Method for detecting cTn I by using platinum nanodot probe based on uncoupled self-enrichment signal amplification

Abstract

The invention relates to a method for detecting cTn I by using a platinum nanodot probe based on uncoupled self-enrichment signal amplification. The tyramine modified platinum nanodot is prepared by a one-step hydrothermal method without any coupling reagent. In the presence of hydrogen peroxide, tyramine on the surfaces of the platinum nanodots can be activated into free radicals with high reaction characteristics, and the free radicals are further covalently bound with protein in plate holes under mild conditions. Due to the characteristics of enzyme activatability and high catalytic capacity of the platinum nanodots, the cTnI is quantitatively detected and analyzed by combining an elisa plate as a high-flux tyramine signal amplification carrier. The method comprises the following steps: preparation and purification of tyramine modified platinum nanodots, activation and self-enrichment of the platinum nanodots as a tyramine signal amplification unit, and construction of the platinum nanodot probe based on uncoupled self-enrichment signal amplification, wherein the content of cTn I is quantified with high sensitivity. The detection method disclosed by the invention is simple to operate, high in sensitivity and strong in specificity, and can realize high-throughput nondestructive detection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for detecting cTnI using a platinum nanodot probe based on non-coupling self-enrichment signal amplification. Background Art

[0002] As the incidence of cardiovascular diseases continues to rise, the detection technology of myocardial injury markers has also received increasing attention from the medical community. Numerous studies have revealed that once myocardial cells are damaged or necrotic, cardiac troponin will be released into the blood. Among these proteins, cardiac troponin I (cTn I) has played an important role in the diagnosis and risk assessment of cardiovascular diseases since its discovery in the 1990s due to its high sensitivity and specificity. At present, clinical detection of cTn I mainly uses immunological methods, including enzyme-linked immunosorbent assay, chemiluminescent immunoassay, immunochromatography, and fluorescent immunosorbent assay. Although these immunoassay techniques have a series of significant advantages such as high throughput, high specificity, and high sensitivity, conventional immunoassay methods are still incapable of detecting low-level biomarkers. Therefore, further improving the sensitivity of detection technology in order to more accurately monitor subtle changes in cardiac troponin is of great significance for providing patients with more accurate auxiliary diagnostic information.

[0003] Among the current quantitative detection methods, tyramide signal amplification strategy (TSA) has attracted widespread attention due to its ability to significantly improve sensitivity and accuracy. Functional HRP and tyramide converting enzyme based on tyramide and its derivatives have been cleverly designed into signal amplification units and successfully applied to the high-sensitivity detection of markers, providing a powerful tool for biomedical research. However, the preparation process of tyramide-coupled bioenzyme signal amplification units is not smooth sailing. It involves at least three cumbersome steps: pre-activation of bioenzymes, coupling reaction, and purification of the complex. Among them, the coupling reaction requires expensive coupling agents, which undoubtedly increases the cost of the experiment. What is more difficult is that tyramide coupling reactions usually require precise control of pH and temperature, which makes the entire coupling process not only time-consuming and complex, but also easily leads to a decrease in the catalytic activity of bioenzymes, thereby affecting the accuracy of the experimental results. In order to overcome these difficulties, click chemistry coupling technology came into being, which significantly simplified the operation process and improved the experimental efficiency. But even so, the cost of using bioenzymes is still high, and click chemistry reagents need to be modified in advance. These problems have restricted the widespread application of TSA technology in the field of signal amplification detection to a certain extent. Therefore, the ability to circumvent the use of biological enzymes and complex coupling steps has important clinical application prospects for the high-sensitivity detection of cTn I by TSA technology. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for detecting cTn I by using a platinum nanodot probe based on non-coupled self-enrichment signal amplification. Based on the one-step synthesis and modification of platinum nanodots and their efficient peroxide mimic enzyme performance, this method improves the sensitivity of cTn I detection. That is, through the sandwich structure formed by cTnI and its antibody, the platinum nanodots activated by HRP labeled antibodies further bind to and enrich the proteins in the ELISA plate, and the quantitative analysis of cTn I is achieved by combining efficient and rapid catalytic reactions. The method is easy to operate, has high sensitivity, strong specificity, can achieve high throughput, precise targeting and non-destructive detection, etc.

[0005] A method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification, the method comprising the following steps:

[0006] a. One-step preparation of platinum nanodots;

[0007] b. The cTn I capture antibody was coated in the ELISA plate, and different concentrations of cTn I standards were added after blocking. After capture, HRP-labeled detection antibody was added. After washing, platinum nanodots and H2O2 were added. After color development, a self-enrichment signal amplification strategy based on platinum nanodots was established to quantify cTn I.

