Multicolor immunoassay method detection kit for hydroquinone-mediated nanogold cone growth and application of multicolor immunoassay method detection kit

Abstract

The invention relates to a hydroquinone-mediated nanogold cone growth multicolor immunoassay method detection kit and application thereof, and the kit internally comprises an elisa plate, a standard solution, a PB buffer solution, a biotin-labeled antibody and streptavidin-modified horse radish peroxidase, the ELISA plate is coated with a capture antibody specifically combined with a to-be-detected object, the standard solution is a to-be-detected object solution with accurate concentration, the biotin-labeled antibody is a biotin-labeled detection antibody specifically combined with the to-be-detected object, and the kit further comprises an enzymatic hydrolysate and an AuNBPs growth solution; according to the present invention, the HQ-mediated AuNBPs growth method through HRP catalytic oxidation is established, the HQ-mediated AuNBPs growth method is combined with the reagents in the ELISA kit to form the brand new kit, the reagents in the kit interact to form the multi-color colorimetric immunoassay sensor for detecting Tg, and the new method for multi-color immune visual detection of Tg protein is constructed.

Description

Technical Field

[0001] The invention belongs to the field of detection, and specifically relates to a multicolor immunoassay detection kit for hydroquinone-mediated nano-gold cone growth and application thereof. The kit can be used for the detection of thyroglobulin. Background Art

[0002] Thyroglobulin (Tg) is a recognized and effective biomarker that can be used to evaluate the postoperative and prognostic status of patients with differentiated thyroid cancer (DTC) after total thyroidectomy. As the most common endocrine malignancy, thyroid cancer has received increasing attention due to its increasing incidence in recent years. DTC is usually indolent, and the vast majority of DTC patients can achieve good treatment effects. However, some patients still experience recurrence after surgery. According to statistics, more than 10% of DTC patients will develop distant metastases, and 20-30% of patients will develop persistent or recurrent tumors, which significantly increases the risk of death for patients. Therefore, in order to dynamically monitor disease recurrence and make timely interventions, long-term follow-up of DTC patients after surgery is very necessary.

[0003] At present, the detection of Tg mainly relies on radioimmunoassays (RIAs), immunoassays (IMAs) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). LC-MS / MS has high specificity for the detection of Tg, but requires complex pretreatment, expensive instruments and professional operators. In contrast, RIAs and IMAs have the advantages of low cost, easy operation and high throughput. Compared with RIAs, IMAs have better sensitivity and safety, which enables DTC patients to self-detect the level of non-irritating Tg in the blood without the need for recombinant thyroid-stimulating hormone stimulation. However, most of the IMAs kits for detecting Tg on the market rely on chemiluminescence or single color intensity change colorimetry, which either require analytical instruments for quantitative analysis or have the disadvantage of inaccurate naked eye detection. This has hindered the application of IMAs kits in patient self-testing. Therefore, the development of a simple, economical, and highly accurate home self-testing method that does not require analytical instruments may enable home self-testing and personalized care based on Tg concentration risk assessment.

[0004] As a noble metal nanomaterial, gold nanoparticles (AuNBPs) have strong localized surface plasmon resonance and sensitive and tunable aspect ratio, making them an ideal multicolor developer for colorimetric methods. Compared with traditional organic color developers, AuNBPs are more sensitive due to their higher extinction coefficient. In these colorimetric methods, the grown / etched AuNBPs will show multicolor changes related to the target concentration due to the change of their aspect ratio. It has been reported that AuNBPs grown using ALP catalyzed AA have high uniformity and dispersion, and can show 9 color changes within a narrow target concentration range (about one order of magnitude), which improves the accuracy of naked eye inspection, especially for determining whether the target exceeds a specific threshold. However, commercial immunocolorimetric methods usually use horseradish peroxidase (HRP) to catalyze signal generation, and the AA-regulated AuNBPs growth method is difficult to perfectly combine with the HRP catalytic system. The enzymes in some commercial kits are already coupled, and ALP cannot replace all HRP, so this method is not universal. The optimal pH value for ALP enzymatic hydrolysis is usually alkaline, and the pH value for AA to regulate the growth of AuNBPs is acidic, so it needs to undergo extreme acid-base changes, which is not conducive to commercial operation. In addition, the instability of the color of AuNBPs grown by AA also greatly limits its practical application in immunocolorimetry.

[0005] In summary, it is necessary to develop a multicolor immunoassay detection reagent or kit based on the horseradish peroxidase-catalyzed regulation of the stable growth of AuNBPs, which can be used in conjunction with the existing ELISA kit that utilizes horseradish peroxidase-catalyzed signal generation for the visualization detection of some proteins, such as thyroglobulin. Summary of the invention

[0006] The object of the present invention is to provide a multicolor immunoassay detection kit for AuNBPs growth mediated by hydroquinone (HQ). The enzymatic hydrolysate, AuNBPs growth solution, streptavidin-modified horseradish peroxidase in the kit interact with an ELISA plate coated with a specific capture antibody for a protein to be detected and a biotinylated detection antibody, thereby realizing visualized semi-quantitative and instrumental quantitative detection of the protein to be detected, such as the detection of thyroglobulin (Tg).

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A multicolor immunoassay detection kit for hydroquinone-mediated nanogold cone growth, the kit comprising an ELISA plate, a standard solution, a PB buffer, a biotin-labeled antibody, and a streptavidin-modified horseradish peroxidase, wherein the ELISA plate is coated with a capture antibody that specifically binds to an object to be detected, the standard solution is a solution of the object to be detected with a precise concentration, the biotin-labeled antibody is a detection antibody that specifically binds to the object to be detected and is labeled with biotin, and the kit also comprises an enzymatic solution and an AuNBPs growth solution;

[0009] The enzymatic hydrolysis solution comprises the following components: acetate buffer, H2O2, hydroquinone and secondary water; the pH of the enzymatic hydrolysis solution is 5.5, the final concentration of the acetate buffer in the enzymatic hydrolysis solution is 10mM, the final concentration of H2O2 is 1mM, and the final concentration of hydroquinone is 0.21mM;

[0010] The AuNBPs growth solution includes the following components: CTAB, HAuCl4, AgNO3, NADH, acetate buffer and gold seed; the pH of the AuNBPs growth solution is 6.0, and in the AuNBPs growth solution, the final concentration of CTAB is 180mM, the final concentration of HAuCl4 is 0.5mM, the final concentration of AgNO3 is 0.2mM, the final concentration of NADH is 0.7mM, the final concentration of acetate buffer is 10mM, and each 75μL of AuNBPs growth solution contains 3.0μL of gold seed. The preparation method of the enzymatic solution is:

[0011] 7.5 μL of 100 mM acetate buffer solution with a pH of 5.5, 7.5 μL of 10 mM H2O2 aqueous solution, and 7.5 μL of 2.1 mM hydroquinone aqueous solution were sequentially added to 52.5 μL of secondary water, and mixed to obtain 75 μL of the enzymatic hydrolyzate.

