WO2022198790A1

WO2022198790A1 - Oligonucleotide aptamer of cardiac troponin and uses thereof

Info

Publication number: WO2022198790A1
Application number: PCT/CN2021/097357
Authority: WO
Inventors: 严鹏科
Original assignee: 严鹏科
Priority date: 2021-03-25
Filing date: 2021-05-31
Publication date: 2022-09-29
Also published as: CN113073101A; CN113073101B

Abstract

Provided are an oligonucleotide aptamer of cardiac troponin and the uses thereof, wherein the sequence of the aptamer is one of SEQ ID NOs: 1-10, or the aptamer is a variant which has a function equivalent to one of the sequences of SEQ ID NOs: 1-10. The aptamer can be used in the preparation of a drug comprising the aptamer, in the preparation of a kit comprising the aptamer, in the non-diagnostic detection of cardiac troponin, and in the separation and purification of cardiac troponin. The aptamer is suitable for oligonucleotide detection methods for various proteins, has a larger detection interval, eliminates the hook effect and has higher detection sensitivity.

Description

An oligonucleotide aptamer of cardiac troponin and its application

technical field

The invention relates to an oligonucleotide aptamer of cardiac troponin and its application, belonging to the technical field of molecular biology.

Background technique

Troponin (Tn) is a regulatory protein of striated muscle contraction and a structural protein of skeletal and cardiac muscle. It is a complex composed of three subunits, TnT, TnC and TnI. Protein-bound, 90% located on striated muscle filaments, involved in regulating muscle contraction. Among them, TnT is a tropomyosin-binding subunit that regulates the interaction between troponin complexes and thin filaments; TnC is a calcium-binding subunit that plays a major role in regulating calcium-dependent muscle contraction, while TnI It is an inhibitory protein in the troponin-myosin regulatory complex that prevents muscle contraction. When myocardial ischemia leads to myocardial injury, a small amount of free cardiac troponin I (cTnI) in the cytoplasm is rapidly released into the blood circulation, and the concentration in peripheral blood increases rapidly. With the continuous degradation of myocardial filaments, cTnI was continuously released into the blood, reaching a peak after 12-18 hours, and the duration of cTnI elevation was 4-10 days. cTnI is not expressed in skeletal muscle, and its biochemical properties determine its high specificity and sensitivity as an indicator of myocardial injury, especially when there is no abnormality in ECG detection, cTnI index can be used as an auxiliary judgment. Therefore, it is necessary to further study and improve the detection of cTnI.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide an oligonucleotide aptamer for cardiac troponin, which is suitable for oligonucleotide detection methods of various proteins, and has The larger detection interval eliminates the hook effect and the detection sensitivity is higher.

The second object of the present invention is to provide an application of the above-mentioned cardiac troponin oligonucleotide aptamer.

The first object of the present invention can be achieved by adopting the following technical solutions: a kind of oligonucleotide aptamer of cardiac troponin, the sequence of the aptamer is one of SEQ ID NO.1-10, or the aptamer A variant is a functionally equivalent variant to one of the sequences SEQ ID NO. 1-10.

Further, the cardiac troponin is cardiac troponin I.

The second object of the present invention can be achieved by adopting the following technical solutions: the application of an oligonucleotide aptamer of cardiac troponin, and the preparation of a medicine comprising the aptamer.

The second object of the present invention can be achieved by adopting the following technical solution: preparing a kit including an aptamer.

The second object of the present invention can be achieved by adopting the following technical solution: to use the aptamer for the detection of cardiac troponin for non-diagnostic purposes.

The second object of the present invention can be achieved by adopting the following technical solution: the aptamer is used for the separation and purification of cardiac troponin.

Further, the isolation and purification of cardiac troponin includes:

Blocking step: block the incubated samples with blocking solution;

Binding step: adding biotin-modified aptamer to the blocked sample, and removing the liquid after incubation to obtain a combination of aptamer and protein;

Washing step: The conjugate is washed.

Further, the isolation and purification of cardiac troponin includes:

Blocking step: add biotin-modified aptamer to streptavidin, remove the liquid after incubation, and block the aptamer with blocking solution;

Binding step: adding a sample to the blocked aptamer for incubation, and then adding a digoxin-modified aptamer for incubation to obtain a combination of the aptamer and the protein;

Washing step: The conjugate is washed.