[0008] c. Draw a standard curve with the logarithm of the cTn I standard concentration as the horizontal axis and the absorbance intensity value as the vertical axis, so as to quantitatively detect the concentration of cTn I.

[0009] The linear equation of the standard curve is Y=1.92+0.93C, where Y represents the ultraviolet absorption intensity and C represents the concentration of cTn I. The correlation coefficient R2=0.992, the linear range is 0.012-0.19 ng / mL, and the detection limit is 3 pg / mL.

[0010] The step a specifically comprises the following steps:

[0011] a-1. Preparation of platinum nanodots: First, centrifuge potassium chloroplatinate to remove particulate matter. Then, filter and separate 1 mL of tyrosine solution. Mix 28 mL of 20 mM potassium chloroplatinate with 2 mL of 60 mM tyrosine, stir at room temperature for 10 min, continue to heat to boiling, quickly inject 0.1 mL of 0.5% sodium borohydride, stir vigorously for 5 min, and cool to room temperature to obtain tyrosine-functionalized platinum nanodots.

[0012] a-2. Purification of platinum nanodots: Place the platinum nanodot solution in a centrifuge tube and centrifuge at 20,000 rpm / min for 30 min to remove unreacted precursors and other ions in the supernatant. Disperse the obtained platinum nanodots in ultrapure water and store at 4°C.

[0013] In step a, tyrosine-modified platinum nanodots are synthesized according to a one-step hydrothermal method and used as tyramide signal amplification units; compared with the tyramide units obtained by the traditional coupling method, this method has the characteristics of one-step preparation and the technical advantage of not requiring coupling reagents.

[0014] The step b specifically comprises the following steps:

[0015] b-1. Formation of antibody-cTn I-antibody complex: 10 ng / mL of capture antibody was coated on the ELISA plate, and after blocking, cTn I antigen was added for incubation, and then HRP-labeled capture antibody was added for incubation to form an antibody-cTn I-antibody sandwich structure;

[0016] b-2. Activation of platinum nanodots: Add 0.05 mL of platinum nanodots to the ELISA plate that has formed a sandwich structure, quickly add 0.01 mL of 2 mM H2O2, and activate at room temperature for 2 min;

[0017] b-3. Self-enrichment of platinum nanodots: The activated system was placed in a 37-degree incubator for further reaction for 5 minutes to promote the enrichment of platinum nanodots in the ELISA plate;

[0018] b-4. Detection: Wash with PBST solution three times to remove unbound platinum nanodots, add color developing solution, and measure the optical density of each well at 490 nm using a multifunctional microplate reader.

[0019] In the step b, the analyte cTn I is first formed into a sandwich structure complex in the ELISA plate; HRP-labeled antibodies are used to catalyze H2O2 to produce reactive oxygen species, further activating the platinum nanodots; subsequently, the tyramide-modified platinum nanodots are self-assembled and enriched with the proteins in the wells of the ELISA plate; compared with the traditional enzyme-catalyzed colorimetric method, this method introduces platinum nanodot nanozymes to increase the effective catalytic units of the system, achieves efficient signal amplification, and has the technical advantage of improving detection sensitivity.

[0020] Compared with the prior art, the present invention has the following characteristics:

[0021] (1) Enzyme-activatable platinum nanodots were prepared by one-step hydrothermal method using tyrosine as a preservative and protectant, providing a new method for the facile preparation of tyramide signal amplification units;

[0022] (2) The ELISA plate was used as an effective carrier for signal amplification of tyramide-modified platinum nanodots, which endowed the tyramide signal amplification strategy with universality;

[0023] (3) The high catalytic activity and enzyme-activatable properties of platinum nanodots combined with high-throughput ELISA plate carriers provide a new strategy for the highly sensitive detection of cTnI.

[0024] (4) This method is easy to operate, highly sensitive, and specific, and can achieve high-throughput nondestructive quantitative detection.