[0012] The preparation method of the AuNBPs growth solution is:

[0013] 3.75 μL of 200 mM acetate buffer solution with a pH of 6.0, 3.75 μL of 10 mM HAuCl4 aqueous solution, 1.5 μL of 10 mM AgNO3 aqueous solution, and 3 μL of 17.5 mM NADH aqueous solution were sequentially added to 60 μL of 225 mM CTAB aqueous solution, mixed at room temperature for 1 min, and then 3.0 μL of gold seed was added to obtain 75 μL of the AuNBPs growth solution;

[0014] The preparation method of the gold seed is:

[0015] First, 10 mL of a solution containing 50 mM CTAC, 5 mM citric acid, and 0.25 mM HAuCl4 was added to a glass bottle. Then, under room temperature and vigorous stirring at 1000 rpm, a freshly prepared NaBH4 solution (0.25 mL, 25 mM) was quickly added to the solution. NaBH4 3+ Rapidly reduced to gold atoms; 2 minutes later, the solution was incubated at 80°C for 90 minutes to form gold seeds, and the seed solution was stored at 4°C in the dark.

[0016] The enzymatic hydrolysate and AuNBPs growth solution in the kit of the present invention can interact with the horseradish peroxidase modified with streptavidin, the ELISA plate coated with the specific capture antibody of the protein to be detected, and the biotin-labeled antibody, so as to realize the visual semi-quantitative and instrumental quantitative detection of the protein to be detected. As described in the multicolor immunoassay detection kit, it is a kit for detecting thyroglobulin, in which case the ELISA plate is coated with the capture antibody that specifically binds to thyroglobulin, the standard solution is a thyroglobulin solution of precise concentration, and the biotin-labeled antibody is a detection antibody that specifically binds to thyroglobulin and is labeled with biotin.

[0017] The method for detecting thyroglobulin by the multicolor immunoassay detection kit comprises the following steps:

[0018] (1) Antibody-antigen-antibody-horseradish peroxidase binding:

[0019] First, add 100 μL of Tg standard solution or sample solution diluted with PB buffer to different concentrations into the reaction well of the enzyme label plate coated with the capture antibody, seal the well plate with a sealing film, incubate it on a 37°C oscillator for 90 minutes, and then rinse it with PB buffer three times; then add 100 μL of biotin-labeled antibody working solution into the well, seal the well plate with a sealing film, incubate it at 37°C for 60 minutes, and then rinse it with PB buffer three times; then add 100 μL of streptavidin-modified horseradish peroxidase conjugate, seal the well plate with a sealing film, incubate it at 37°C for 30 minutes, and then rinse it with PB buffer three times;

[0020] The reagents used in this step can be the reagents in the commercial human thyroglobulin ELISA kit, including: an enzyme-labeled plate coated with a capture antibody, a Tg standard solution, a biotin-labeled antibody, and a streptavidin-modified horseradish peroxidase; and the operation method of this step is also similar to the operation method of the commercial human thyroglobulin ELISA kit. Of course, the enzyme-labeled plate in the kit can also be coated with other capture antibodies that specifically bind to thyroglobulin, and the biotin-labeled antibody can also be other self-developed detection antibodies that specifically bind to thyroglobulin and are labeled with biotin.

[0021] (2) Horseradish peroxidase-catalyzed oxidation of hydroquinone:

[0022] Add 75 μL of enzymatic solution (10 mM acetate buffer, 1 mM H2O2 and 0.21 mM HQ, pH = 5.5) to the reaction wells of the ELISA plate after step (1), seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0023] (3) AuNBPs growth:

[0024] Finally, add 75 μL of AuNBPs growth solution (180 mM CTAB, 0.5 mM HAuCl4, 0.2 mM AgNO3, 0.7 mM NADH, 10 mM acetate buffer and 3.0 μL gold seed, pH = 6.0) to the enzymatic solution, mix well and react at 50°C oscillator for 20 minutes; after the reaction, take out the ELISA plate and observe the color of the solution with the naked eye, take pictures and record, and use an ELISA instrument to measure the extinction spectrum of the solution in the range of 400-1000 nm; calculate the Tg content based on the color observed with the naked eye or based on the wavelength of the maximum absorption peak.

[0025] The multicolor immunoassay detection kit is used for detecting thyroglobulin.

[0026] Compared with the prior art, the advantages of the present invention are:

[0027] 1. In the present invention, the inventors first optimized the conditions for the growth of AuNBPs by AA and hydroquinone reducing agents, and investigated the stability and sensitivity of the growth of AuNBPs by AA and hydroquinone reducing agents. Since the pH required for the growth of AuNBPs by HQ is close to the pH of the HRP catalytic reaction, the inventors further constructed a reaction based on the HRP catalytic oxidation of HQ to mediate the growth of AuNBPs. In this reaction, the enzymatic solution obtained by the oxidation of HQ by HRP catalyzed by H2O2 can be directly used for the growth of AuNBPs without changing the pH. Finally, the inventors combined the HRP-regulated AuNBPs growth system with a commercial ELISA kit for detecting Tg, and constructed a multicolor immunosensor that exhibits multicolor changes and is stable in a narrow linear range, achieving high-precision on-site detection of Tg concentration in serum. It is worth noting that the constructed HRP-regulated AuNBPs growth system can be perfectly combined with the reagents in the commercial ELISA kit to form a new kit, and can also provide stable multicolor visual detection results in liquid for ELISA detection of other targets.

[0028] 2. The present invention takes Tg as the analysis object, and aims at the actual detection demand of Tg concentration (narrow concentration detection range), and constructs a multicolor colorimetric immunosensor based on in-depth research and optimization of the AuNBPs growth method. The present invention establishes an HRP-catalyzed oxidation HQ-mediated AuNBPs growth method, and combines it with the reagents in a commercial ELISA kit to form a brand-new kit. The reagents in the kit interact with each other to form a multicolor colorimetric immunoassay sensor for detecting Tg, thereby constructing a new method for multicolor immunovisual detection of Tg protein. Under optimal conditions, the constructed multicolor immunosensor shows 10 color changes related to Tg concentration within the Tg concentration range of 0.1-1.3ng / mL. In this Tg concentration range, its concentration is related to the Δλ of AuNBPs. LLSPR It shows good linear correlation, with a visual detection limit of 0.1 ng / mL and a spectral detection limit of 0.06 ng / mL. Using this method, the content of Tg in serum spiked samples was successfully detected by naked eye colorimetry and UV-visible spectrophotometry, with a spike recovery rate of 94.1-105% and a relative standard deviation (RSD, n=5) of <4%. Compared with the reported multicolor immunoassay detection method based on ALP-catalyzed production of AA-mediated AuNBPs growth, the multicolor immunoassay detection method constructed by the present invention does not require replacing HRP in the traditional ELISA kit with ALP, thereby improving the universality of the method; allowing the enzyme-catalyzed reaction and AuNBPs growth to be carried out at the same pH value, thereby reducing the experimental difficulty and experimental cost; improving the stability of the AuNBPs signal and ensuring the accuracy of the detection; improving the simplicity of the experimental operation, without relying on a paper-based analytical device to stabilize the AuNBPs signal, eliminating the operation of assembling PAD, and can be directly completed in the liquid. Therefore, the successful establishment of this method not only provides a reliable and highly accurate multicolor immunosensor for the dynamic monitoring of Tg concentration and prognostic detection, but also provides a multicolor analysis method with simple operation, stable signal and high sensitivity for other commercial ELISA kits.

