Solid-state self-enhanced ECL aptamer thrombin sensor, construction method and application

Abstract

The invention discloses a solid-state self-enhanced ECL aptamer thrombin sensor as well as a construction method and application of the solid-state self-enhanced ECL aptamer thrombin sensor. The method comprises the following steps: (1) synthesizing conjugated polymer points PDC; (2) incubating a PDC / dicarboxyl bipyridine ruthenium solid self-enhanced ECL working electrode; and (3) construction of the conjugate heme-G-quadruplex / AuNPs (AuNPs). The branched chain polyethyleneimine and tri-n-propylamine are similar in multi-tertiary amine structure, so that the branched chain polyethyleneimine and a polymer point PDC formed by hydrothermal synthesis of NH2BDC also have the capability of serving as a bipyridyl ruthenium co-reaction reagent, the detection range of the ultra-sensitive TB aptamer sensor of a sandwich structure constructed based on the PDC is 1015-108 M, the fluctuation range of TB in a blood sample is included, and the sensitivity of the TB aptamer sensor is improved. The detection limit is 3.6 * 10 <-16 > M, and the probe has an application prospect in thrombin medical detection.

Description

Technical Field

[0001] The present invention relates to a solid-state self-enhanced ECL aptamer thrombin sensor, a construction method and an application, belonging to the field of electrochemiluminescence detection. Background Art

[0002] Thrombin (TB) is a specific serine endoprotease that plays a key role in hemostasis. It naturally acts as a coagulation factor, solidifying fibrin clots in terms of strength and elasticity. The concentration of thrombin in blood can vary from picomolar to micromolar levels, corresponding to normal conditions and the coagulation process. As a key enzyme in pathological processes such as leukemia, arterial thrombosis, and liver diseases, the detection and quantitative detection of thrombin in biological sera or other complex samples have clinical significance. Detecting thrombin, especially thrombin in the picomolar range in blood, is of great significance for the clinical diagnosis of related diseases.

[0003] Existing detection methods for thrombin include: direct detection and indirect detection. Direct detection means that thrombin directly acts on the substrate peptide segment to produce signal changes, including fluorescence method and ultraviolet method. The advantages are high precision and sensitivity, but the disadvantages are high cost. Indirect detection has the advantages of simplicity and low cost, but the disadvantages are low accuracy and sensitivity. Electrochemiluminescence (ECL) combines the advantages of high sensitivity, good reproducibility, and simple use of chemiluminescence and electrochemistry, providing new ideas for the determination of thrombin activity.

[0004] Electrochemiluminescence refers to substances on the electrode surface being excited by a power source and forming excited-state products through electron transfer. The excited-state products then release energy and return to the ground state in the form of light emission, combining the characteristics of electrochemistry and chemiluminescence. Since the discovery of ECL, its applications have expanded from the initial analysis of conventional inorganic and organic substances to the biological and medical fields. To improve the sensitivity of ECL, some auxiliary methods and amplification strategies are needed. And how to efficiently immobilize the luminophore is the key point in constructing an ECL biosensor, which is directly related to the reaction amount and luminescence efficiency of the luminophore. In the early stage of ECL detection, directly putting the luminophore into the detection solution may waste reagents and increase costs, but the effect is limited. To overcome these disadvantages, in most subsequent studies, the luminophore is usually immobilized on the electrode surface, especially for the most commonly used ECL luminophore of ruthenium terpyridine derivatives. For example, ruthenium terpyridine derivatives can be loaded on the electrode surface and assisted by some organic solvents with excellent film-forming properties, such as nafion, chitosan, and N,N-dimethylformamide. In addition, using nanomaterials or DNA as a support platform, different structures of ruthenium terpyridine derivatives can be immobilized on the electrode surface through methods such as adsorption, crosslinking, doping, and embedding. Yuan Ruo et al. proposed a thrombin sensor constructed based on the immobilized Ru(bpy)3 2+ and its linear range is 10-13 ~10 -8 M, with a detection limit of 0.03 pM, has limited detection performance. And since Ru(bpy)3 2+ is immobilized by monomer complexation, its long-term detection stability also does not meet the actual requirements (Lin-Ru, Hong, Ya-Qin, et al. Highlyefficient electrogenerated chemiluminescence quenching of PEI enhanced Ru(bpy)3 2+ nanocomposite by hemin and Au@CeO2 nanoparticles[J]. Biosensors&Bioelectronics, 2015.). SUMMARY OF THE INVENTION

[0005] An object of the present invention is to provide a solid-state self-enhanced ECL aptamer thrombin sensor, a construction method and an application thereof.

