is 1μl. That is, as the thickness of the titanium film layer and the silver film layer changes, the amount of the detection antibody remains unchanged overall, and the concentration and dosage of the detection antibody can be adjusted randomly. The coating antibody used, as well as the CBS buffer, the detection antibody of the target, the standard of the target, the PBST buffer, the protective agent, and the blocking agent mentioned in the chip microplate all use common reagent raw materials that can be purchased on the market.

[0030] The above-mentioned nanoplasmonic resonance biochip can target various qualitative and quantitative detection fields, coat the corresponding coated antibodies or proteins on the substrate, and react with the corresponding enzyme-labeled secondary antibodies and TMB solutions. The technical means provided by the existing Chinese patent application CN101617229A is based on LSPR technology, using fixed enzymes to react with enzyme substrates to form insoluble precipitates, which will affect the spectrum of the chip and cause the generated extinction spectrum to shift in the direction of long wavelengths, thereby being able to qualitatively detect whether the target exists; there is no special requirement for the thickness of the gold-coated nanoparticle array, 5-1000nm is fine; and the existing technology mainly detects the reflection spectrum. The present invention is obviously different from the above-mentioned prior art. The technical principle of the present invention is to use TMB ions to etch metals on the surface of the NanoSPR biochip, causing the spectral peak to weaken or even disappear. In order to observe the change of this spectral peak and improve the detection sensitivity, the thickness of the metal layer on the NanoSPR biochip must be very thin; during detection, a blue TMB ion peak is first generated, and the blue peak disappears after the etching reaction, and the absorption spectrum peak OD value of the chip will also decrease significantly; the greater the concentration of TMB ions produced by enzyme catalysis, the more obvious the etching reaction with the precious metals on the chip, and the lower the absorption peak of the chip. Therefore, the technical principles of the two are obviously different, and the technical effects produced are also obviously different.

[0031] Preferably, in step P3, after adding TMB solution and vibrating the plate for reaction, a stop solution is also added, and then the plate is placed in an ELISA instrument to record the endpoint value. After adding the stop solution, the detection accuracy can be improved to a certain extent and the detection limit can be reduced.

[0032] Compared with the prior art, the advantages of the present invention are as follows: the present invention provides a method for rapid quantitative detection of different target analytes by combining a sandwich ELISA method with a microporous plasma resonance chip method using a one-step sample addition method. The NanoSPR chip provided by the present invention can significantly improve the detection sensitivity of the target analyte, reduce the detection limit, greatly simplify the operation steps, and improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a full spectrum graph of the response values ​​of alpha-fetoprotein at 8 concentration gradients in Example 1;

[0034] Figure 2 The OD value bar graph at 580 nm of alpha-fetoprotein with eight concentration gradients in Example 1;

[0035] Figure 3 This is a schematic diagram of the standard curve of alpha-fetoprotein in Example 1;

[0036] Figure 4It is a full spectrum diagram of the response values ​​of prostate specific antigen at 8 concentration gradients in Example 2;

[0037] Figure 5 The OD value bar graph at 720 nm of the prostate specific antigen of 8 concentration gradients in Example 2;

[0038] Figure 6 is a schematic diagram of the standard curve of prostate specific antigen in Example 2;

[0039] Figure 7 This is a full spectrum graph of the response values ​​of African swine fever virus at 8 concentration gradients in Example 3;

[0040] Figure 8 The OD value bar graph of the African swine fever virus at 720 nm at 8 concentration gradients in Example 3;

[0041] Fig. 9 This is a schematic diagram of the standard curve of African swine fever virus in Example 3;

[0042] Fig.10 This is a schematic diagram of the standard curve of the African swine fever serum sample of Example 4;

[0043] Fig.11 This is a bar graph of the OD values ​​at 720 nm of the African swine fever serum sample of Example 4;

[0044] Fig.12 It is the full spectrum of the reaction values ​​of sulfanilamide of 8 concentration gradients in Example 5;

[0045] Fig.13 It is a graph showing the difference in OD values ​​of sulfonamides of eight concentration gradients in Example 5 at dual wavelengths of 615 nm and 650 nm;

[0046] Fig.14 This is a schematic diagram of the standard curve of sulfonamide in Example 5. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The method for rapid qualitative and quantitative detection of target objects by nanoplasma based on sandwich ELISA provided by the present invention has a very high detection effect on tumor markers, animal diseases, and food small molecules. The following examples of the detection method of the present invention are given for tumor markers, animal diseases, and food small molecules. Tumor markers are represented by alpha-fetoprotein (AFP) and prostate-specific antigen (PSA), animal diseases are represented by African swine fever virus, and food small molecules are represented by sulfonamide.