Further, the isolation and purification of cardiac troponin includes:

Binding step: adding a sample to the blocked aptamer for incubation, and then adding a cardiac troponin antibody for incubation to obtain a conjugate of aptamer, protein and antibody;

Washing step: The conjugate is washed.

Further, the isolation and purification of cardiac troponin includes:

Blocking step: block cardiac troponin antibody with blocking solution;

Binding step: adding a sample to the blocked cardiac troponin antibody for incubation, and then adding a digoxin-modified aptamer for incubation to obtain a conjugate of antibody, protein and aptamer;

Washing step: The conjugate is washed.

The design principle of the present invention is as follows:

Aptamer (also translated as aptamer or aptamer), commonly referred to as "chemical antibody", is short single-stranded DNA or RNA, and is currently recognized as the most potential targeting substance. The preparation process of aptamers is controllable and can be produced and synthesized completely by in vitro procedures, with low batch-to-batch variation, no pollution or low risk of contamination, and has the advantages of convenience, rapidity, and low cost. It can be used in diseases through various modifications or couplings. Diagnosis and treatment.

At present, the most sensitive clinical detection method for cTnI is the protein antibody kit Beckman chemiluminescence detection method. effect). This effect refers to false negative results due to an inappropriate ratio of antigen to antibody, in which excess antibody is called prozone effect, and excess antigen is called postzone effect. That is to say, when the antigen content is higher than 50ng/mL, accurate results cannot be obtained by this method, and false negative results are prone to occur, which will give clinicians or patients wrong prompts, delay treatment or affect the judgment of efficacy. Therefore, the aptamer was further developed with the elimination of the HOOK effect as the starting point of the research.

Compared with the prior art, the beneficial effects of the present invention are:

1. The aptamer of the present invention can be used for single aptamer ELONA detection, double aptamer sandwich ELONA detection, aptamer-antibody sandwich ELONA detection, antibody-aptamer sandwich ELONA detection method, and has a larger detection method. interval, eliminating the hook effect and higher detection sensitivity;

2. The aptamer of the present invention has small volume, good stability, and easy production. The aptamer can be bound to the second molecule, and the result of the aptamer binding to the second molecule is a complex, showing two functional binding of the first molecule, i.e. a complex with specific binding capacity and activity associated with the second molecule;

3. The aptamer of the present invention has various uses, especially for the separation and purification of cardiac troponin, and the purification effect is good.

Description of drawings

1-10 are schematic diagrams of the secondary structure of the aptamer of Example 1;

Figure 11 is a dot blot of aptamer;

Figure 12 is the dose-response curve of the 96bp library of Example 2;

Figure 13-22 is the dose-response curve of the aptamer of Example 2 SEQ ID NO.1-10;

Figure 23 is the standard curve of embodiment 3;

Figure 24 is the standard curve of embodiment 4;

Figure 25 is the standard curve of embodiment 5;

Figure 26 is the standard curve of embodiment 6;

Figure 27 is the standard curve of embodiment 7;

Figure 28 is the standard curve of embodiment 8;

Figure 29 is the standard curve of embodiment 9;

Figure 30 is the standard curve of embodiment 10;

FIG. 31 is the standard curve of Example 11. FIG.

Detailed ways

Below, in conjunction with accompanying drawing and specific embodiment, the present invention is further described:

An oligonucleotide aptamer of cardiac troponin I, the sequence of the aptamer is one of SEQ ID NO.1-10, or the aptamer is functionally equivalent to one of the sequence SEQ ID NO.1-10 variant of .

A functionally equivalent variant refers to a sequence substantially similar to one of SEQ ID NOs. 1-10 that retains the ability to specifically bind cardiac troponin I. It can also be a nucleic acid sequence derived from one of SEQ ID NO. 1-10, including additions, substitutions or modifications of multiple nucleotides. For example, it can be modified at the 3' end or 5' end of one of SEQ ID NO. 1-10 by at least 1 oligonucleotide and retain at least 50% binding specificity to cardiac troponin I. The above-mentioned modifications include, but are not limited to, modification of nucleic acid backbones, substitution bonds, modification of nucleotides, modification of nucleic acid peptide chains, and the like. The modified aptamer has at least 50% specific binding capacity for cardiac troponin I.

