A kind of detection reagent and method of cardiac troponin I

Abstract

The present invention provides a detection reagent for cardiac troponin I, comprising: a cTnI aptamer labeled with a first marker and a horseradish peroxidase covalent linker labeled with a second marker; the first marker and the second marker can be combined. The present invention also provides the application of the detection reagent, a detection kit and a detection method. The present invention uses a cTnI aptamer instead of a signal antibody to detect cTnI. As a molecular recognition element, nucleic acid aptamers have many excellent properties such as simple in vitro synthesis, good stability, and easy sequence design and modification, thereby reducing the difficulty of preparation while ensuring sensitivity. There is no need for complex and high-cost DNA amplification technology and nanomaterial synthesis and modification, and the method is simple and low-cost. Furthermore, the present application adds albumin as a supporting agent to the signal aptamer solution, which significantly improves the sensitivity, repeatability, applicability to complex samples and stability of the method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a detection reagent and method for cardiac troponin I. Background Art

[0002] Early diagnosis of cardiovascular diseases (such as myocardial infarction) is crucial to improving patient survival and quality of life after illness. In the past decade, people have continuously developed simple, sensitive and rapid detection methods for various cardiovascular disease biomarkers. The antibody-based enzyme-linked immunosorbent assay (ELISA) is the most common disease marker detection strategy on the market. Its basic steps are: an antibody (capture antibody) is coated on the stationary phase by adsorption, and the target antigen to be tested and another antibody labeled with a biological enzyme (signal antibody) are added in turn to form a capture antibody-antigen-signal antibody sandwich structure. Finally, the enzyme-catalyzed reaction is used to produce a colored product and the antigen content is calibrated with the help of a UV spectrophotometer or an enzyme marker.

[0003] Cardiac troponin I (cTnI) is one of the important biomarkers that can reflect early myocardial injury. In recent years, people have screened and obtained a number of cTnI aptamers, reported aptamer sensing methods and devices for detecting cTnI, and adopted a variety of signal reading methods such as ultraviolet absorption, fluorescence, Raman, electrochemiluminescence, and electrochemistry. In addition, the introduction of nanomaterials and DNA amplification technology further improved the detection sensitivity. However, while pursuing high sensitivity, people also face many problems, such as complex interface modification and operation processes, unstable micro-nanomaterial preparation, modification and use, etc., which seriously weaken the repeatability of aptamer methods or devices and the possibility of future commercialization. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a detection reagent and method for cardiac troponin I with high sensitivity, good repeatability and good stability.

[0005] Aptamers are artificially screened DNA or RNA chains that can specifically bind to ligands (such as proteins, small molecules, etc.). Their binding constants with ligands can reach nanomolar or even picomolar levels, which is equivalent to monoclonal antibodies. They can recognize and bind to target ligands simultaneously with antibodies, which can also be called hybrid recognition.

[0006] Compared with the antibody-based ELISA method, nucleic acid aptamers as molecular recognition elements have many excellent properties such as simple in vitro synthesis, good stability, easy sequence design and modification, etc. Therefore, combined with mature nucleic acid chemical synthesis technology, people have developed a biomarker detection method based on nucleic acid aptamers. Based on the mature enzyme-linked immunosorbent assay method, this application replaces the signal antibody with a signal aptamer, and constructs an analytical method based on hybrid recognition of antibodies and aptamers for detecting cTnI. For the first time, by additionally introducing supporting proteins into the signal aptamer solution, an effective path that can effectively improve the sensitivity, repeatability and practicality of the hybrid detection method for complex samples is obtained.

[0007] The present invention provides a detection reagent for cardiac troponin I, comprising: a cTnI aptamer labeled with a first marker and a horseradish peroxidase covalent linker labeled with a second marker;

[0008] The first label and the second label can bind.

[0009] The present invention uses cTnI aptamers instead of signal antibodies to detect cTnI, which has high sensitivity, good repeatability and good stability.