[0025] The present invention provides a method for detecting cTnI by using platinum nanodot probes based on non-coupled self-enrichment signal amplification, which combines the excellent catalytic activity of platinum nanozymes and the characteristics of enzyme activation-initiated tyramine-modified platinum nanodots self-enrichment to achieve high-sensitivity detection of cTnI. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The standard curve is established with the concentration of cTn I standard as the horizontal axis and the absorbance intensity as the vertical axis;

[0027] Figure 2 This is a transmission electron microscope characterization image of platinum nanodots;

[0028] Figure 3 The size characterization diagram of platinum nanodots

[0029] Figure 4 X-ray powder diffraction characterization of platinum nanodots

[0030] Figure 5 X-ray photoelectron spectroscopy characterization of platinum nanodots

[0031] Figure 6 Fourier transform infrared spectroscopy characterization of platinum nanodots

[0032] Figure 7 Transmission electron microscopy characterization of the enrichment of platinum nanodots in the presence of hydrogen peroxide

[0033] Figure 8 This is the UV-visible spectrum of platinum nanodots catalyzing the color development reaction of hydrogen peroxide-o-phenylenediamine system. DETAILED DESCRIPTION

[0034] The platinum nanodots used in the experiment were synthesized by our laboratory, and other reagents can be purchased from commercial manufacturers.

[0035] The preparation method of the buffer solution and washing solution involved in the present invention is:

[0036] Phosphate buffer solution (1 mM, pH = 7.4): weigh 1 g of sodium chloride, 0.125 g of potassium chloride, 0.625 g of sodium dihydrogen phosphate, and 0.4 g of disodium hydrogen phosphate and dissolve them in deionized water and make up to 125 mL;

[0037] Carbonate buffer (0.5 mM, pH = 9.6): weigh 4.5 g of sodium bicarbonate and 2 g of sodium carbonate, dissolve in distilled water and make up to 125 mL;

[0038] Phosphate wash solution: Take 0.1 mL of Tween 20 and slowly push it into 1 mM, 200 mL of phosphate buffer. Shake well and set aside.

[0039] Example 1

[0040] A method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification, comprising the following steps:

[0041] a. One-step preparation of platinum nanodots:

[0042] a-1. Purification of potassium chloroplatinate: transfer 1 mL of potassium chloroplatinate into a 5 mL centrifuge tube, centrifuge at 15000 rpm / min for 30 min, and take 0.8 mL into a clean glass bottle for later use;

[0043] a-2. Preparation of tyrosine stock solution: Take 1 mg of tyrosine and dissolve it into a 1 mg / mL solution with freshly prepared acetate buffer and store at 4°C for later use.

[0044] a-3. One-step preparation of platinum nanodots: 28 mL of 20 mM potassium chloroplatinate and 2 mL of 60 mM tyrosine were mixed, stirred at room temperature for 10 min, and then heated to boiling. 0.1 mL of 0.5% sodium borohydride was quickly injected, and the mixture was stirred vigorously for 5 min. After cooling to room temperature, tyrosine-modified platinum nanodots were obtained.

[0045] b. The capture antibody is coated in the ELISA plate, and different concentrations of cTnI standards are added after blocking. After incubation, HRP-labeled detection antibodies are added. In the presence of H2O2, the platinum nanodots are activated and further self-enriched in the ELISA plate. The platinum nanodots are used as signal amplification units to establish a platinum nanodot-catalyzed sandwich immunoquantitative detection method for cTn I. The enzyme-activated self-assembly characteristics of tyramide-modified platinum nanodots and the high specificity of antigen-antibody reactions are utilized to achieve the purpose of quantitative detection of cTn I by detecting the signal of substrate color development. This method has higher sensitivity than the detection of biological enzyme methods.

[0046] The step b specifically comprises the following steps:

[0047] b-1. Capture antibody coating: dilute the capture antibody with carbonate buffer, add 100 μL to each well, and incubate in a 37°C oven for 1 to 2 hours;

[0048] b-2. Add standard samples: After washing with PBS solution for 3 times, add 1.56, 0.78, 0.39, 0.19, 0.097, 0.049, 0.024, 0.012, 0.006 and 0.003 ng / mL of cTn I standard to each well, respectively, and incubate in a 37°C oven for 1 to 2 hours;

[0049] b-3. Add detection antibody: After washing, add 100 μL of detection antibody to each well and incubate in a 37°C oven for 1 to 2 hours to form a sandwich structure;

[0050] b-4. Activation of platinum nanodots: Add 0.05 mL of platinum nanodots to the ELISA plate that has formed a sandwich structure, quickly add 0.01 mL of 2 mM H2O2, and activate at room temperature for 2 min;

[0051] b-5. Self-enrichment of platinum nanodots: The activated system was placed in a 37-degree incubator and continued to react for 5 minutes to promote the binding of platinum nanodots with proteins in the ELISA plate;

[0052] b-6. Color development and detection: Wash with PBS solution three times to remove unbound platinum nanodots, add color development solution, and measure the optical density of each well at 490 nm using a multifunctional microplate reader.