[0049] Alpha-fetoprotein (AFP) is a glycoprotein with a high concentration in fetal blood but a very low content in adult serum. AFP is closely related to the occurrence and development of liver cancer and various tumors. It is present in high concentrations in various tumors and can be used as a positive detection indicator for various tumors. At present, it is mainly used as a serum marker for primary liver cancer in clinical practice, and is used for the diagnosis and efficacy monitoring of primary liver cancer.

[0050] Prostate-specific antigen (PSA) only exists in the cytoplasm of human prostate acinar and ductal epithelial cells and is not expressed in other cells. Prostatitis, benign prostatic hyperplasia and prostate cancer can all lead to increased total PSA levels (free and composite PSA).

[0051] The substrates used in the following examples were all produced by Liangzhun (Shanghai) Medical Device Co., Ltd.; the coating antibody and detection antibody (HRP-modified secondary antibody) of alpha-fetoprotein and prostate-specific antigen were purchased from Sino Biological Co., Ltd.; the alpha-fetoprotein and prostate-specific antigen standards were purchased from Sino Biological Co., Ltd. The African swine fever virus standards (African swine fever protein P54, CD2V protein) and their corresponding coating antibodies and detection antibodies (HRP-modified secondary antibodies) were purchased from Keqian Biological Co., Ltd., and the sulfonamide standards and their corresponding coating antibodies and detection antibodies (HRP-modified secondary antibodies) were purchased from Guangzhou Youkangduo Biotechnology Co., Ltd.

[0052] The raw material composition of CBS buffer solution is: 8-12g potassium carbonate, 15-20g sodium bicarbonate, 5-10ml preservative Proclin300, and 500ml purified water.

[0053] The raw material composition of PBST buffer is: 20-30 g of disodium hydrogen phosphate dodecahydrate, 2-5 g of sodium dihydrogen phosphate, 1-3 g of potassium chloride, 60-80 g of sodium chloride, 5-10 ml of Tween-20, 5-10 ml of preservative Proclin300, and 10 L of double distilled water.

[0054] The blocking agent is a compound solution of 10-100 μg / ml prepared by dissolving bovine serum albumin in CBS solution. The protective agent is a compound solution of 100-1000 μg / ml prepared by dissolving sucrose in PBST solution. The washing solution is PBST buffer.

[0055] Example 1

[0056] The present embodiment provides a detection method for detecting alpha-fetoprotein (AFP). Before the detection, a NanoSPR chip is integrated into a microplate to make a chip microplate. The integrated manufacturing method is: ① a 12-inch silicon wafer nanopore mold is photolithographically manufactured on an oxidized silicon wafer; ② the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; ③ a 5nm titanium film layer, a 10nm silver film layer, and a 5nm gold film layer are sequentially plated on the surface to make a substrate; ④ the substrate is then integrated into the bottom of a 96-well microplate by pasting to make a chip microplate.

[0057] (1) Wash the substrate in each microwell of the chip microplate, dilute the coating antibody of alpha-fetoprotein with 10 mM CBS buffer to obtain 2 μg / ml coating antibody working solution, coat the substrate with the coating antibody working solution at a dosage of 50 μl / well, and incubate in a refrigerator at 4°C for 12 h;

[0058] (2) Wash the plate three times with 150 μl PBST buffer, pat dry, add 150 μl blocking agent, and incubate in an oven at 37°C for 60 min.