[0006] The technical solution for achieving the object of the present invention is as follows:

[0007] A construction method of a solid-state self-enhanced ECL aptamer thrombin sensor, comprising the following steps:

[0008] (1) According to the mass ratio of branched polyethyleneimine (PEI) to 2-aminoterephthalic acid (NH2BDC) being 1:1.8 - 2.0, ultrasonically dissolve branched polyethyleneimine in water, add 2-aminoterephthalic acid, stir until evenly mixed, then carry out a hydrothermal reaction at 100 - 120 °C. After the reaction ends, centrifuge, collect the supernatant, add an excessive amount of absolute ethanol, precipitate the product, centrifuge, collect the precipitate, wash it with absolute ethanol multiple times, and vacuum dry to obtain PEI-based polymer dots (PDC);

[0009] (2) Incubation of the electrode: According to the mass ratio of PDC to ruthenium dicarboxy bipyridine being 1:0.2 - 0.3, sequentially dropwise add a PDC solution and a ruthenium dicarboxy bipyridine solution on the surface of a clean electrode to complete mixing, dry at room temperature, dropwise add a gold nanoparticle (AuNPs) solution, dry at room temperature, dropwise add a thiolated TBA 1 strand (SH-TBA 1) solution, incubate at 4 °C, and finally dropwise add an aqueous bovine serum albumin (BSA) solution for blocking to obtain a working electrode;

[0010] (3) Construction of the conjugate: Suspend AuNPs in an aqueous BSA solution, stir and adsorb at 4 °C, wash the product with water by centrifugation, then add a SH-TBA 1 solution, stir at 4 °C, and add a hemin solution to obtain hemin-G-quadruplex / AuNPs.

[0011] Further, in step (1), the Mw of branched polyethyleneimine is 600.

[0012] Further, in step (1), the hydrothermal reaction time is 6 - 8 h.

[0013] Further, in step (1), the centrifugation speed is 8000 - 10000 rpm, and the centrifugation time is 5 - 15 min.

[0014] Further, in step (1), the vacuum drying temperature is 25 - 30 °C, and the vacuum drying time is 1 - 2 h.

[0015] Further, in step (2), the mass concentration of the AuNPs solution is 0.1%, the sequence of SH - TBA 1 is: 5'-SH-(CH2)6 - GGTTGGTGTGGTTGG - 3', its concentration is 2 μM, the mass concentration of the BSA aqueous solution is 0.25%; the loading amount of PDC on the working electrode is 0.20 mg / cm 2 。

[0016] Further, in step (2), the drying time at room temperature is 3 - 5 h, and the incubation time is 8 - 14 h.

[0017] Further, in step (3), the stirring adsorption time is 2 - 4 h, the centrifugation speed is 10000 - 12000 rpm, and the centrifugation time is 5 - 15 min.

[0018] Further, in step (3), the concentration of the SH - TBA 1 solution is 2 - 3 μM, the stirring time is 10 - 12 h, the concentration of the hemin solution is 1 mM, and the molar ratio of SH - TBA 1 to hemin is 2 - 3:1000.

[0019] Further, in step (3), the centrifugation speed is 10000 - 12000 rpm, and the centrifugation time is 5 - 15 min.

[0020] The present invention provides the application of the above - mentioned solid - state self - enhanced ECL aptamer thrombin sensor in the electrochemical luminescence detection of thrombin, and the specific steps are as follows:

[0021] Incubate the working electrode with the solution to be measured at room temperature. After the incubation is completed, add hemin - G - quadruplex / AuNPs, dry and then drop - add naphthol solution for blocking. Using a three - electrode system, with Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and a phosphate buffer solution with a pH of 8 - 9 as the electrolyte, perform electrochemical luminescence signal detection. According to the linear relationship between the thrombin concentration and the electrochemical luminescence signal, calculate the thrombin concentration in the solution to be measured.