One of the applications of the above-mentioned cardiac troponin I oligonucleotide aptamer is to prepare a medicine comprising the aptamer.

One of the applications of the above-mentioned cardiac troponin I oligonucleotide aptamer is to prepare a kit including the aptamer.

One of the applications of the oligonucleotide aptamer for cardiac troponin I is to use the aptamer for the detection of cardiac troponin I for non-diagnostic purposes.

One of the applications of the above-mentioned cardiac troponin I oligonucleotide aptamer is to use the aptamer for the separation and purification of cardiac troponin I, which is one of the following four methods.

1) The separation and purification of cardiac troponin I includes:

Blocking step: Incubate 20-2000μL of the sample in an ELISA plate, overnight at 4°C, remove the liquid, and block with blocking solution (2-5wt% nonfat dry milk, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) 1-2h incubated samples;

Binding step: Add 20-200 μL of biotin-modified 1-200nM aptamer to the blocked sample, cover the microplate plate, incubate at 37°C for 0.5-2h, remove the liquid, and obtain a mixture of aptamer and protein. conjugate;

Washing step: Wash each well with 350 μL of washing solution (0.01-0.5wt% PBST), soak the conjugate for 1-2 min, tap the ELISA plate on absorbent paper to remove all the liquid in the well, and repeat the plate washing 5 times.

After obtaining cardiac troponin I by this method, it can also be detected: add Streptavidin/HRP 20-200μL (1:5000, prepared before use) to each well, cover the microplate with membrane, and incubate at 37°C for 30min-1h; Discard the liquid in the well, spin dry, and wash the plate 5 times; add 20-200 μL of TMB substrate solution to each well, cover the ELISA plate, and develop color at 37°C for about 15-30 min; add 50 μL of stop solution to each well to stop After the reaction, the blue turns to yellow; the optical density (OD) of each well is measured with a microplate reader at a wavelength of 450 nm.

2) The separation and purification of cardiac troponin I includes:

Blocking step: Add 20-200 μL of aptamer modified with 50-200nM biotin to the microtiter plate pre-coated with streptavidin, incubate at 37°C for 0.5-2h, remove the liquid, and use the blocking solution (2- 5wt% skimmed milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) blocking aptamer for 1-2h; the aptamer added in the concentration range of 50-200nM can maximize the combination of streptavidin and biological The aptamer can be firmly combined with the aptamer, and the aptamer can be firmly adsorbed on the ELISA plate under the maximum cost-effectiveness; when the incubation temperature is 37 °C, it is suitable for the reaction; if the blocking time is less than 1 h, the blocking effect is insufficient, and the non-specific sites are not blocked. It is completely blocked, resulting in non-specific binding of other substances in the sample; if the blocking time is longer than 2 hours, the blocking is excessive, resulting in the weak binding of the protein that can be bound to the aptamer and falling off;

Binding step: Add 20-200 μL of sample to the blocked aptamer, incubate at 37°C for 1-4 hours, then add 50-200 μL of 50-200nM digoxigenin-modified aptamer, and coat the ELISA plate , and incubated at 37°C for 0.5-2h to obtain the aptamer-protein conjugate;

The aptamer in this concentration range can fully bind to the target protein, so that the aptamer and the target protein are in the optimum ratio, avoiding the HOOK effect caused by antigen-antibody binding;

Washing step: wash each well with 350 μL washing solution (0.01-0.5wt% PBST), soak the conjugate for 1-2 min, tap the ELISA plate on absorbent paper to remove all the liquid in the well, and repeat the plate washing 5 times;

The washing solution can remove other non-specifically bound proteins and impurities, and maximize the retention of the binding complex between the aptamer and the target protein.