[0010] In some possible implementations, the detection reagent further includes a support agent, and the support agent is selected from bovine serum albumin. The inventors of the present application unexpectedly discovered that, on the basis of replacing the signal antibody with the cTnI aptamer, adding albumin, especially bovine serum albumin (BSA) to the cTnI aptamer solution can greatly promote the molecular recognition of cTnI by the aptamer and the capture antibody, thereby further improving the sensitivity of the detection.

[0011] In some possible implementations, the support agent is selected from albumin, such as bovine serum albumin, human serum albumin, etc. In some possible implementations, the working concentration of the support agent is 0.002% to 5%. In some possible implementations, the working concentration of the support agent is 0.02% to 5%. In some possible implementations, the working concentration of the support agent is 0.2% to 2%. In some possible implementations, the working concentration of the support agent is 2%.

[0012] The detection reagent provided by the present invention also includes: capture antibody; enzyme catalysis reactant; diluent; cleaning agent; blocking agent and reaction termination solution.

[0013] In some possible implementations, the first label is selected from biotin; the second label is selected from streptavidin. Biotin and streptavidin can be combined so that the cTnI aptamer is linked to horseradish peroxidase to generate a signal aptamer.

[0014] The present application has no special restrictions on the cTnI aptamer, which preferably has a base sequence shown in SEQ ID No. 1 or SEQ ID No. 2. The sequence shown in SEQ ID No. 1 is specifically:

[0015] 5'-TTTTTTCGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3';

[0016] The specific sequence of SEQ ID No.2 is:

[0017] 5'-TTTTTTCGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'.

[0018] In some possible implementation schemes, the enzyme-catalyzed reactant is selected from hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB); the diluent is selected from PBS diluent; the cleaning agent is selected from Tween 20; the blocking agent is selected from bovine serum albumin; and the reaction termination solution is selected from 2M sulfuric acid.

[0019] In some specific implementation schemes, a typical composition of the reagent composition for detecting cTnI is: capture antibody; biotin-labeled cTnI aptamer (Biotin-apt); streptavidin-labeled horseradish peroxidase covalent linker (S-HRP); enzyme catalytic reactants TMB and H2O2; diluent PBS solution; cleaning agent Tween 20; blocking agent bovine serum albumin, reaction termination solution 2M sulfuric acid and supporting agent bovine serum albumin.

[0020] Wherein, the PBS solution includes 10 mM phosphate and 137 mM NaCl, and the pH value is 7.2-7.4;

[0021] The working concentration of the capture antibody is 3 μg / mL, and the solvent is PBS solution;

[0022] The working concentration of the biotin-labeled cTnI aptamer is 20 nM, and the solvent is PBS solution;

[0023] The working concentration of the streptavidin-labeled horseradish peroxidase covalent linker is 0.55 μg / mL, and the solvent is PBS solution;

[0024] The working concentration of the enzyme-catalyzed reactant TMB is 2.16 mM, and the solvent is 20 mM H2SO4; the working concentration of H2O2 is 0.035%, and the solvent is acetic acid buffer solution (0.14 M, pH 5.8);

[0025] The working concentration of the cleaning agent is 0.05%, and the solvent is PBS solution;

[0026] The working concentration of the blocking agent bovine serum albumin was 10%, and the solvent was PBS solution;

[0027] The working concentration of the supporting agent bovine serum albumin is 2%, and the solvent is PBS solution.

[0028] In the above reagents, bovine serum albumin is used as a blocking agent and a supporting agent at the same time, and its use time and role are different: bovine serum albumin is added as a supporting agent to the signal aptamer solution formed by the biotin-labeled cTnI aptamer and the streptavidin-labeled horseradish peroxidase covalent linker to improve the detection sensitivity; it is added as a blocking agent to the high-binding well plate bound with the capture antibody to block the unreacted binding sites on the well plate.