[0029] 3. The present invention establishes a method for HRP-catalyzed oxidation of HQ to mediate the growth of AuNBPs, which can be combined with the reagents in the commercial ELISA kit for the detection of the corresponding protein. Therefore, the enzymatic hydrolysate, the AuNBPs growth solution and the reagents in the commercial ELISA kit can be combined to form a new multicolor immunoassay detection kit. Or it can be combined with a self-developed capture antibody that specifically binds to the object to be detected, a biotin-labeled detection antibody and a streptavidin-modified horseradish peroxidase to form a new multicolor immunoassay detection kit for visual semi-quantitative and instrumental quantitative detection of the object to be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the detection principle of the multicolor immunoassay detection kit of the present invention.

[0031] Figure 2 is the extinction spectra of AuNBPs grown at different growth pH values ​​using reducing agents AA, CC, HQ and RS (A), and the optimal pH value of each reducing agent is indicated in red font; LLSPR TEM images of AuNBPs grown using four reducing agents (λ = 750 nm) (B); LLSPR Changes over time (C).

[0032] Figure 3 The photos (A), extinction spectra (B) and λ of AuNBPs at different AA concentrations in the AA-mediated AuNBPs growth system are shown in Figure 2. LLSPR Linear relationship with AA concentration (C)

[0033] Figure 4 The photos (A), extinction spectra (B) and λ of AuNBPs at different CC concentrations in the CC-mediated AuNBPs growth system. LLSPR Logarithmic relationship with CC concentration (C).

[0034] Figure 5 The photos (A), extinction spectra (B) and λ of AuNBPs at different HQ concentrations in the HQ-mediated AuNBPs growth system are shown in Figure 2. LLSPR Linear relationship with HQ concentration (C).

[0035] Figure 6 The photos (A), extinction spectra (B) and λ of AuNBPs at different RS concentrations in the RS-mediated AuNBPs growth system. LLSPR Logarithmic relationship with RS concentration (C).

[0036] Figure 7 This is the feasibility analysis result of HRP catalyzing HQ to regulate the growth of AuNBPs. The experimental conditions are 5U / LHRP, 1mM H2O2 and 0.21mM HQ.

[0037] Figure 8 are TEM images of AuNBPs at different HRP concentrations.

[0038] Fig. 9 Optimization of HQ concentration (A), H2O2 concentration (B), and pH value (C). Tg concentration was 1.3 ng / mL.

[0039] Fig.10 The color (A) and extinction spectrum (B) of AuNBPs in the presence of different concentrations of Tg, as well as the Δλ of AuNBPs LLSPR (C) Graph showing the linear relationship between the concentration of Tg.

[0040] Fig.11 This is a graph showing the specificity of Tg detection using a multicolor immunoassay kit.

[0041] Fig.12 This is a graph showing the analysis results of Tg in serum samples. DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0043] Experimental materials and tools:

[0044] Glacial acetic acid and catechol (CC) were purchased from Shanghai Aladdin Reagent Co., Ltd., China. Resorcinol (RS) was purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. Human TG ELISA Kit EH0420 was purchased from Wuhan Fine Biotechnology Co., Ltd. Chloroauric acid tetrahydrate (HAuCl4·4H2O), cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO3), sodium borohydride (NaBH4), reduced nicotinamide adenine dinucleotide I (NADH), H2O2, human serum albumin (BSA), hydroquinone (HQ), and L-ascorbic acid (AA) were purchased from Sigma-Aldrich, USA. Hexadecyl trimethyl ammoniumbromide (CTAC), citric acid, HCl, and sodium hydroxide (NaOH) were purchased from China Pharmaceutical Chemical Reagent Co., Ltd. (Shanghai, China). Horseradish peroxidase (HRP) was purchased from Sangon Biotech (Shanghai) Co., Ltd. The PB buffer mentioned in this article is composed of 10 mM NaH2PO4 and 10 mM Na2HPO4, with a pH of 7.5.

[0045] The UV-visible extinction spectra of AuNBPs were measured using a Spark10M multifunctional microplate reader (Tecan, Switzerland). All photos in the experiment were recorded using a 12pro max mobile phone (iPhone, USA). Transmission electron microscopy (TEM) images were obtained using a HT7700 transmission electron microscope (Hitachi, Japan). Ultrapure water was obtained by a Millipore purification system (Millipore, USA). The BE-9008 microplate oscillator used for incubation was purchased from Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0046] Embodiment 1: Synthesis of gold seed:

[0047] First, 10 mL of a solution containing 50 mM CTAC, 5 mM citric acid, and 0.25 mM HAuCl4 was added to a glass bottle. Then, under room temperature and vigorous stirring at 1000 rpm, a freshly prepared NaBH4 solution (0.25 mL, 25 mM) was quickly added to the solution. NaBH4 3+ Rapidly reduced to gold atoms; 2 minutes later, the solution was incubated at 80°C for 90 minutes to form gold seeds, and the seed solution was stored at 4°C in the dark.

[0048] Example 2: Preparation of reducing agent-mediated AuNBPs growth

[0049] The present invention firstly studies the growth of AuNBPs mediated by AA and different hydroquinone reducing agents. The specific experimental operation is as follows:

[0050] Preparation of AuNBPs with AA: First, 15 μL of AA aqueous solution with different concentrations was mixed with 135 μL of AuNBPs growth solution (100 mM CTAB, 0.28 mM HAuCl4, 0.11 mM AgNO3, 0.39 mM NADH, 6.7 mM HCl and 1.5 μL gold seed) and incubated at 50 ° C for 15 minutes. Finally, the color of the solution was recorded by taking a photo with a mobile phone and observed with the naked eye, and the extinction spectrum of AuNBPs was recorded using a microplate reader, ranging from 400-1000 nm.

[0051] Preparation of AuNBPs by CC: First, 15 μL of CC aqueous solution with different concentrations was mixed with 135 μL of AuNBPs growth solution (100 mM CTAB, 0.28 mM HAuCl4, 0.11 mM AgNO3, 0.39 mM NADH, 11.1 mM CH3COOH and 1.5 μL gold seed, pH = 4.5) and incubated at 50 ° C for 15 minutes. Finally, the color of the solution was observed with the naked eye and recorded with a mobile phone, and the extinction spectrum of AuNBPs was recorded using a microplate reader, ranging from 400-1000 nm.

[0052] Preparation of AuNBPs with HQ: First, 15 μL of HQ aqueous solution with different concentrations was mixed with 135 μL of AuNBPs growth solution (100 mM CTAB, 0.28 mM HAuCl4, 0.11 mM AgNO3, 0.39 mM NADH, 11.1 mM CH3COOH and 1.5 μL gold seed, pH = 6) and incubated at 50 ° C for 20 minutes. Finally, the color of the solution was observed with the naked eye and recorded with a mobile phone, and the extinction spectrum of AuNBPs was recorded using a microplate reader, ranging from 400-1000 nm.