[0059] (3) adding 150 μl of protective agent, incubating in an oven at 37° C. for 30 min, and then drying to remove the protective agent and dry the chip surface to prepare the SPR chip;

[0060] (4) Wash the microplate three times with 150 μl of PBST buffer in each microwell, then add 50 μl of PBST buffer, place it in the microplate reader to record the initial value, and discard the liquid in the microwell;

[0061] (5) Add 50 μl of 8 concentration gradients (0, 5, 10, 20, 4000, 80, pg / ml) of AFP standard and 50 μl of 2 pg / ml AFP detection antibody to each microwell, shake the plate at 700 rpm for 45 min, and discard the liquid in the microwell; wash each well three times with 150 μl PBST buffer and shake the plate dry;

[0062] (6) Add 50 μl of TMB solution to each microwell, shake the plate at 700 rpm for 15 min, and then place it in an ELISA reader to record the endpoint value. The reaction value is calculated by subtracting the endpoint value from the initial value. The full spectrum of the reaction value of alpha-fetoprotein is shown in the figure below. Figure 1 As shown, the OD value at a wavelength of 580nm is Figure 2 As shown, draw a standard curve of the response value of different concentration gradients, such as Figure 3 As shown, the standard curve equation is: y=(AD) / [1+(x / C)^B]+D, A=0.11769, B=-1.43939, C=29.52081, D=0.00043, r 2 =0.99968. Example 2

[0063] The present embodiment provides a detection method for detecting prostate-specific antigen (PSA). Before the detection, a NanoSPR chip is integrated into a microplate to make a chip microplate. The integrated manufacturing method is: ① photolithography is used to make a 12-inch silicon wafer nanopore mold on an oxidized silicon wafer; ② the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; ③ a 10nm titanium film layer, a 10nm silver film layer, and a 10nm gold film layer are sequentially plated on the surface to make a substrate; ④ the substrate is then integrated into the bottom of a 96-well microplate by pasting to make a chip microplate.

[0064] (1) Wash the substrate in each microwell of the chip microplate, dilute the coating antibody of prostate-specific antigen with 10 mM CBS buffer to obtain 2 μg / ml coating antibody working solution, coat the substrate with the coating antibody working solution at a dosage of 50 μl / well, and incubate in a refrigerator at 4°C for 12 h;

[0065] (2) Wash the plate three times with 150 μl PBST buffer, pat dry, add 150 μl blocking agent, and incubate in an oven at 37°C for 60 min.

[0066] (3) adding 150 μl of protective agent, incubating in an oven at 37° C. for 30 min, and then drying to remove the protective agent and dry the chip surface to prepare the SPR chip;

[0067] (4) Wash the microplate three times with 150 μl of PBST buffer in each microwell, then add 50 μl of PBST buffer, place it in the microplate reader to record the initial value, and discard the liquid in the microwell;

[0068] (5) Add 50 μl of 8 different concentrations of prostate-specific antigen standard (0, 10, 20, 40, 80, 160, 320, 640 pg / ml) and 50 μl of 2 pg / ml prostate-specific antigen detection antibody to each microwell, shake the plate at 700 rpm for 45 min, and discard the liquid in the microwell; wash each well three times with 150 μl PBST buffer and pat the plate dry;

[0069] (6) Add 50 μl of TMB solution to each microwell, shake the plate at 700 rpm for 15 min, and then place it in an ELISA reader to record the endpoint value. The reaction value is calculated by subtracting the endpoint value from the initial value. The full spectrum of the reaction value of prostate-specific antigen is shown in the figure below. Figure 4 As shown, the OD value at a wavelength of 720nm is Figure 5 As shown, draw a standard curve of the response value of different concentration gradients, such as Figure 6 As shown, the standard curve equation is: y=(AD) / [1+(x / C)^B]+D, A=0.49448, B=-0.89995, C=451.49116, D=-0.00511, r 2 =0.99712. Example 3

[0070] The present embodiment provides a detection method for qualitatively detecting antibodies to African swine fever virus. Before the detection, a NanoSPR chip is integrated into a microplate to make a chip microplate. The integrated manufacturing method is: ① photolithography is performed on a silicon oxide wafer to produce a 12-inch silicon wafer nanopore mold; ② the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; ③ a 6nm titanium film layer, an 8nm silver film layer, and a 6nm gold film layer are sequentially plated on the surface to make a substrate; ④ the substrate is then integrated into the bottom of a 96-well microplate by pasting to make a chip microplate.

[0071] (1) Wash the substrate in each microwell of the chip microplate, dilute the coating antibody of African swine fever protein P54 with 10 mM CBS buffer to obtain 2 μg / ml coating antibody working solution, coat the substrate with the coating antibody working solution at a dosage of 50 μl / well, and incubate in a refrigerator at 4°C for 12 h;

[0072] (2) Wash the plate three times with 150 μl PBST buffer, pat dry, add 150 μl blocking agent, and incubate in an oven at 37°C for 60 min.