[0029] The present invention also provides a kit, comprising the reagent described in the above technical solution.

[0030] The detection principle of the reagent and kit provided by the present invention is as follows Figure 1 As shown, Figure 1 Schematic diagram of the principle of detection by the reagent provided by the present invention: the capture antibody is coated on the substrate of a high-binding force 96-well plate by adsorption, and then a mixed solution of cTnI and a signal aptamer is added to form a sandwich structure of capture antibody-cTnI-signal aptamer, wherein the signal aptamer is generated by the biotin-labeled cTnI aptamer (Biotin-apt) and the streptavidin-horseradish peroxidase covalent linker (S-HRP) through the binding action of biotin and streptavidin, and the horseradish peroxidase is used to catalyze hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce a colored product (TMB 2+ ), and the cTnI content was calibrated with the aid of an enzyme marker.

[0031] The present invention also provides a method for detecting cardiac troponin I, which uses the detection reagent or detection kit described in the above technical solution for detection.

[0032] According to the traditional ELISA operation steps, the antigen must first bind to the capture antibody and then react with the signal antibody to form a sandwich structure. However, for the hybrid recognition system of the present invention, the recognition order of the biomarker cTnI and the molecular recognition element (antibody and aptamer), that is, the sample addition method, directly affects the response strength of the method. Therefore, in addition to the traditional sample addition method, that is, cTnI first reacts with the capture antibody on the well plate substrate and then introduces the signal aptamer, the inventors of the present application creatively discovered that the second sample addition method is more conducive to the response of the reagent or kit to low concentration (≤40ng / mL) of cTnI, and the linear response range obtained is 5 to 40ng / mL, wherein the second sample addition method is: cTnI interacts with the signal aptamer in a homogeneous solution, and then is injected into the well plate incubated with the capture antibody to bind to the capture antibody.

[0033] In a large number of interface sensing methods, bovine serum albumin (BSA) is often used as an interface blocker to remove non-specific adsorption, reduce detection background and improve sensitivity. In the present invention, the well plate for incubating antibodies is also fully blocked with 10% BSA (well plate capacity 300 μL), and the supporting protein BSA is additionally introduced into the signal aptamer solution, which significantly improves the sensitivity, repeatability and applicability of the method to complex samples.

[0034] The present invention detects cTnI by replacing the signal antibody with a cTnI aptamer. As a molecular recognition element, the nucleic acid aptamer has many excellent properties such as simple in vitro synthesis, good stability, and easy sequence design and modification, thereby reducing the difficulty of preparation while ensuring sensitivity. It does not require complex and costly DNA amplification technology and nanomaterial synthesis and modification, and the method is simple and low-cost. Furthermore, the present application adds albumin as a supporting agent to the signal aptamer solution, which significantly improves the sensitivity, repeatability, applicability to complex samples, and stability of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of the principle of detection by the reagent provided by the present invention, wherein Biotin-apt represents a biotin-labeled cTnI aptamer sequence, S-HRP is a streptavidin-horseradish peroxidase covalent linker, and TMB is a commonly used enzyme reaction substrate 3,3',5,5'-tetramethylbenzidine;

[0036] Figure 2 The test results of cTnI under different sample addition methods;

[0037] Figure 3 The test results of cTnI before and after the introduction of BSA into the signal aptamer solution;

[0038] Figure 4The response changes of cTnI (20 ng / mL) after introducing different concentrations of BSA into the signal aptamer solution;

[0039] Figure 5 Response to potential interfering substances;

[0040] Figure 6 The responses to cTnI (20 ng / mL) before and after the introduction of BSA into different signal aptamer solutions. DETAILED DESCRIPTION

[0041] The present invention provides a detection reagent and method for cardiac troponin I. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] In the following examples, the capture antibody is a mouse cTnI monoclonal antibody purchased from Shanghai Lingchao Biotechnology Co., Ltd.;

[0043] The sequence of cTnI aptamer 1 is shown in SEQ ID No. 1, specifically: 5′-TTTTTTCGCATGCCAAACGTTGCCTCATAGTTCCCTCCCCGTGTCC-3′;

[0044] The sequence of cTnI aptamer 2 is shown in SEQ ID No. 2, specifically: 5′-TTTTTTCGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3′;

[0045] The biotin-labeled cTnI aptamer was synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

[0046] Streptavidin-horseradish peroxidase covalent conjugate was prepared and provided by Sangon Biotechnology (Shanghai) Co., Ltd.