[0053] Preparation of AuNBPs by RS: First, 15 μL of RS aqueous solution with different concentrations was mixed with 135 μL of AuNBPs growth solution (100 mM CTAB, 0.28 mM HAuCl4, 0.11 mM AgNO3, 0.39 mM NADH, 11.1 mM Tris-HCl and 1.5 μL gold seed, pH = 8.3), and incubated at 50 ° C for 25 minutes. Finally, the color of the solution was observed with the naked eye and recorded with a mobile phone, and the extinction spectrum of AuNBPs was recorded using an ELISA instrument, ranging from 400-1000 nm.

[0054] Afterwards, the pH value of AuNBPs growth mediated by AA and different hydroquinone reducing agents was optimized with reference to the above method. The experimental results showed that AA, CC, HQ and RS were able to grow AuNBPs under acidic, acidic, neutral and alkaline conditions, respectively. In the subsequent performance test, it was found that compared with AA and CC (acidic pH growth conditions), the AuNBPs morphology obtained by HQ (neutral pH growth conditions) and RS (alkaline pH growth conditions) mediated the growth of AuNBPs was sharper and the solution color was more stable, and the amount of reducing agent consumed was also less. Afterwards, the sensitivity of AuNBPs grown by AA and hydroquinone reducing agents was also investigated. It was concluded that HQ is expected to replace AA to mediate the growth of AuNBPs and build a more sensitive and stable multicolor colorimetric sensor under neutral conditions. Therefore, on this basis, the present invention continues to analyze the feasibility of HRP-catalyzed oxidation of HQ, and the specific operation is shown in Example 3; then a method for HRP-catalyzed oxidation of HQ-mediated AuNBPs growth was established, and it was combined with a commercial ELISA kit to construct a new method for multi-color immunovisualization detection of Tg protein, as shown in Example 4:

[0055] Example 3: Feasibility analysis of HRP catalytic oxidation of HQ:

[0056] 3.1 HQ-mediated growth of AuNBPs without H2O2 and HRP: 75 μL of 0.21 mM HQ aqueous solution (containing 10 mM CH3COOH, pH = 5.5) was mixed with 75 μL of AuNBPs growth solution (180 mM CTAB, 0.5 mM HAuCl4, 0.2 mM AgNO3, 0.7 mM NADH, 10 mM acetate buffer and 3.0 μL gold seed, pH = 6.0), and incubated in a 50°C constant temperature metal bath for 20 minutes. Finally, the color of the solution was recorded by taking a photo with a mobile phone, and the extinction spectrum of the solution was measured in the range of 400 to 1000 nm using an ELISA reader; the AuNBPs growth solution was the best AuNBPs growth solution obtained by the inventor after optimizing the concentration of each substance and the pH of the solution, and the AuNBPs growth solution was used for all subsequent experiments.

[0057] 3.2 HQ-mediated growth of AuNBPs with H2O2 and without HRP: 75 μL of 0.21 mM HQ aqueous solution (containing 1 mM H2O2 and 10 mM CH3COOH, pH = 5.5) was incubated at 37°C for 30 minutes, then mixed with 75 μL of AuNBPs growth solution (180 mM CTAB, 0.5 mM HAuCl4, 0.2 mM AgNO3, 0.7 mM NADH, 10 mM acetate buffer and 3.0 μL gold seed, pH = 6.0), and incubated in a 50°C constant temperature metal bath for 20 minutes. Finally, the color of the solution was recorded by taking a photo with a mobile phone, and the extinction spectrum of the solution was measured in the range of 400 to 1000 nm using an enzyme reader.

[0058] 3.3 H2O2 and HRP both present HQ-mediated AuNBPs growth: 75 μL 0.21 mM HQ aqueous solution (containing 1 mM H2O2, 5 U / L HRP and 10 mM CH3COOH, pH = 5.5) was incubated at 37°C for 30 minutes, then mixed with 75 μL AuNBPs growth solution (180 mM CTAB, 0.5 mM HAuCl4, 0.2 mM AgNO3, 0.7 mM NADH, 10 mM acetate buffer and 3.0 μL gold seed, pH = 6.0), and incubated in a 50°C constant temperature metal bath for 20 minutes. Finally, the color of the solution was recorded by taking a photo with a mobile phone, and the extinction spectrum of the solution was measured in the range of 400-1000 nm using an enzyme marker.

[0059] Embodiment 4:

[0060] 4.1 Multicolor immunoassay kit for detecting Tg

[0061] Combining the method of HRP-catalyzed oxidation of HQ to mediate the growth of AuNBPs with an ELISA kit for detecting Tg, a new method for multicolor immuno-visual detection of Tg protein can be constructed, and at the same time, it can be reassembled into a multicolor immunoassay detection kit for visual detection of Tg. The reagents in the kit interact to form a multicolor colorimetric immunoassay sensor for detecting Tg, realizing visual semi-quantitative and instrumental quantitative detection of Tg.

[0062] The kit is a multicolor immunoassay detection kit based on HRP catalysis and regulation of AuNBPs growth, and the kit includes an ELISA plate, a PB buffer, a standard solution, a biotin-labeled antibody, and a streptavidin-modified horseradish peroxidase, wherein the ELISA plate is coated with a capture antibody that specifically binds to the object to be detected, the standard solution is a solution of the object to be detected with a precise concentration, the biotin-labeled antibody is a detection antibody that specifically binds to the object to be detected and is labeled with biotin, and the kit also includes an enzymatic solution and an AuNBPs growth solution;

[0063] The enzymatic hydrolysis solution comprises the following components: acetate buffer, H2O2, hydroquinone and secondary water; the pH of the enzymatic hydrolysis solution is 5.5, the final concentration of the acetate buffer in the enzymatic hydrolysis solution is 10mM, the final concentration of H2O2 is 1mM, and the final concentration of hydroquinone is 0.21mM;

[0064] The AuNBPs growth solution includes the following components: CTAB, HAuCl4, AgNO3, NADH, acetate buffer and gold seed; the pH of the AuNBPs growth solution is 6.0, and in the AuNBPs growth solution, the final concentration of CTAB is 180mM, the final concentration of HAuCl4 is 0.5mM, the final concentration of AgNO3 is 0.2mM, the final concentration of NADH is 0.7mM, the final concentration of acetate buffer is 10mM, and each 75μL of AuNBPs growth solution contains 3.0μL of gold seed.

[0065] The preparation method of the enzymatic hydrolysate may be (taking the preparation of 75 μL of enzymatic hydrolysate as an example):

[0066] 7.5 μL of 100 mM acetate buffer solution with a pH of 5.5, 7.5 μL of 10 mM H2O2 aqueous solution, and 7.5 μL of 2.1 mM hydroquinone aqueous solution were sequentially added to 52.5 μL of secondary water, and mixed to obtain 75 μL of the enzymatic hydrolyzate.

[0067] The preparation method of the AuNBPs growth solution can be (taking the preparation of 75 μL AuNBPs growth solution as an example):

[0068] 3.75 μL of 200 mM acetate buffer solution with a pH of 6.0, 3.75 μL of 10 mM HAuCl4 aqueous solution, 1.5 μL of 10 mM AgNO3 aqueous solution, and 3 μL of 17.5 mM NADH aqueous solution were sequentially added to 60 μL of 225 mM CTAB aqueous solution, mixed at room temperature for 1 min, and then 3.0 μL of gold seed was added to obtain 75 μL of the AuNBPs growth solution;

[0069] The synthesis method of the gold seed is prepared by referring to the method described in Example 1.