[0073] (3) adding 150 μl of protective agent, incubating in an oven at 37° C. for 30 min, and then drying to remove the protective agent and dry the chip surface to prepare the SPR chip;

[0074] (4) Wash the microplate three times with 150 μl of PBST buffer in each microwell, then add 50 μl of PBST buffer, place it in the microplate reader to record the initial value, and discard the liquid in the microwell;

[0075] (5) Add 50 μl of 8 positive sample concentration gradients (1k, 2k, 4k, 8k, 16k times) of African swine fever antibody serum samples and 50 μl of African swine fever protein P54 detection antibody at a concentration of 2 pg / ml to each microwell, shake the plate at 700 rpm for 45 min, and discard the liquid in the microwell; wash each well with 150 μl PBST buffer three times, and tap the plate to dry;

[0076] (6) Add 50 μl of TMB solution to each microwell, shake the plate at 700 rpm for 15 min, and then place it in an ELISA reader to record the endpoint value. The reaction value is calculated by subtracting the endpoint value from the initial value. The full spectrum of the reaction value of African swine fever antibody is shown in the figure below: Figure 7 As shown, the OD value at a wavelength of 720nm is Figure 8 As shown, the fitting curves of different sample dilution multiples and reaction values ​​are drawn, such as Fig. 9 As shown, the equation of the fitting curve is: y=(AD) / [1+(x / C)^B]+D, A=0.37569, B=0.98332, C=1461.17596, D=0.01639, r 2 =0.99902. Example 4

[0077] This example provides a method for qualitatively detecting antibodies to African swine fever virus. The method is basically the same as that in Example 3. This example selects 29 serum samples of African swine fever for testing (all samples have been determined to be negative or positive by ELISA), including 19 negative samples and 10 positive samples. The results are 100% consistent with the ELISA results. The full spectrum of 29 serum samples of African swine fever is shown in Figure 2. Fig.10 As shown, the bar graph of 720nm wavelength is as follows Fig.11 . Example 5

[0078] This embodiment provides a method for detecting sulfonamide by competitive method based on NanoSPR biochip. Before detection, the NanoSPR chip is integrated into a microplate to make a chip microplate. The integrated production method is: ① Photolithography is performed on an oxide silicon wafer to make a 12-inch silicon wafer nanopore mold; ② The nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET sheet substrate) by a nanoimprinting method to form an inverse wafer-level nanodevice structure; ③ A 9nm titanium film layer, a 30nm silver film layer, and a 50nm gold film layer are sequentially plated on the surface to make a substrate; ④ The substrate is then integrated into the bottom of a 96-well microplate by pasting to make a chip microplate.

[0079] (1) Wash the substrate in each microwell of the chip microplate, dilute the sulfonamide antigen with 10 mM CBS buffer to obtain a 2 μg / ml coating antigen protein working solution, coat the substrate with the coating antigen protein working solution at a dosage of 50 μl / well, and incubate in a 4°C refrigerator for 12 h;

[0080] (2) Wash the plate three times with 150 μl PBST buffer, pat dry, add 150 μl blocking agent, and incubate in an oven at 37°C for 60 min.

[0081] (3) adding 150 μl of protective agent, incubating in an oven at 37° C. for 30 min, and then drying to remove the protective agent and dry the chip surface to prepare the SPR chip;

[0082] (4) Wash the microplate three times with 150 μl of PBST buffer in each microwell, then add 50 μl of PBST buffer, place it in the microplate reader to record the initial value, and discard the liquid in the microwell;

[0083] (5) Add 50 μl of sulfonamide standards with eight concentration gradients (0, 500, 1000, 2000, 4000, 8000, 16000, 32000 pg / ml) and 50 μl of anti-sulfonamide detection antibody with a concentration of 2 pg / ml to each microwell, shake the plate at 700 rpm for 45 min, and discard the liquid in the microwell; wash each well three times with 150 μl PBST buffer and shake the plate dry;

[0084] (6) Add 50 μl of TMB solution to each microwell, shake the plate at 700 rpm for 15 min, and then place it in an ELISA reader to record the endpoint value. The reaction value is calculated by subtracting the endpoint value from the initial value. The full spectrum of the reaction value of sulfonamide is shown in the figure below. Fig.12 As shown in the figure, the OD value difference at dual wavelengths of 615nm and 650nm is as follows Fig.13 As shown, draw a standard curve of the response value of different concentration gradients, such as Fig.14As shown, the standard curve equation is: y=(AD) / [1+(x / C)^B]+D, A=0.57281, B=0.81894, C=963.49161, D=-0.01152, r2=0.99336.