Cardiac troponin I can also be detected by this method: add anti-digoxigenin antibody/HRP 20-200μL (1:500-5000, prepared before use) to each well, cover with ELISA plate, 37°C Incubate for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; add 20-200 μL of TMB substrate solution to each well, cover the ELISA plate, and develop color at 37°C for about 15-30 min in the dark; stop by adding to each well 50 μL of solution was added to stop the reaction, at which point the blue turned to yellow; the optical density value (OD) of each well was measured with a microplate reader at a wavelength of 450 nm;

The method uses the biotin-modified aptamer as a substrate to capture the target protein, and uses the digoxigenin-modified aptamer to perform qualitative and quantitative analysis of the target protein to form an aptamer-protein-aptamer sandwich structure; , using different systems for immobilization and enzyme labeling, which can effectively avoid cross-reaction and avoid false positives. In this method, the concentration range of aptamer binding to the target protein is large, which effectively avoids the HOOK effect caused by not reaching the optimal concentration ratio between antigen and antibody in the antigen-antibody reaction. At the same time, aptamer acts as a The self-stability and low immunogenicity of various DNA sequences are advantages that cannot be achieved by antibodies.

3) The separation and purification of cardiac troponin I includes:

Blocking step: Add 100 μL of 50-200nM biotin-modified aptamer to the pre-coated streptavidin ELISA plate, incubate at 37°C for 0.5-2h, remove the liquid, and use the blocking solution (2-5wt%) to remove the liquid. Skim milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) blocking aptamer for 1-2h;

The aptamer added in the concentration range can bind streptavidin and biotin firmly to the greatest extent, so that the aptamer can be firmly adsorbed on the ELISA plate with the greatest cost-effectiveness; when the incubation temperature is 37 °C, it is suitable for A reaction occurs; if the blocking time is less than 1h, the blocking effect is insufficient, and the non-specific sites are not completely blocked, resulting in non-specific binding of other substances in the sample; if the blocking time is greater than 2h, the blocking is excessive, resulting in The ligand-bound protein is not firmly bound and falls off;

Binding step: Add 20-200μL of sample to the blocked aptamer and incubate at 37°C for 1-4h, then add cardiac troponin antibody (1:100-1000 dilution) 50-200μL at 37°C Incubate for 0.5-2h, discard the liquid, and obtain the conjugate of aptamer, protein and antibody;

Cardiac troponin I can also be detected by this method: add secondary antibody/HRP 50-200μL (1:500-10000, prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30min ; Discard the liquid in the well, spin dry, and wash the plate 5 times; add 50-200 μL of TMB substrate solution to each well, cover the ELISA plate, and develop color at 37°C for about 15-30 min; add 50 μL of stop solution to each well, The reaction was terminated, and the blue color turned to yellow; the optical density value (OD) of each well was measured with a microplate reader at a wavelength of 450 nm.

In the method, the aptamer is used as the substrate to capture the target protein, and the antibody is used as the enzyme marker for qualitative and quantitative analysis of the target protein to form an aptamer-protein-antibody sandwich structure. This method realizes the joint action of aptamer and antibody, and exerts their respective advantages.

4) The separation and purification of cardiac troponin I includes:

Blocking step: The ELISA plate pre-coated with cardiac troponin antibody is blocked with blocking solution (2-5wt% skim milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) for 0.5-2h;

If the blocking time is less than 1 h, the blocking effect is insufficient, and the non-specific sites are not completely blocked, resulting in non-specific binding of other substances in the sample; if the blocking time is more than 2 h, the blocking effect is excessive, resulting in the aptamer that could have been combined. The protein binding is not firm and falls off;

Binding step: Add 20-200μL of sample to the blocked cardiac troponin antibody for 1-4h incubation, and then add 50-200nM aptamer modified with digoxigenin and 50-200μL of enzyme-labeled plate to cover the membrane, 37℃ Incubate for 1 h to obtain a conjugate of antibody, protein and aptamer;

After obtaining cardiac troponin I by this method, it can also be detected: add anti-digoxigenin antibody/HRP 50-200μL (1:500-5000, prepared before use) to each well, cover with ELISA plate, 37℃ Incubate for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; add 50-200 μL of TMB substrate solution to each well, cover the ELISA plate, and develop color at 37°C for about 15-30 min in the dark; stop adding to each well 50 μL of the solution was added to terminate the reaction, at which point the blue turned yellow; the optical density (OD) of each well was measured with a microplate reader at a wavelength of 450 nm.

In the method, the antibody is used as the substrate to capture the target protein, and the aptamer is used as the enzyme marker for qualitative and quantitative analysis of the target protein to form an antibody-protein-aptamer sandwich structure. This method realizes the joint action of aptamer and antibody, and exerts their respective advantages.