[0022] Further, the linear relationship between thrombin concentration and electrochemiluminescence signal is y = -4360.15 - 589.74x, where x = LgC and C is the thrombin concentration.

[0023] Further, the electrochemiluminescence signal detection conditions are as follows: the photomultiplier voltage is 600 V, the amplification factor is 2, and the sweep rate is 10 mV / s.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) A brand-new conjugated polymer dot PDC is green-synthesized, which has the ability to act as a ruthenium bipyridine ECL luminescence coreactant;

[0026] (2) PDC and ruthenium bipyridine dicarboxylate are covalently linked on the electrode by covalent bonds, and a solid-state self-enhanced ECL system is successfully constructed;

[0027] (3) Using the enhanced electrochemiluminescence signal, a sandwich-structured ultrasensitive thrombin biosensing system is constructed, and its detection linear range is 10 -15 to 10 -8 M, and the detection limit is 0.36 fM. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural concept diagram of the sandwich-structured sensor;

[0029] Figure 2 It is a diagram for verifying the feasibility of ECL enhancement. Among them, (A) is a 3D diagram of the feasibility verification ECL, (B) is a TV spectrum diagram of the feasibility verification ECL. a is the mixture of dried PDC and ruthenium bipyridine dicarboxylate on the electrode, b is the sequential drying of ruthenium bipyridine dicarboxylate and PDC on the electrode, c is the sequential drying of PDC and ruthenium bipyridine dicarboxylate on the electrode, d is the modification of ruthenium bipyridine dicarboxylate on the electrode, and e is the modification of PDC on the electrode;

[0030] Figure 3 It is a diagram for regulating the loading amount of ECL enhancement. Among them, (A) is a diagram of the ECL intensity regulated by the loading amount, and (B) is a bar chart of the error bars of the loading amount regulation;

[0031] Figure 4 It is a diagram for regulating the pH of the electrolyte during the ECL process. Among them, (A) is a diagram of the ECL intensity regulated by the pH of the electrolyte, and (B) is a bar chart of the error bars of the pH regulation of the electrolyte;

[0032] Figure 5 It is a diagram of the stability during the ECL enhancement process;

[0033] Figure 6It is a linear relationship diagram of TB sensing. Among them, (A) is the ECL intensity diagram of the sensing linear relationship, (B) is the error bar histogram of the sensing linear relationship, (C) is the fitting curve diagram of the sensing linear relationship, and a - h successively represent the TB concentration of the sample as 10 -15 -10 -8 M;

[0034] Figure 7 It is a verification diagram of the anti - interference ability of the sensor. Among them, (A) is the ECL intensity diagram of anti - interference verification, (B) is the error bar histogram of the quenching effect of the interferent. a is the blank sample, b is the 100 nM hemoglobin sample, c is the 0.25% BSA sample, d is the 10 -11 MTB sample, e is the mixture sample of the three;

[0035] Figure 8 It is a diagram of the stability of the sensing process;

[0036] Figure 9 It is a fluorescence verification diagram of material synthesis;

[0037] Figure 10 It is a comparison diagram of the raw material ratio of PDC synthesis. Among them, (A) to (D) respectively correspond to the dosages of 2 - aminoterephthalic acid of 330 mg, 660 mg, 990 mg, and 1320 mg;

[0038] Figure 11 It is a comparison diagram of the hydrothermal synthesis temperature of PDC;

[0039] Figure 12 It is a comparison diagram of the hydrothermal synthesis time of PDC;

[0040] Figure 13 It is a transmission electron microscope characterization diagram of PDC. Specific embodiments

[0041] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the protection scope of the present invention is not limited to the content described.

[0042] Example 1

[0043] The construction of the solid - state self - enhanced ECL aptasensor is as follows:

[0044] (1) Hydrothermal synthesis of PDC: Weigh 500 mg of branched polyethyleneimine (PEI, Mw = 600) and dissolve it in 10 mL of ultrapure water. Sonicate for 2 minutes to dissolve. Weigh 990 mg (5.5 mmol) of NH2BDC and pour it into the solution. Stir at room temperature for 15 minutes to disperse the solution evenly. After sonication, pour the mixture into a 20 ml Teflon liner and place it in a forced-air drying oven to react at 120 °C for 6 h. After the reaction, centrifuge at 8000 rpm for 5 min using a high-speed centrifuge, collect the supernatant, discard the precipitate, then add excess absolute ethanol to the supernatant and let it stand for 1 day to wait for PDC to precipitate. Centrifuge the standing solution at 8000 rpm for 5 min using a high-speed centrifuge, collect the precipitate, wash it 4 times with absolute ethanol, and centrifuge at 8000 rpm for 5 min to collect the sample after each wash. Place the collected sample in a vacuum drying oven at 30 °C and dry for 1 h. The obtained PDC is stored at room temperature in a ventilated and cool place for standby. The TEM image of PDC is as shown in Figure 13 shown.

[0045] (2) Incubation of the electrode: Ultrasonically clean the electrode with ultrapure water for 2 min, then polish it on suede with particle sizes of 0.3 μm and 0.05 μm for 5 min in sequence, then ultrasonically clean the polished electrode with ultrapure water for 5 min, and finally let the electrode dry naturally at room temperature. Drop 10 μL of 4 mg / ml PDC and 10 μL of 1 mg / ml ruthenium bipyridine dicarboxylate on the smooth and dry electrode and composite-dry at room temperature. Then drop 10 μL of AuNPs solution onto the Ru-PDC composite material to form a covalent bond with PEI and dry in air for 3 h. Subsequently, incubate the nano-Au / Ru-PDC modified electrode with 10 μL of 2 μM MSH-TBA1 solution at 4 °C for 13 h. To reduce non-specific adsorption, block the modified electrode with 10 μL of 0.25% BSA aqueous solution and let it stand for 40 min.

[0046] (3) Construction of the conjugate: The conjugate of the quenching probe in the "sandwich" structure is composed of heme (HE) / SH-TBA1 / AuNPs. First, suspend AuNPs in 2 mL of 1% BSA aqueous solution and stir at 4 °C for 4 h to adsorb the nano-gold. Then, centrifuge the product at 12000 rpm for 15 min and wash it 3 times with deionized water. Add 15 μL of SH-TBA1 to the above-mentioned dispersed product solution and stir at 4 °C for 12 h. Finally, introduce 10 μL of 1 mM heme solution to allow the DNA rich in G in the TBA1 sequence to fold into a G-quadruplex structure, thus obtaining heme-G-quadruplex / AuNPs. The product is continuously purified by centrifugation at 12000 rpm and washed 3 times with deionized water. The obtained heme-G-quadruplex / AuNPs can be stored in a refrigerator at 4 °C when not in use.

[0047] (4) Respectively, the electrodes obtained in step (2) were incubated with 10 μL of 10 -11 MTB solution at 37 °C for 40 minutes. After that, the treated conjugate was dropped onto the obtained electrode for 40 minutes to form a sandwich-type complex. To remove physically adsorbed substances, each step of the electrode treatment was rinsed with deionized water.

[0048] (5) A three-electrode system was adopted. The reference electrode was a silver / silver chloride electrode (Ag / AgCl electrode), the counter electrode was a platinum wire, and the electrolyte was a nitrogen-saturated PB buffer solution (0.1 M) with a pH of 9. When performing the ECL test, the photomultiplier tube of the instrument was set at 600 V, the amplification factor was 2, and the scanning rate was 10 mV / s.

[0049] The ECL spectra obtained by taking 10 consecutive potential cyclic scans were as follows Figure 8 shown. It can be seen that the relative standard deviation was 0.90%. In addition, the overall detection and analysis performance did not significantly decline, indicating that the aptamer sensor had good stability.

[0050] Comparative Example 1

[0051] This comparative example was basically the same as Example 1, except that in step (2), only 10 μL of 4 mg / ml PDC was dropped onto the smooth and dry electrode to construct an electrode with PDC as the luminescent material.

[0052] Comparative Example 2

[0053] This comparative example was basically the same as Example 1, except that in step (2), only 10 μL of 1 mg / ml ruthenium dicarboxy bipyridine was dropped onto the smooth and dry electrode to construct an electrode with ruthenium dicarboxy bipyridine as the luminescent material.

[0054] Comparative Example 3

[0055] This comparative example was basically the same as Example 1, except that in step (2), 10 μL of 4 mg / ml PDC and 10 μL of 1 mg / ml ruthenium dicarboxy bipyridine were successively modified on the smooth and dry electrode.