[0085] It can be seen from the above embodiments that the NanoSPR biochip provided by the present invention and the detection method using the biochip can achieve rapid qualitative and quantitative detection of various types of antigens such as tumor markers, animal diseases, and food small molecules, with a low detection limit and very high sensitivity.

Claims

1. A nanoplasmonic resonance biochip based on sandwich ELISA, characterized in that: The invention comprises a substrate and a coated antibody modified on the substrate; the substrate comprises a base and a titanium film layer, a silver film layer, and a gold film layer with a thickness of 2-5 nm, which are sequentially plated on the surface of the base from bottom to top, or comprises a base and a titanium film layer, a silver film layer, and a silver film layer with a thickness of 2-5 nm, which are sequentially plated on the surface of the base from bottom to top, and which are sequentially plated on the surface of the base from bottom to top, and the coated antibody is used for specific binding with a target; the surface of the base is imprinted with nanopores arranged in a matrix; The method for preparing the nanoplasmon resonance biochip comprises the following steps: S1, preparing and cleaning the substrate, diluting the coating antibody with CBS buffer to obtain a coating antibody working solution, coating the substrate with the coating antibody working solution, and incubating at 4-37° C. for 2-24 h; S2, wash the plate with PBST buffer, tap the plate, add 150 μl blocking agent, and incubate at 25-37°C for 30-120 min; S3. Then, 150 μl of a protective agent is added, and the mixture is incubated at 25-37° C. for 1-60 min. The protective agent is removed by drying to prepare the nanoplasmon resonance biochip.

2. A chip microplate, characterized in that: The nanoplasmonic resonance biochip according to claim 1 is integrated.

3. A detection kit for rapid qualitative and quantitative detection of a target, characterized in that: A nanoplasmonic resonance biochip according to claim 1, or a chip microplate according to claim 2.

4. The detection kit according to claim 3, characterized in that The invention also comprises a washing solution, a target standard substance, and a detection antibody for the target. The detection antibody is an enzyme-labeled secondary antibody. The enzyme used in the enzyme-labeled secondary antibody is horseradish peroxidase, and the enzyme substrate is at least one of 3,3',5,5'-tetramethylbenzidine, 4-chloro-l-naphthol, and 3,3'-diaminobenzidine tetrahydrochloride.

5. A method for rapid qualitative and quantitative detection of a target object not for the purpose of diagnosis and treatment, characterized in that: The detection is performed using the nanoplasmon resonance biochip described in claim 1, or the chip microplate described in claim 2, or the detection kit described in any one of claims 3-4.

6. The method for rapid qualitative and quantitative detection of a target object according to claim 5, characterized in that: The following steps are involved: P1. After washing the nanoplasmon resonance biochip with washing solution, add washing solution again, put it into the microplate reader to record the initial value, and discard the remaining liquid; P2. Add 30-150 μl of target standard with different concentration gradients, or add the sample to be tested after being diluted by a specific multiple, and the enzyme-labeled detection antibody with appropriate concentration of the target to several nanoplasmonic resonance biochips, shake the plate to react, and discard the liquid in the microwells; then wash with washing solution, tap the plate and spin dry; P3. Add 25-100 μl TMB solution to each nanoplasmon resonance biochip. After vibrating the plate for 10-20 minutes, place the chip in a microplate reader to record the endpoint value. Calculate the reaction value by subtracting the endpoint value from the initial value. Draw a standard curve of the reaction values ​​of target standards with different concentration gradients. Record the endpoint value of the diluted sample to be tested, and read the concentration of the sample to be tested against the standard curve.

7. The method for rapid qualitative and quantitative detection of a target object according to claim 6, characterized in that: In step P3, after adding TMB solution to vibrate the plate for reaction, a stop solution is added, and then the plate is placed in an ELISA reader to record the endpoint value.

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