Example 1:

Obtainment of aptamers:

The two ends of the 96bp ssDNA library (500-600pmol) sequence are fixed sequences, and the middle 60 nucleotides are random sequences. Design upstream primers and downstream primers. The upstream primers are labeled with Biotin, and the downstream primers are unlabeled. The dosage of cTnI as positive sieve protein is 10-200 μg, and the dosage of normal human serum as reverse sieve protein is 200-600 μL;

ssDNA was amplified by ePCR. The amplification system was: water phase, ssDNA template 0.1-3.3μg, 1x PCR Mix buffer 25μL, upstream primer 50pmol, downstream primer 50pmol, supplemented with deionized water to 50μL; oil phase, centrifuge in 15mL Add 5mL mineral oil, 270μL Span80, 24μL Tween80, 3μL Triton X-100 to the tube, add mineral oil to 6mL, vortex and mix well and store at 4°C; prepare PCR amplification system according to the ratio of water phase:oil phase=2:1 The amplification conditions were: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 40 s, annealing at 68 °C for 1 min, and extension at 72 °C for 7 min.

The DNA after each round of isolation was re-enriched by ePCR: DNA was dissolved in PBS, bound to mycovidin beads through a biotin-labeled strand, denatured, and biotin-coated beads were eluted from the streptavidin beads The labeled chain was neutralized with a small amount of hydrochloric acid, then precipitated by adding 2 volumes of absolute ethanol overnight in a -80°C refrigerator, centrifuged at 14,000 rpm/min for 40 min, removed the supernatant, washed with 600 μL 70vt% ethanol, and centrifuged for 20 min, and dried in air. Dissolved in a small amount of PBS, the concentration of ssDNA was determined by ultra-micro UV spectrophotometer;

The single-stranded secondary library is then prepared for the next round of SELEX screening, ePCR can reduce the appearance of non-specific bands. About 500-600pmol random double-stranded library was added to a 1.5mL centrifuge tube, denatured at 95°C for 5min, renatured at room temperature, added to streptavidin magnetic beads, incubated on a shaker for 30min, and then added NaOH to break the double-stranded Unlabeled strands were shed by hydrogen bonds, and then cardiac troponin I recombinant protein was added to incubate for 1 h. DNA was extracted from the supernatant using the phenol-chloroform method, and 1/10 volume of sodium acetate solution, 2.5 times volume of anhydrous ethanol, -80 The ssDNA was precipitated in the refrigerator overnight; after centrifugation at 14,000 rpm for 40 min, washed once with 75vt% ethanol, dried in air, dissolved in a small amount of TE or sterile water, and the concentration of ssDNA was measured by UV spectrophotometer. the next round of screening;

In each round of screening, the enrichment conditions were selected according to the following ePCR system: L-Mix 25 μL, water 22 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL (concentrations were 1 ng/μL, 0.5 ng/μL); amplification conditions : The annealing temperature was 72°C, 71°C, 70°C, 69°C, and 68°C, and the number of cycles was 13, 11, and 9, respectively.

After repeating the above steps for multiple rounds of screening, the ePCR products were verified and sequenced: polyacrylamide gel electrophoresis was used for detection, and 12wt% polyacrylamide gel was used for electrophoresis at 150v for about 40 minutes, and the integrity of the sample was detected, and the test was qualified. The samples entered the library preparation stage.

It mainly includes taking enough genomic DNA samples to select and purify target fragments using PAGE gel; the purified DNA is subjected to end repair plus "A" and adapter reactions according to the kit instructions, and purified using Agencourt AMPure XP magnetic beads; The resulting product was cut through PAGE gel to recover the target fragment, the purified library was dissolved in Elution Buffer, and the library label was affixed, so far the library construction was completed. Then, the quality of the library is checked, usually using two methods: one is to use the Agilent 2100 Bio analyzer to detect the fragment range of the library; the other is to use the ABI StepOnePlus Real-Time PCR System (TaqMan Probe) to quantify the concentration of the library.