[0047] Example 1

[0048] 1) Prepare the required solution, mainly including:

[0049] PBS diluent: 10 mM phosphate, 137 mM NaCl, pH 7.2-7.4;

[0050] PBST washing solution: PBS solution containing 0.05% Tween 20;

[0051] Enzyme catalytic reaction solution: 3,3',5,5'-tetramethylbenzidine (TMB) (2.16 mM) and H2O2 (0.035%);

[0052] BSA solution: PBS solution containing 10% BSA;

[0053] Reaction termination solution: 2M sulfuric acid.

[0054] 2) Add 100 μL of PBS solution containing 3 μg / mL capture antibody to the high binding capacity well plate, incubate at 4°C overnight, and then wash three times with PBST.

[0055] 3) Add 300 μL of PBS solution containing 10% BSA for interface blocking treatment, incubate at 37° C. for 2 hours, and wash three times with PBST for later use.

[0056] 4) A 10 μM biotin-labeled cTnI aptamer 1 (Biotin-apt) solution was prepared using PBS diluent, heated at 95°C for 3 minutes and then cooled to room temperature; then a signal aptamer solution was prepared, wherein the DNA chain concentration of the biotin-labeled cTnI aptamer was 160 nM, and the concentration of the streptavidin-horseradish peroxidase covalent linker (S-HRP) was 4.4 μg / mL, and the two were mixed and shaken at room temperature for 1 hour.

[0057] 5) A certain concentration of cTnI was mixed with the signal aptamer solution in step 4) above, with the final concentrations of DNA chain and S-HRP being 20 nM and 0.55 μg / mL, respectively, and reacted at room temperature for 1 hour.

[0058] 6) 100 μL of the solution in step 5) above was injected into the wells coated with the capture antibody obtained in step 3), reacted at 37° C. for 1 hour, and then washed four times with PBST.

[0059] 7) adding enzyme-catalyzed reaction substrates, including 50 μL TMB (2.16 mM) and 50 μL H2O2 (0.035%), reacting at room temperature for 8 minutes, adding 50 μL of reaction stop solution, and obtaining the absorbance value at 450 nm using a microplate reader.

[0060] Example 2

[0061] The difference from Example 1 is:

[0062] 5) Inject 100 μL of a certain concentration of cTnI solution into the wells coated with the capture antibody obtained in step 3), react at 37° C. for 1 hour, and then wash four times with PBST;

[0063] 6) Inject the signal aptamer solution in step 4) into the wells in step 5) and mix them. The final concentrations of DNA chain and S-HRP are 20 nM and 0.55 μg / mL, respectively. React at room temperature for 1 hour.

[0064] Results Figure 2 , Figure 2 The test results of cTnI under different loading methods in Examples 1 and 2 are shown, wherein method a is that cTnI first interacts with the capture antibody on the well plate substrate, and then the signal aptamer is introduced (i.e., the loading method in Example 2), and b is that cTnI first interacts with the signal aptamer in a homogeneous solution, and then is injected into the well plate incubated with the antibody (i.e., the loading method in Example 1). Line b' reflects the linear response relationship of the detection system to cTnI under the new loading method b (5-40 ng / mL). The error bars in the figure are derived from the standard deviation of three sets of parallel samples, and the error bars in the following figures are the same.