[0070] The multicolor immunoassay detection kit is a kit for detecting thyroglobulin, wherein the ELISA plate is coated with a capture antibody that specifically binds to thyroglobulin, the standard solution is a thyroglobulin solution of precise concentration, and the biotin-labeled antibody is a biotin-labeled detection antibody that specifically binds to thyroglobulin. Of course, the ELISA plate, standard solution, biotin-labeled antibody and streptavidin-modified horseradish peroxidase can be reagents in a commercial human thyroglobulin ELISA kit.

[0071] The method for detecting thyroglobulin by the multicolor immunoassay detection kit described in 4.2, the specific operation steps are as follows:

[0072] First, 100 μL of Tg standard solution or sample solution diluted with PB buffer to different concentrations was added to the reaction wells of the ELISA plate coated with the capture antibody, the well plate was sealed with a sealing film, incubated on a 37°C oscillator for 90 minutes, and then rinsed with PB buffer three times; then 100 μL of biotin-labeled antibody working solution was added to the well, the well plate was sealed with a sealing film, incubated at 37°C for 60 minutes, and rinsed with PB buffer three times; then 100 μL of streptavidin-modified horseradish peroxidase conjugate was added, the well plate was sealed with a sealing film, incubated at 37°C for 30 minutes, and rinsed with PB buffer three times.

[0073] The reagents used in this step are the reagents in the commercial human thyroglobulin ELISA kit (Wuhan Fine Biotechnology Co., Ltd.), including: an ELISA plate covered with capture antibodies, Tg standard solution, biotin-labeled antibodies, and streptavidin-modified horseradish peroxidase; and the operation method of this step is also similar to that of the commercial human thyroglobulin ELISA kit.

[0074] Afterwards, 75 μL of enzymatic solution (10 mM CH3COOH, 1 mM H2O2, and 0.21 mM HQ, pH = 5.5) was added to the reaction wells of the ELISA plate, and the plate was sealed with a sealing film and incubated at 37 °C for 30 min;

[0075] Finally, add 75 μL of AuNBPs growth solution (180 mM CTAB, 0.5 mM HAuCl4, 0.2 mM AgNO3, 0.7 mM NADH, 10 mM CH3COOH and 3.0 μL gold seed, pH = 6), mix well and react for 20 minutes at 50 ° C oscillator. After the reaction, take out the ELISA plate and observe the color of the solution with the naked eye, take pictures with a mobile phone, and use an ELISA reader to measure the extinction spectrum of the solution in the range of 400-1000 nm. Calculate the Tg content based on the color observed by the naked eye or the wavelength of the maximum absorption peak.

[0076] Each condition in the above detection method is the optimized Tg optimal detection condition. According to the above method, Tg standard solutions of different concentrations were detected respectively, and the working performance and sensitivity of the detection method established by the present invention were evaluated. At the same time, the above method was used to detect Tg in serum.

[0077] Example 5: Detection of Tg in serum

[0078] The steps of processing serum samples are as follows: first, the purchased serum samples were heated at 90°C for 10 minutes, then centrifuged at 4000 rpm for 10 minutes, and then diluted ten times with PB buffer. Finally, the spiked serum samples were detected according to the method described in Example 4.

[0079] VI. Results and Discussion

[0080] 6.1 Principle of Tg detection by multicolor immunoassay kit

[0081] The detection principle of the multicolor immunoassay detection kit of the present invention is as follows Figure 1 As shown. When Tg is present, the capture antibody (Ab1) coated on the microplate can specifically bind to Tg, and then the biotinylated detection antibody specifically attaches to Tg. Finally, HRP labeled with streptavidin (SA) binds to the biotinylated detection antibody, and finally forms an HRP-labeled sandwich immune complex. HRP catalyzes H2O2 to oxidize HQ to generate benzoquinone (BQ) under the condition of pH 5.5. Next, a gold seed and Au + The AuNBPs growth solution was added to the ELISA plate and incubated at pH 6 and 50°C for 20 min. + Reduction to Au 0 , Au 0Anisotropically deposited on the surface of the gold seed. With the increase of HQ concentration, the gold seed gradually grows from a sphere to an ellipsoid, a bi-truncated cone and a pointed bi-pyramid, and the color of the solution changes from pink to purple, green and burgundy. Since the higher the Tg concentration, the less HQ is obtained after the enzymatic hydrolysis reaction, so with the increase of Tg concentration, the color of the AuNBPs solution changes from burgundy to pink. The above experimental principle converts the general strategy of HRP-catalyzed TMB color development in commercial ELISA kits into a strategy of HRP-catalyzed oxidation of HQ to regulate the growth of AuNBPs, so that the detection signal is directly transformed from the single-color intensity change of TMB to the multi-color change of AuNBPs, thereby realizing high-sensitivity and high-precision multi-color visualization detection of Tg concentration.

[0082] 6.2 Performance evaluation of AuNBPs growth mediated by AA and hydroquinone reducing agents

[0083] 6.2.1 Effect of pH on the Growth of AuNBPs Mediated by AA and Hydroxyphenol Reductants

[0084] The present invention explores the effect of pH value on the growth of AuNBPs with AA and hydroquinone reducing agents (the method of reducing agent-mediated AuNBPs growth is carried out with reference to Example 2, and the pH value in the system is adjusted by changing the buffer in the reaction system while keeping the concentrations of AA, CC, HQ, and RS unchanged). Figure 2 As shown in A, when the pH value increases from 1.5 to 2.2 under the condition of constant AA concentration, the λ LLSPR From nothing to something and red shift to 782nm, it shows that the increase of pH value in the range of 1.5-2.2 is conducive to the growth of AuNBPs by AA. When the pH value continues to increase, the LLSPR peak begins to become wider and shorter, and the lateral LSPR peak begins to gradually increase, indicating that the increase of pH value is not conducive to the growth of AuNBPs by AA, and other non-AuNBPs nanogold particles begin to appear. The above experimental results show that the optimal pH value for AA to regulate the growth of AuNBPs is about 2.2. Similarly, pH value has a similar influence trend on the growth of AuNBPs by CC, HQ and RS. The optimal pH values ​​for the growth of AuNBPs by the three are 4.5, 6 and 8.3, respectively.