On-machine sequencing: First, inject the acrylamide solution into the capillary, and the acrylamide will polymerize under the ionization effect of ultraviolet light to become a polyacrylamide gel. Voltage is applied to both ends for electrophoresis, and the end of the positive electrode of the capillary is irradiated with laser light, and the fluorescent signal is recorded through an optical sensor. Finally, specific oligonucleotide aptamers are selected through specific procedures and criteria:

The sequences of the aptamers are SEQ ID NO.1-10:

The secondary structures of the aptamers whose sequences are SEQ ID NO.1-10 are shown in Figures 1-10 respectively;

An aptamer can also be a functionally equivalent variant of one of the sequences SEQ ID NO. 1-10.

Detection of specificity of aptamer binding to human cardiac troponin I by dot blot:

The human cardiac troponin I standard was immobilized on the nc membrane, 1 mL of biotin-modified aptamer was added, and incubated for 1 h at room temperature on a shaker, and the corresponding secondary antibody and chromogenic reagent were added to observe in a gel imager. The amount of human cardiac troponin I standard was 0.3 μg/μL, and the amount of aptamer added was 50 nM. The dot blots of the aptamers with sequences of SEQ ID NO. 1-10 are shown in Figure 11.

Example 2:

Single aptamer ELONA assay

1) Separation and purification of cardiac troponin I:

Blocking step: The standard 5ng/μL cardiac troponin I was incubated in an ELISA plate overnight at 4°C, the liquid was removed, and a blocking solution (2-5wt% nonfat milk powder, 250-1000nM 20bp DNA, 0.5-5wt % Tween 20) closed for 1h;

Binding step: Add 100 μL of biotin-modified 1-200nM aptamer SEQ ID NO.1-10 to the blocked sample, cover the ELISA plate, and incubate at 37°C for 1 h to remove the liquid to obtain the aptamer. body and protein conjugates;

Washing step: wash each well with 350 μL of washing solution (0.01-0.5wt% PBST), soak the conjugate for 1-2 min, tap the ELISA plate on absorbent paper to remove all liquid in the well, and repeat the plate washing 5 times.

2) Detection steps:

Add 100 μL of Streptavidin/HRP (1:5000, prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; add TMB bottom to each well Add 100 μL of the solution solution, cover the microplate plate with a film, and develop color at 37°C for about 15 minutes in the dark; add 50 μL of stop solution to each well to stop the reaction, at which point the blue turns to yellow; use a microplate reader to measure the optical density of each well at a wavelength of 450 nm value (OD).

Fig. 12 is the dose-response curve of 96bp library, under the situation that other detection conditions remain unchanged, the aptamer is replaced with dna library to carry out dose-response curve measurement, Kd value is calculated according to this curve, the Kd calculated by the dose-response curve of dna library The value of P>0.05 indicates that the Kd value is not statistically significant, and the curve fitting is unsuccessful, while the Kd value represents the affinity of the DNA. The failure of the curve fitting indicates that the binding affinity of the DNA library to cardiac troponin I is extremely low. The dose-response curves of aptamers of SEQ ID NO.1-10 are shown in Figures 13-22 respectively. All 10 aptamers have good affinity, and the dissociation equilibrium constant (Kd value) ranges from 0.2558 to 9.2814n mol/ Between L, it shows that the affinity of the obtained aptamer for cardiac troponin I is significantly improved, and the affinity of SEQ ID NO.10 and SEQ ID NO.8 is the highest, and the Kd values are 0.2558 nmol/L and 0.6813 nmol/ L.

Example 3:

Double aptamer sandwich ELONA assay

1) The separation and purification of cardiac troponin I includes:

Blocking step: Add 100 μL of 100nM biotin-modified aptamer SEQ ID NO.3 to the ELISA plate pre-coated with streptavidin, incubate at 37°C for 1 h, remove the liquid, and use the blocking solution (2-5wt % skimmed milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) blocking aptamer for 1h;

Binding step: Add 100 μL of gradient concentration of cardiac troponin I standard and human serum sample (with human serum protein as negative control) to the blocked aptamer, incubate at 37°C for 2h, and then add 50nM Digoxigenin-modified aptamer SEQ ID NO.6 100 μL, coated with microplate, and incubated at 37°C for 1 h to obtain the combination of aptamer and protein;

2) Detection steps:

Add 100 μL of anti-digoxigenin antibody/HRP (1:5000, prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; each well Add 100 μL of TMB substrate solution, cover the ELISA plate, and develop color at 37°C for about 30 minutes in the dark; add 50 μL of stop solution to each well to stop the reaction, at which point the blue turns to yellow; measure each well with a microplate reader at a wavelength of 450 nm The optical density value (OD) of the obtained standard curve is shown in Figure 23.