[0056] Comparative Example 4

[0057] This comparative example was basically the same as Example 1, except that in step (2), 10 μL of 1 mg / ml ruthenium dicarboxy bipyridine and 10 μL of 4 mg / ml PDC were successively modified on the smooth and dry electrode.

[0058] Comparative Example 5

[0059] This comparative example is basically the same as Example 1, except that in step (2), 10 μL of 4 mg / ml PDC and 10 μL of 41 mg / ml dicarboxydipyridyl ruthenium are simultaneously modified on a smooth and dry electrode.

[0060] The ECL test results of the aptamer sensors constructed in Comparative Examples 1-5 are as Figure 2 shown. Pure PDC basically has no ECL ability; dicarboxydipyridyl ruthenium itself emits light, but in the absence of the catalysis of a co-reactant, there is only about 600 a.u. of luminescence; when PDC and dicarboxydipyridyl ruthenium are modified on the electrode in sequence, their spatial order on the electrode is different, and the ECL enhancement is also different, which shows that the enhancement is limited by the contact area between them; therefore, when PDC and dicarboxydipyridyl ruthenium solutions are mixed and titrated on the electrode, it can be seen that its intensity is about 20 times that of pure dicarboxydipyridyl ruthenium, showing excellent ECL enhancement ability.

[0061] The ECL enhancement system constructed in Comparative Example 5 was subjected to ECL testing according to the optimal loading amount and electrolyte pH, and the ECL spectra obtained from 10 consecutive potential cycles were taken. The results are as Figure 5 shown. It can be seen that its relative standard deviation is 2.05%, and the good stability of its ECL enhancement is significantly lower than that of Example 1.

[0062] Example 2

[0063] This example is basically the same as Example 1, except that in step (5), the pH of the electrolyte is adjusted to 7, 8, 9, 10, and 11 respectively.

[0064] The results are as Figure 4 shown. It can be seen that the optimal pH of the electrolyte is 9, and the ECL enhancement effect is the strongest at this pH.

[0065] Example 3

[0066] This example is basically the same as Example 1, except that in step (2), the concentrations of PDC dropped on the smooth and dry electrode are adjusted to 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, and 5 mg / ml respectively.

[0067] The results are as Figure 3 shown. It can be seen that the optimal loading amount for ECL enhancement is 10 μL of 4 mg / ml PDC solution, that is, 0.20 mg / cm 2 .

[0068] Example 4

[0069] This example is basically the same as Example 1, except that in step (5), the concentrations of the TB solution are 10- 8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M.

[0070] The results are as Figure 6 shown. It can be seen that the ECL intensity decreases with the increase of TB concentration, and the detection deviation fluctuates slightly within the acceptable range. The linear range of TB is 10 -15 ~10 -8 M, covering the fluctuation range of TB in blood samples, and its detection limit is 3.6×10 -16 M (S / N = 3). The change of ECL intensity is proportional to the logarithm of TB concentration, and the correlation coefficient is 0.9989.

[0071] Example 5

[0072] This example is basically the same as Example 1, except that the TB solution is replaced with blank samples, 100 nM hemoglobin, 0.25% BSA, 10 -11 M TB, and the mixture of the first three.

[0073] The results are as Figure 7 shown. It can be seen that the ΔI value of the foreign protein is much smaller than that of thrombin, indicating that the sensor has strong anti-interference ability. At the same time, the high ECL quenching signal obtained from the mixed sample indicates that the aptamer sensor has acceptable detection selectivity.

[0074] Example 6

[0075] This example is basically the same as Example 1, except that the addition amount of NH2BDC in step (1) is 330 mg, 660 mg, 990 mg, 1320 mg, and PDCs with different mass ratios are obtained.

[0076] Prepare PEI solution, NH2BDC solution, and PDC solutions with different mass ratios at a mass concentration of 5 mg / mL with ultrapure water, ultrasonically disperse for 10 min, then use a pipette to add 100 μL into a fluorescence cuvette filled with 3 ml of deionized water, mix for 5 min and then perform fluorescence testing. Each sample is tested three times, the excitation wavelength is 331 nm, and the grating is set to 6 lines / mm.