[0065] Depend on Figure 2 It can be seen that compared with the traditional sample addition method (a), the sample addition method (b) in which cTnI first binds to the aptamer is more conducive to the response of the system to low concentration (≤40 ng / mL) cTnI, and the linear response range obtained is 5-40 ng / mL ( Figure 2 b'), the linear equation is y = 0.2660 + 0.0127x (x represents the concentration of cTnI, y is the absorbance value of the system under different concentrations of cTnI, R 2 =0.9925), and the detection limit was calculated to be 3.59 ng / mL (signal-to-noise ratio was 3, and the number of background samples was 11). This result shows that the binding of antibody to cTnI will seriously affect the recognition of cTnI by aptamer in space, while the aptamer has little effect on the recognition of cTnI by antibody.

[0066] Example 3

[0067] The difference from Example 1 is:

[0068] 5) A certain concentration of cTnI solution and supporting protein BSA were mixed with the signal aptamer solution in step 4) above, the final concentrations of DNA chain and S-HRP were 20 nM and 0.55 μg / mL respectively, the concentration of BSA was 2%, and the reaction was carried out at room temperature for 1 hour.

[0069] Results Figure 3 , Figure 3The test results of cTnI before and after the introduction of BSA into the signal aptamer solution, where b is the interaction between cTnI and the signal aptamer in a homogeneous solution before injection into the well plate incubated with antibodies, and c is the introduction of 2% BSA, a supporting protein, on the basis of b. Line b' reflects the linear response relationship of the detection system to cTnI under the new sample addition method b (5-40 ng / mL), and line c' reflects the linear response relationship of the detection system to cTnI with the assistance of BSA (0.5-10 ng / mL).

[0070] Depend on Figure 3 It can be seen that the detection sensitivity of the system for cTnI has been significantly improved with the assistance of 2% BSA (b→c), and the linear equation of the fitting standard curve c' is y=0.2401+0.1612x(R 2 =0.9905), the linear range is 0.5-10 ng / mL, and the detection limit is calculated to be 0.24 ng / mL (signal-to-noise ratio is 3, the number of background samples is 11). From the linear response slope, it can be seen that the sensitivity of the system after adding the supporting protein BSA is 12.7 times that of the system without BSA, which reduces the detection limit of cTnI by one order of magnitude. This phenomenon shows that the supporting protein BSA can greatly promote the molecular recognition of cTnI by aptamers and antibodies.

[0071] The average relative standard deviation of the five concentration points within the linear range of the test (the number of samples at each concentration point was 5) was used to characterize the repeatability of the method. Figure 3 The average relative standard deviations of b' and c') were 11.5%±0.05 and 8.3%±0.04, respectively, which confirmed that BSA assisted in improving the repeatability of the detection system.

[0072] Example 4

[0073] The difference from Example 1 is:

[0074] 5) 20 ng / mL cTnI solution and supporting protein BSA were mixed with the signal aptamer solution in step 4) above. The final concentrations of DNA chain and S-HRP were 20 nM and 0.55 μg / mL, respectively. The concentrations of BSA were 0%, 0.002%, 0.02%, 0.2%, 2% and 5%, and the reaction was carried out at room temperature for 1 hour.

[0075] Results Figure 4 , Figure 4 The response changes of cTnI (20 ng / mL) after different concentrations of BSA were introduced into the signal aptamer solution. Figure 4It can be seen that the introduction of BSA into the signal aptamer solution significantly improves the detection effect of this method, especially in the presence of BSA concentration above 2%, the response of the hybrid recognition system to cTnI reaches the maximum.