[0085] 6.2.2 Morphological Characterization of AuNBPs Grown by AA and Hydroxyphenol Reductants

[0086] Next, the present invention characterized the morphology of AuNBPs grown by AA and hydroquinone reducing agents by transmission electron microscopy (TEM). Four reducing agents were used to grow AuNBPs with similar λ LLSPR AuNBPs. Figure 2As shown in Figure B, the grown AuNBPs are all biconical in shape, with good dispersion and uniform particle size. It can also be seen from the figure that compared with AA, the AuNBPs grown by CC, HQ and RS are sharper. Next, the present invention further analyzed the size of AuNBPs grown by each reducing agent. As shown in Table 1, when the concentration of AA is 150μM, the concentrations of CC, HQ and RS are 120μM, 110μM and 115μM respectively, the four reducing agents each grow to λ LLSPR The size of AuNBPs is between 747-754 nm. The statistical analysis of the size of AuNBPs shows that the length is HQ>AA>RS>CC, and the width is AA>CC>HQ>RS. + The Au in the solution is reduced and anisotropically deposited on the gold seed surface to complete the growth of AuNBPs. + Au can be spontaneously disproportionated on the surface of AuNBPs 3+ and Au 0 . 3+ The AuNBPs tip is specifically etched, and the Au 0 The deposition on the side of AuNBPs caused the length of AuNBPs to become shorter and the width to become wider, thus hindering the anisotropic growth of AuNBPs. Therefore, when AA and hydroquinone reducing agents grow under their respective optimal pH conditions with the same λ LLSPR AuNBPs: Since the pH value of AA and CC-mediated growth of AuNBPs is acidic, it is favorable for Au + The disproportionation reaction hindered the longitudinal growth of AuNBPs and promoted the lateral growth of AuNBPs, resulting in the need for more reducing agents for the longitudinal growth of AuNBPs and the acquisition of wider AuNBPs. The pH of the AuNBPs mediated by HQ and RS was neutral or alkaline. + The disproportionation reaction of AuNBPs is inhibited, the reducing agent required to grow AuNBPs is reduced, and narrower AuNBPs are obtained. In summary, as the pH value of AuNBPs increases, the growth to the same λ LLSPR The concentration of reducing agent required for AuNBPs decreased, and the morphology of the obtained AuNBPs was sharper.

[0087] Table 1 Synthesis conditions and λ of AuNBPs grown by different reducing agents LLSPR and size statistical analysis

[0088]

[0089] 6.2.3 Stability of AuNBPs grown by AA and hydroquinone reducing agents

[0090] As the signal of multicolor colorimetric analysis, the stability of AuNBPs is an important factor affecting the accuracy of the results. Previous research results in this field have shown that AuNBPs grown by AA are unstable in CTAB solution. + In the CTAB solution, Au + The disproportionation of AuNBPs specifically reduces the aspect ratio of AuNBPs, resulting in their λ LLSPR Therefore, the present invention then investigated the stability of AuNBPs mediated by AA and hydroquinone reducing agents (HQ, CC, RS) at their respective optimal pH values. Figure 2 As shown in C, as the storage time of the growing AuNBPs increases, the λ of the growing AuNBPs mediated by AA (pH 2.2) LLSPR A significant blue shift occurred, and the λ of AuNBPs grown under CC (pH 4.5) LLSPR The blue shift was relatively small, compared with the λ of AuNBPs grown under HQ (pH 6) and RS (pH 8.3). LLSPR The above results show that the higher the pH value of the solution where the AuNBPs are grown, the better their stability. The reason may be that when the pH value of the growth solution is in a neutral / alkaline condition, the Au + Therefore, HQ and RS can be used as reducing agents to mediate the growth of AuNBPs to construct sensors, thereby obtaining stable color signals and λ LLSPR Signal.

[0091] 6.2.4 Working curve and sensitivity study of AuNBPs grown by AA and hydroquinone reducing agents

[0092] The present invention analyzes the performance of AuNBPs grown by AA and hydroquinone reducing agents. Under the respective optimal pH conditions, referring to the method of Example 2, AA and hydroquinone reducing agents (CC, HQ, RS) were used to grow AuNBPs, and their color and λ LLSPR Changes. Figure 3 As shown in Figure 2, when the AA concentration increased from 30 μM to 130 μM, AuNBPs exhibited 10 bright colors (pink, magenta, purple, blue-purple, blue, blue-green, green, gray-green, red-brown, and wine red), λ LLSPR The wavelength red-shifted from 550nm to 735nm and showed a good linear relationship with the AA concentration.

[0093] like Figure 4 As shown, when the CC concentration increased from 30 μM to 100 μM, the λ of AuNBPs LLSPRThe AuNBPs exhibited 10 bright colors (pink, magenta, purple, blue-purple, blue, blue-green, green, gray-green, red-brown, and wine-red) from 548 nm to 751 nm. The CC concentration in the range of 30–100 μM was significantly different from the λ LLSPR There is a good logarithmic relationship.

[0094] like Figure 5 As shown, when the HQ concentration increased from 25 μM to 95 μM, the λ LLSPR From 550nm to 735nm, AuNBPs present 10 bright colors (pink, magenta, purple, blue-purple, blue, blue-green, green, gray-green, red-brown, wine red) and the λ LLSPR There was a good linear relationship with HQ concentration in the range of 25-95μM.

[0095] like Figure 6 As shown, when the RS concentration increased from 24 μM to 100 μM, the λ LLSPR The AuNBPs exhibited 10 bright colors (pink, magenta, purple, blue-purple, blue, blue-green, green, gray-green, red-brown, and wine-red) from 535 nm to 740 nm. The RS concentration ranged from 24 to 100 μM and the λ LLSPR There is a good logarithmic relationship.

[0096] The performance parameters of AuNBPs grown by the above four reducing agents are shown in Table 2. Similar to AA, the AuNBPs solution grown by the hydroquinone reducing agent exhibited similar 10 color changes, and within a certain concentration range, they were consistent with the λ LLSPR There is a good linear / logarithmic relationship. Comparing the linear working curves of HQ and AA, the slope of the HQ working curve is greater than that of AA, indicating that AuNBPs grown by HQ are more sensitive than AuNBPs grown by AA. Therefore, HQ is expected to replace AA to build a more sensitive and stable multicolor colorimetric sensor under neutral conditions.

[0097] Table 2 Calibration curves, correlation coefficients, linear ranges, number of colors and detection limits of AuNBPs growth mediated by 4 reducing agents

[0098]

[0099] 6.2.5 Feasibility Analysis of HRP Catalytic Oxidation of HQ

[0100] The present invention investigates the feasibility of HRP catalyzing the oxidation of HQ to mediate the growth of AuNBPs. Figure 7As shown in the figure, when there is no H2O2 in the sample, the solution color of the AuNBPs growth mediated by HQ is wine red, and the λ LLSPR When HQ and H2O2 were incubated at 50℃ for 30 minutes, the color of the grown AuNBPs did not change significantly, and the λ LLSPR There is only a slight blue shift (724nm), indicating that HQ does not react easily with H2O2. However, in the presence of HRP, the color of AuNBPs turns pink, and the λ LLSPR The obvious blue shift (535nm) indicates that HRP catalyzes the oxidation of HQ by H2O2. In addition, the results of TEM show that with the increase of HRP concentration, AuNBPs gradually change from pointed bipyramids to bitruncated pyramids and spheres, with good dispersion and uniform particle size ( Figure 8 ). The above results show that the method of using HRP to catalyze the oxidation of HQ to mediate the growth of AuNBPs is feasible.