Example 4:

The difference between embodiment 4 and embodiment 3 is:

In the blocking step, add aptamer modified with 50nM biotin;

In the combining step, the amount of cardiac troponin I standard substance and human serum sample added is 50 μL;

The remaining steps and parameters are the same as in Example 3, and the obtained standard curve is shown in Figure 24.

Example 5:

The difference between embodiment 5 and embodiment 3 is:

In the combining step, add 100nM aptamer modified by digoxin;

The remaining steps and parameters are the same as in Example 3, and the obtained standard curve is shown in Figure 25.

The results of Examples 3-5 show that the aptamer has no specific binding to human serum protein, and the linear range of this method for detecting cTnI is 0.2-100 ng/mL.

Example 6:

Aptamer-antibody sandwich ELONA assay

1) The separation and purification of cardiac troponin I includes:

Blocking step: add 100nM biotin-modified aptamer SEQ ID NO.3 100μL to the pre-coated streptavidin ELISA plate, incubate at 37°C for 1h, remove the liquid, and use the blocking solution (2-5wt % skimmed milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) blocking aptamer for 1h;

Binding step: Add gradient concentration of cardiac troponin I standard and human serum protein (with human serum protein as negative control) 100 μL to the blocked aptamer, incubate at 37°C for 2 hours, and then add myocardial muscle Calcin antibody (1:1000 dilution) 100 μL was incubated at 37°C for 1 h, the liquid was discarded, and the conjugate of aptamer, protein and antibody was obtained;

2) Detection steps:

Add 100 μL of secondary antibody/HRP (1:10000, prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; add TMB bottom to each well Add 100 μL of the solution solution, cover the microplate plate with a film, and develop color at 37°C for about 30 minutes in the dark; add 50 μL of stop solution to each well to stop the reaction, at this time, the blue turns to yellow; the optical density of each well is measured with a microplate reader at a wavelength of 450 nm. value (OD), the resulting standard curve is shown in Figure 26.

Example 7:

The difference between embodiment 7 and embodiment 6 is:

In the blocking step, add 50nM biotin-modified aptamer;

In the binding step, add cardiac troponin I standard and 50 μL of human serum protein;

The remaining steps and parameters are the same as in Example 6, and the obtained standard curve is shown in Figure 27.

Example 8:

The difference between embodiment 8 and embodiment 6 is:

In the blocking step, add 50nM biotin-modified aptamer;

In the binding step, add 50 μL of cardiac troponin antibody;

The remaining steps and parameters are the same as in Example 6, and the obtained standard curve is shown in Figure 28.

Example 9:

Antibody-aptamer sandwich ELONA assay

Blocking step: The ELISA plate pre-coated with cardiac troponin antibody was blocked with blocking solution (2-5wt% nonfat milk powder, 250-1000nM 20bp DNA, 0.5-5wt% Tween 20) for 1h;

Binding step: Add 100 μL of gradient concentration of cardiac troponin I standard and human serum sample (with human serum protein as negative control) to the blocked cardiac troponin antibody, incubate at 37°C for 2h, and then add 50nM Digoxigenin-modified aptamer SEQ ID NO.6 100μL microplate was covered with membrane, and incubated at 37°C for 1 h to obtain a conjugate of antibody, protein and aptamer;

2) Detection steps:

Add 100 μL of anti-digoxigenin antibody/HRP (1:5000, prepared before use) to each well, cover the ELISA plate, and incubate at 37°C for 30 min; discard the liquid in the well, spin dry, and wash the plate 5 times; each well Add 100 μL of TMB substrate solution, cover the ELISA plate, and develop color at 37°C for about 30 minutes in the dark; add 50 μL of stop solution to each well to stop the reaction, at which point the blue turns to yellow; measure each well with a microplate reader at a wavelength of 450 nm The optical density value (OD) of the obtained standard curve is shown in Figure 29.

Example 10:

The difference between embodiment 10 and embodiment 9 is:

The remaining steps and parameters are the same as in Example 10, and the obtained standard curve is shown in Figure 30.