[0053] c. Establish a standard curve with the concentration of cTn I standard solution as the horizontal axis and the absorbance intensity as the vertical axis. The obtained standard curve is as follows: Figure 1 As shown, the linear equation of the standard curve is: Y = 1.92 + 0.93C, where Y represents the UV absorbance intensity and C is the cTnI concentration. The correlation coefficient R2 = 0.992, the linear range is 0.012-0.19 ng / mL, and the detection limit is 3 pg / mL.

[0054] Repeat the above steps, except that the cTn I standard solutions of different concentrations in step b-2 are replaced with cTn I test samples of unknown concentration, and then the absorbance intensity of each well at 490 nm is measured on a microplate reader, and the average absorbance intensity is calculated. The concentration of cTn I in the test sample can be calculated based on the above standard curve.

[0055] The above method is the best experimental method after multiple experimental verifications. The standard curve obtained by this method has the best linear relationship and the widest linear range.

[0056] refer to Figure 2-8 As shown: Figure 5 The successful preparation of platinum nanodots can be demonstrated;

[0057] Figure 6 It was demonstrated that the surface of platinum nanodots contained tyrosine;

[0058] Figure 7 It was demonstrated that platinum nanodots can be activated and enriched by enzymes;

[0059] Figure 8 It was demonstrated that platinum nanodots have the properties of mimicking enzyme catalysis.

[0060] The detailed description of the method for detecting cTn I by using a platinum nanodot probe with non-coupled self-enrichment signal amplification with reference to the above-mentioned embodiment is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification, characterized in that: The following steps are involved: a. One-step preparation of platinum nanodots; b. The cTn I capture antibody was coated on the ELISA plate, and different concentrations of cTn I standard were added. After the antigen-antibody reaction, the HRP-labeled detection antibody was added to form a sandwich structure. After washing, platinum nanodots and H2O2 were added to establish a platinum nanodot self-enrichment signal amplification strategy for quantifying cTn I. c. Draw a standard curve with the logarithm of the cTn I standard concentration as the horizontal axis and the absorbance intensity value as the vertical axis, so as to quantitatively detect the concentration of the cTn I to be tested.

2. The method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification according to claim 1, characterized in that: Step a specifically includes the following steps: a-1. Purification of potassium chloroplatinate: transfer 1 mL of potassium chloroplatinate into a 5 mL centrifuge tube, centrifuge at 15000 rpm / min for 30 min, and take 0.8 mL into a clean glass bottle for later use; a-2. Preparation of tyrosine mother solution: Take 1 mg of tyrosine and dissolve it into a 1 mg / mL solution with freshly prepared acetate buffer and store at 4°C for later use; a-3. One-step preparation of platinum nanodots: Mix 28 mL, 20 mM potassium chloroplatinate and 2 mL, 60 mM tyrosine, stir at room temperature for 10 min, continue heating to boiling, quickly inject 0.1 mL, 0.5% sodium borohydride, stir vigorously for 5 min, and cool to room temperature to obtain tyrosine-functionalized platinum nanodots.

3. The method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification according to claim 2, characterized in that: Step b specifically includes the following steps: b-1. Activation of platinum nanodots: Add 0.05 mL of platinum nanodots to the ELISA plate that has formed a sandwich structure, quickly add 0.01 mL of 2 mM H2O2, and activate at room temperature for 2 min; b-2. Self-enrichment of platinum nanodots: The activated system was placed in a 37-degree incubator for 5 minutes to promote the binding of platinum nanodots with proteins in the ELISA plate; b-3. Color development and detection: Wash the ELISA plate three times with PBST solution to remove unbound nanodots, add o-phenylenediamine colorimetric solution, terminate the reaction, and measure the optical density of each well on a multifunctional ELISA reader.

4. The method for detecting cTn I based on platinum nanodot probes based on uncoupled self-enrichment signal amplification according to claim 3, characterized in that: The linear equation of the standard curve in step c is Y=1.92+0.93C, wherein Y is the absorbance intensity value and C is the cTn I concentration.

5. The method for detecting cTn I based on platinum nanodot probes with uncoupled self-enrichment signal amplification according to claim 4, characterized in that: The correlation coefficient of the linear equation is R2=0.992, the linear range is 0.012-0.19 ng / mL, and the detection limit is 3 pg / mL.

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