[0101] 6.2.6 Optimization of Tg Detection Conditions Using Multicolor Immunoassay Kit

[0102] As described in the experimental principle, the constructed multicolor immunosensor is based on the HRP catalytic oxidation of HQ, whose content is positively correlated with the Tg concentration, to mediate the growth of AuNBPs, thereby presenting multicolor changes. Therefore, in order to obtain the best detection sensitivity and more color changes, the present invention optimizes the HQ concentration, H2O2 concentration and pH value in the enzymatic hydrolysis reaction. The specific experimental method during optimization refers to Section 4.2 of Example 4, and the corresponding single factor changes can be satisfied. For example, when optimizing the HQ concentration, the HQ concentration in the enzymatic hydrolyzate is adjusted while ensuring that other conditions remain unchanged, and the extinction spectra of the corresponding solutions are detected respectively.

[0103] First, the present invention optimizes the HQ concentration. Fig. 9 As shown in A, when the HQ concentration increased from 0.19 mM to 0.21 mM, the λ LLSPR The solution color changed from blue-green to wine red. When the HQ concentration was greater than 0.21 mM, the λ LLSPR The red shift will continue, but the solution color will only become darker without further color changes. Therefore, considering the sensitivity and the number of colors, 0.21 mM was selected as the optimal HQ concentration.

[0104] Secondly, the concentration of H2O2 was optimized. As a catalytic substrate, the concentration of H2O2 affects the efficiency of HRP catalytic oxidation of HQ. Fig. 9 As shown in B, as the H2O2 concentration increased from 0.2 mM to 1.0 mM, the λ LLSPR Displacement (Δλ LLSPR) increases and reaches the highest point, and then as the H2O2 concentration continues to increase, Δλ LLSPR Therefore, the optimal H2O2 concentration is 1mM.

[0105] Finally, the present invention optimizes the pH value of the enzymatic solution, and the pH value of the solution affects both the HRP activity and the growth of AuNBPs. Fig. 9 As shown in C, when the pH value is 5.5, Δλ LLSPR The maximum value is reached, so the optimum pH value is 5.5. 6.2.7 Performance and sensitivity of the established detection method for detecting Tg

[0106] Under the optimal conditions, the present invention used the established detection method to detect different concentrations of Tg (the detection method refers to Section 4.2 of Example 4). The results are as follows Fig.10 As shown in Figure 2, as the Tg concentration increases from 0 to 1.3 ng / mL, the color of AuNBPs gradually changes from wine red to reddish brown, gray-green, green, blue-green, blue, blue-purple, purple, magenta, and pink. LLSPR Gradually blue shifts from 725nm to 560nm, and Δλ LLSPR It has a good linear relationship with Tg concentration in the range of 0.1-1.3ng / mL. The detection limit is calculated to be 0.06ng / mL using 3σ / S, where σ is the standard deviation of the blank sample and S is the slope of the linear regression equation. Compared with other optical methods for detecting Tg (see Table 3), this detection method presents more colors and has a higher detection sensitivity for Tg. The results once again prove that this detection method has good color resolution and detection sensitivity.

[0107] Table 3 Comparison of optical methods for Tg detection

[0108]

[0109] 6.2.8 Specificity of the established assay for detecting Tg

[0110] In order to evaluate the specificity of the thyroglobulin detection method established by the present invention, the present invention used 1 ng / mL Tg and 10ng / mL of IgG (immunoglobulin G), HSA (human serum albumin), CEA (carcinoembryonic antigen), HER2 (human epidermal growth factor receptor 2 (HER2) The experiment was conducted with IgG, HSA, CEA, HER2 and PB buffer as the detection object (the detection method refers to Example 4, only the samples to be detected were replaced with IgG, HSA, CEA, HER2 and PB buffer respectively). The results are as follows Fig.11 As shown in Figure 2, compared with the blank control group (Blank), the AuNBPs obtained from the sample containing 1 ng / mL Tg produced obvious color changes and Δλ LLSPRFor the other four proteins, even when the concentration was much higher than Tg, the color of the AuNBPs obtained was the same as that of the blank control group (Blank), and the corresponding Δλ LLSPR All are close to 0. The above experimental results show that the multicolor immunosensor constructed using enzymatic hydrolysate, AuNBPs growth solution and commercial human thyroglobulin ELISA detection reagent has good specificity for Tg.

[0111] 6.2.9 Analysis and determination of serum samples

[0112] In order to evaluate the practicality of the multicolor immunosensor of the present invention, the Tg in two serum samples was measured and verified by ELISA kit detection. Fig.12 As shown, samples with different concentrations of Tg added all showed colors corresponding to the Tg concentrations contained in the samples. For example, when the concentration of Tg added was 500pg / mL, the concentration measured by our multicolor immunosensor was 518pg / mL. The results of the other five serum-added samples were basically consistent, with a recovery rate of 94.1-105% and a relative standard deviation (RSD, n=5) of <4%, which was similar to the results measured by the ELISA kit. The experimental results show that the constructed multicolor immunosensor has high accuracy, strong practicality, and reliability, and can be actually used for the detection of Tg concentration in serum samples.

[0113] summary

[0114] In the present invention, the inventors combined the HRP-regulated AuNBPs growth method with a commercial ELISA kit to construct a multicolor immunosensor for visual detection of Tg. First, by optimizing the conditions for growing AuNBPs with AA and hydroquinone reducing agents (CC, HQ and RS) and investigating their performance, it was proved that AA, CC, HQ and RS can grow AuNBPs under acidic, acidic, neutral and alkaline conditions, respectively. As the pH value increases, the grown AuNBPs will be more stable and sensitive, and the morphology will be sharper. Then, Ab1 and Bio-Ab2 in the commercial ELISA kit are used to specifically bind to Tg to form a sandwich structure of Ab1 / Tg / Bio-Ab2. After further modification with St-HRP, the formed Ab1 / Tg / Bio-Ab2 / St-HRP conjugate can catalyze H2O2 oxidation of HQ under the condition of pH 5.5. The obtained enzymatic solution is mixed with the AuNBPs growth solution, and AuNBPs can be directly grown without adjusting the pH. Different concentrations of Tg can consume different concentrations of HQ, resulting in the growth of AuNBPs with different morphologies and multi-color changes, thus realizing multi-color visual detection of Tg. Under optimal conditions, when the Tg concentration is in the range of 0.1-1.3 ng / mL, AuNBPs will show 10 color changes corresponding to the concentration, and the Δλ of AuNBPs is LLSPR It has a good linear relationship with Tg concentration. The visual detection limit of this method is 0.1ng / mL, and the spectral detection limit is 0.06ng / mL. The established immunoassay method can detect the content of Tg in serum spiked samples, with a recovery rate of 94.1-105% and RSD (n=5) <4%. This method has the advantages of simple operation, stable signal, high sensitivity, and can present multi-color changes in a narrow linear range. It can provide a reliable and high visual accuracy multi-color immunoassay method for dynamic monitoring of Tg concentration and prognostic detection. In addition, the successful construction of this multi-color immunosensor also provides a universal strategy for multi-color immunoassay of other targets based on commercial ELISA kits.