Example 11:

The difference between embodiment 11 and embodiment 9 is:

In the combining step, add 100nM aptamer modified by digoxin;

The remaining steps and parameters are the same as in Example 9, and the obtained standard curve is shown in Figure 31.

Linearity of double aptamer sandwich ELONA assay (Example 3-5), aptamer-antibody sandwich ELONA assay (Example 6-8), and antibody-aptamer sandwich ELONA assay (Example 9-11) Comparing the range results, it was found that the double aptamer sandwich method has the smallest detection limit and the largest linear slope k, which proves that this method can detect lower than the aptamer-antibody sandwich method and the antibody-aptamer sandwich method. The concentration of cTnI content is more sensitive to the change of cTnI concentration.

The detection linear ranges of the three methods in the present invention are all larger than those of the kits currently used clinically. While ensuring the detection rate of positive samples, the detectable concentration range is expanded, and clinically, it can provide the basis for the follow-up treatment of critically ill patients.

Since the double aptamer sandwich method completely adopts the method of binding the aptamer to the target protein without antigen-antibody binding, the HOOK effect caused by the antigen-antibody reaction is effectively avoided, and it will not produce immunogenicity in clinical application. This leads to deviations in results; at the same time, aptamers are more stable than antibodies in the preparation process, and will not cause batch-to-batch differences in products to affect the detection results. Both the aptamer-antibody sandwich method and the antibody-aptamer sandwich method achieve a synergistic effect in the same detection system. Although the detection effect is slightly inferior to the double aptamer sandwich method, it plays an important role in the performance of aptamer and antibody. On the basis of their respective advantages, there is no antagonistic effect, which also has certain clinical significance.

For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

An oligonucleotide aptamer for cardiac troponin, wherein the sequence of the aptamer is one of SEQ ID NO.1-10, or the aptamer is the same as the sequence SEQ ID NO.1-10. A functionally equivalent variant of one of 1-10.
The oligonucleotide aptamer of cardiac troponin according to claim 1, wherein the cardiac troponin is cardiac troponin I.
An application of an oligonucleotide aptamer for cardiac troponin, characterized in that a medicine comprising the aptamer is prepared.
An application of an oligonucleotide aptamer for cardiac troponin, characterized in that a kit comprising the aptamer is prepared.
An application of an oligonucleotide aptamer for cardiac troponin, characterized in that the aptamer is used for the detection of cardiac troponin for non-diagnostic purposes.
An application of an oligonucleotide aptamer for cardiac troponin, characterized in that the aptamer is used for the separation and purification of cardiac troponin.
The application of the oligonucleotide aptamer of cardiac troponin according to claim 6, wherein the separation and purification of the cardiac troponin comprises:

Blocking step: block the incubated samples with blocking solution;

Binding step: adding biotin-modified aptamer to the blocked sample, and removing the liquid after incubation to obtain a combination of aptamer and protein;

Washing step: The conjugate is washed.
The application of the oligonucleotide aptamer of cardiac troponin as claimed in claim 6, is characterized in that, the separation and purification of described cardiac troponin comprises:

Blocking step: add biotin-modified aptamer to streptavidin, remove the liquid after incubation, and block the aptamer with blocking solution;

Binding step: adding a sample to the blocked aptamer for incubation, and then adding a digoxin-modified aptamer for incubation to obtain a combination of aptamer and protein;

Washing step: The conjugate is washed.
The application of the oligonucleotide aptamer of cardiac troponin according to claim 6, wherein the separation and purification of the cardiac troponin comprises:

Blocking step: add biotin-modified aptamer to streptavidin, remove the liquid after incubation, and block the aptamer with blocking solution;

Binding step: adding a sample to the blocked aptamer for incubation, and then adding a cardiac troponin antibody for incubation to obtain a conjugate of aptamer, protein and antibody;

Washing step: The conjugate is washed.
The application of the oligonucleotide aptamer of cardiac troponin according to claim 6, wherein the separation and purification of the cardiac troponin comprises:

Blocking step: block cardiac troponin antibody with blocking solution;

Binding step: adding a sample to the blocked cardiac troponin antibody for incubation, and then adding a digoxin-modified aptamer for incubation to obtain a conjugate of antibody, protein and aptamer;

Washing step: The conjugate is washed.