[0076] Example 5

[0077] The difference from Example 3 is that the 20 ng / mL cTnI solution in step 5) was replaced with blank, VEGF165, CEA, AFP, copeptin, hFABP, PSA, MUC1, thrombin, lysozyme, and myoglobin. The results are shown in Figure 5 , Figure 5 The response of potential interfering substances, among which hFABP is heart-type fatty acid binding protein, similar to cTnI and myoglobin, are all biomarkers of cardiovascular disease; VEGF165 is a subtype of vascular endothelial growth factor A (VEGF-A), CEA is carcinoembryonic antigen, AFP is alpha-fetoprotein, PSA is prostate-specific antigen, and MUC1 is a glycoprotein, all of which are tumor-related biomarkers. Figure 5 As shown, only the addition of cTnI can make the system produce obvious ultraviolet signals, while other biomarkers have no response. The system has good selectivity under the final optimized conditions.

[0078] Example 6

[0079] The difference from Example 3 is that the PBS solution in the cTnI solution in step 5) is replaced with 50-fold diluted human serum, which is provided free of charge by the First Hospital of Jilin University. The results are shown in Table 1, which shows the detection results of cTnI in 50-fold diluted serum.

[0080] Table 1 Detection results of cTnI in 50-fold diluted serum

[0081]

[0082] The detection of cTnI in blood is an important means of diagnosing cardiovascular diseases. However, the complex blood components make many designed cTnI aptamer sensing methods ineffective. The introduction of the supporting protein BSA in the signal aptamer solution in this technical solution not only improves the sensitivity and repeatability of the hybrid recognition method, but also enhances its anti-serum interference ability and improves the adaptability of the hybrid recognition method to complex samples. The good recovery rate and relative standard deviation in Table 1 confirm that the detection system can detect cTnI in diluted serum, indicating that the system is expected to be applied to the detection of actual samples in the future.

[0083] Example 7

[0084] Compared with Example 1, the difference is that the biotin-labeled cTnI aptamer 2 is used to replace the biotin-labeled cTnI aptamer 1. The results are shown in Figure 6 , Figure 6 The responses to cTnI (20 ng / mL) before and after the introduction of BSA into different signal aptamer solutions.

[0085] Example 8

[0086] Compared with Example 3, the difference is that the biotin-labeled cTnI aptamer 2 is used to replace the biotin-labeled cTnI aptamer 1. The results are shown in Figure 6 , Figure 6 The responses to cTnI (20 ng / mL) before and after the introduction of BSA into different signal aptamer solutions. Figure 6 The data showed that the introduction of BSA into the signal aptamer solution significantly improved the response to cTnI in both hybrid recognition systems based on aptamer 1 and aptamer 2.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection reagent for cardiac troponin I, comprising: A cTnI aptamer labeled with a first marker, a covalent linker of horseradish peroxidase labeled with a second marker, and a supporting agent; The first label and the second label can bind; The supporting agent is selected from albumin, and the supporting agent is added to the signal aptamer solution formed by the cTnI aptamer labeled with the first marker and the horseradish peroxidase covalent linker labeled with the second marker.

2. The detection reagent according to claim 1, characterized in that The supporting agent is selected from bovine serum albumin.

3. The detection reagent according to claim 2, characterized in that The working concentration of the supporting agent is 0.002% to 5%.

4. The detection reagent according to any one of claims 1 to 3, characterized in that Also includes: Capture antibody; enzyme-catalyzed reactant; diluent; cleaning agent; blocking agent and reaction termination solution.

5. The detection reagent according to claim 4, characterized in that The first label is selected from biotin; The second label is selected from streptavidin.

6. The detection reagent according to claim 5, characterized in that The cTnI aptamer has a base sequence shown in SEQ ID No.1 or SEQ ID No.

2.

7. The detection reagent according to claim 6, characterized in that The enzyme-catalyzed reactant is selected from hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine; The diluent is selected from PBS diluent; The cleaning agent is selected from Tween 20; The blocking agent is selected from bovine serum albumin; The reaction termination solution is selected from 2M sulfuric acid.

8. Use of the detection reagent according to any one of claims 1 to 7 in the preparation of a kit for detecting cardiac troponin I.

9. A detection kit for cardiac troponin I, comprising the detection reagent according to any one of claims 1 to 7.

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