[0115] in conclusion

[0116] The present invention takes Tg as the analysis object, and aims at the actual detection demand of Tg concentration (narrow concentration detection range). On the basis of in-depth research and optimization of AuNBPs growth method, a stable, sensitive and multicolor colorimetric immunosensor is constructed, and used for visual detection of Tg concentration in human serum. First, the pH value of AuNBPs growth mediated by AA and hydroquinone reducing agents is optimized. The experimental results show that AA, CC, HQ and RS can grow AuNBPs under acidic, acidic, neutral and alkaline conditions, respectively. In the subsequent performance investigation, it is proved that compared with AA and CC (acidic pH growth conditions), HQ (neutral pH growth conditions) and RS (alkaline pH growth conditions) mediate AuNBPs growth. The AuNBPs morphology is sharper and the solution color is more stable, and the amount of reducing agent consumed is also less. On this basis, HRP catalytic oxidation HQ mediated AuNBPs growth method is established, and it is combined with a commercial ELISA kit to construct a new method for multicolor immunovisual detection of Tg protein. Under optimal conditions, the constructed multicolor immunosensor showed 10 Tg concentration-related color changes in the Tg concentration range of 0.1-1.3 ng / mL. In this Tg concentration range, its concentration was related to the Δλ of AuNBPs. LLSPR It showed good linear correlation, with a visual detection limit of 0.1 ng / mL and a spectral detection limit of 0.06 ng / mL. Using this method, we successfully detected the content of Tg in serum spiked samples by naked eye observation and UV-visible spectrophotometry, with a spike recovery rate of 94.1-105% and a relative standard deviation (RSD, n=5) of <4%. Compared with the reported multicolor immunoassay detection method based on ALP-catalyzed production of AA-mediated AuNBPs growth, the multicolor immunoassay detection method constructed in this chapter does not require the replacement of HRP in the traditional ELISA kit with ALP, which improves the universality of the method; enables the enzyme-catalyzed reaction and AuNBPs growth to be carried out at the same pH value, reducing the experimental difficulty and experimental cost; improves the stability of the AuNBPs signal and ensures the accuracy of the detection; improves the simplicity of experimental operation, does not need to rely on paper-based analytical devices to stabilize the AuNBPs signal, eliminates the operation of assembling PAD, and can be directly completed in the liquid. Therefore, the successful establishment of this method not only provides a reliable and highly visually accurate multicolor immunosensor for the dynamic monitoring of Tg concentration and prognostic detection, but also provides a multicolor analysis method with simple operation, stable signal and high sensitivity for other commercial ELISA kits.

Claims

1. A multicolor immunoassay kit for hydroquinone-mediated nanogold cone growth, the kit comprising an ELISA plate, a PB buffer, a standard solution, a biotin-labeled antibody, and a streptavidin-modified horseradish peroxidase, wherein: The ELISA plate is coated with a capture antibody that specifically binds to the object to be detected, the standard solution is a solution of the object to be detected with a precise concentration, and the biotin-labeled antibody is a detection antibody that specifically binds to the object to be detected and is labeled with biotin. The kit further includes an enzymatic solution and an AuNBPs growth solution; The enzymatic hydrolysis solution comprises the following components: acetate buffer, H2O2, hydroquinone and secondary water; the pH of the enzymatic hydrolysis solution is 5.5, the final concentration of the acetate buffer in the enzymatic hydrolysis solution is 10mM, the final concentration of H2O2 is 1mM, and the final concentration of hydroquinone is 0.21mM; The AuNBPs growth solution includes the following components: CTAB, HAuCl4, AgNO3, NADH, acetate buffer and gold seed; the pH of the AuNBPs growth solution is 6.0, and in the AuNBPs growth solution, the final concentration of CTAB is 180mM, the final concentration of HAuCl4 is 0.5mM, the final concentration of AgNO3 is 0.2mM, the final concentration of NADH is 0.7mM, the final concentration of acetate buffer is 10mM, and each 75μL of AuNBPs growth solution contains 3.0μL of gold seed.

2. The multicolor immunoassay detection kit according to claim 1, characterized in that: The preparation method of the enzymatic hydrolysate is: 7.5 μL of 100 mM acetate buffer solution with a pH of 5.5, 7.5 μL of 10 mM H2O2 aqueous solution, and 7.5 μL of 2.1 mM hydroquinone aqueous solution were sequentially added to 52.5 μL of secondary water, and mixed to obtain 75 μL of the enzymatic hydrolyzate.

3. The multicolor immunoassay detection kit according to claim 1, characterized in that: The preparation method of the AuNBPs growth solution is: 3.75 μL of 200 mM acetate buffer solution with a pH of 6.0, 3.75 μL of 10 mM HAuCl4 aqueous solution, 1.5 μL of 10 mM AgNO3 aqueous solution, and 3 μL of 17.5 mM NADH aqueous solution were sequentially added to 60 μL of 225 mM CTAB aqueous solution, mixed at room temperature for 1 min, and then 3.0 μL of gold seed was added to obtain 75 μL of the AuNBPs growth solution; The preparation method of the gold seed is: First, 10 mL of a solution containing 50 mM CTAC, 5 mM citric acid, and 0.25 mM HAuCl4 was added to a glass bottle. Then, under room temperature and vigorous stirring at 1000 rpm, a freshly prepared NaBH4 solution (0.25 mL, 25 mM) was quickly added to the solution. NaBH4 3+ Rapidly reduced to gold atoms; 2 minutes later, the solution was incubated at 80°C for 90 minutes to form gold seeds, and the seed solution was stored at 4°C in the dark.

4. The multicolor immunoassay detection kit according to claim 1, characterized in that: It is a kit for detecting thyroglobulin. The enzyme label plate is coated with a capture antibody that specifically binds to thyroglobulin, the standard solution is a thyroglobulin solution with a precise concentration, and the biotin-labeled antibody is a biotin-labeled detection antibody that specifically binds to thyroglobulin.

5. A method for detecting thyroglobulin using the multicolor immunoassay detection kit according to claim 4, characterized in that: It includes the following steps: (1) Antibody-antigen-antibody-horseradish peroxidase binding: First, add 100 μL of Tg standard solution or sample solution diluted with PB buffer to different concentrations into the reaction well of the enzyme label plate coated with the capture antibody, seal the well plate with a sealing film, incubate it on a 37°C oscillator for 90 minutes, and then rinse it with PB buffer three times; then add 100 μL of biotin-labeled antibody working solution into the well, seal the well plate with a sealing film, incubate it at 37°C for 60 minutes, and then rinse it with PB buffer three times; then add 100 μL of streptavidin-modified horseradish peroxidase conjugate, seal the well plate with a sealing film, incubate it at 37°C for 30 minutes, and then rinse it with PB buffer three times; (2) Horseradish peroxidase-catalyzed oxidation of hydroquinone: Add 75 μL of enzyme solution to the reaction wells of the ELISA plate after step (1), seal the plate with a sealing film and incubate at 37°C for 30 minutes; (3) AuNBPs growth: Finally, add 75 μL of AuNBPs growth solution to the enzymatic solution, mix well and react at 50°C for 20 minutes on an oscillator. After the reaction, take out the ELISA plate and observe the color of the solution with the naked eye, and use an ELISA reader to measure the extinction spectrum of the solution in the range of 400-1000 nm. Calculate the Tg content based on the color observed with the naked eye or based on the wavelength of the maximum absorption peak.

6. Use of the multicolor immunoassay detection kit as claimed in claim 4 in detecting thyroglobulin.

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