[0077] The results are as Figure 9 shown. It can be seen that PDC shows different fluorescence results from the two raw materials, proving the synthesis of the new substance.

[0078] The results are as follows Figure 10 shown. Based on the peaks of fluorescence, the degree of carbonization of the material was roughly judged, and the material closer to the near-infrared was selected as a better judgment basis (better fluorescence performance and the possibility of fluorescence resonance enhanced ECL with ruthenium derivatives). It can be seen that the optimal addition amount of NH2BDC is 990 mg, that is, the best mass ratio of NH2BDC to PEI is 2:1.

[0079] Example 7

[0080] This example is basically the same as Example 1, except that in step (1), the hydrothermal reaction temperatures are 100 °C, 120 °C, 140 °C, 160 °C, 180 °C, and 200 °C respectively.

[0081] The results are as follows Figure 11 shown. It can be seen that the optimal temperature for hydrothermal synthesis is 120 °C.

[0082] Example 8

[0083] This example is basically the same as Example 1, except that in step (1), the hydrothermal reaction times are 2 h, 4 h, 6 h, 8 h, and 10 h respectively.

[0084] The results are as follows Figure 12 shown. It can be seen that the optimal time for hydrothermal synthesis is 6 h.

[0085] Example 9

[0086] This example is basically the same as Example 1, except that the TB aptamer sensor constructed in Example 1 was used to detect serum samples with different concentrations of TB added by itself, and the results are shown in Table 1.

[0087] Table 1 Detection results of the aptamer sensor for TB serum samples

[0088]

[0089] As can be seen from Table 1, the TB aptamer sensor constructed in Example 1 has a high detection accuracy.

Claims

1. A construction method of a solid-state self-enhanced ECL aptamer thrombin sensor, characterized in that, It includes the following steps: (1) According to the mass ratio of PEI to NH2BDC being 1:1.8 - 2.0, ultrasonically dissolve PEI in water, add NH2BDC, stir until evenly mixed, then carry out a hydrothermal reaction at 100 - 120 °C. After the reaction ends, centrifuge, collect the supernatant, add excessive absolute ethanol, precipitate the product, centrifuge, collect the precipitate, wash it with absolute ethanol multiple times, and dry it under vacuum to obtain PDC; (2) Incubation of the electrode: According to the mass ratio of PDC to ruthenium (II) 2,2'-bipyridine-4,4'-dicarboxylate being 1:0.2 - 0.3, sequentially dropwise add PDC solution and ruthenium (II) 2,2'-bipyridine-4,4'-dicarboxylate solution on the clean electrode surface to complete mixing, dry at room temperature, dropwise add AuNPs solution, dry at room temperature, dropwise add SH-TBA 1 solution, incubate at 4 °C, and finally dropwise add BSA aqueous solution for blocking to obtain the working electrode; (3) Construction of the conjugate: Suspend AuNPs in BSA aqueous solution, stir and adsorb at 4 °C, wash the product by centrifugation with water, then add SH-TBA 1 solution, stir at 4 °C, and add hemin solution to obtain hemin-G-quadruplex / AuNPs.

2. The construction method according to claim 1, characterized in that, In step (1), the Mw of branched polyethyleneimine is 600, and the hydrothermal reaction time is 6 - 8 h.

3. The construction method according to claim 1, characterized in that, In step (1), the centrifugation speed is 8000 - 10000 rpm, the centrifugation time is 5 - 15 min, the vacuum drying temperature is 25 - 30 °C, and the vacuum drying time is 1 - 2 h.

4. The construction method according to claim 1, wherein In step (2), the mass concentration of the AuNPs solution is 0.1%, the sequence of SH-TBA 1 is: 5'-SH-(CH2)6-GGTTGGTGTGGTTGG-3', its concentration is 2 μM, and the mass concentration of the BSA aqueous solution is 0.25%; the loading amount of PDC on the working electrode is 0.20 mg / cm 2 .

5. The construction method according to claim 1, characterized in that In step (2), the drying time at room temperature is 3 - 5 h, and the incubation time is 8 - 14 h.

6. The construction method according to claim 1, characterized in that In step (3), the stirring adsorption time is 2 - 4 h, the centrifugation speed is 10000 - 12000 rpm, and the centrifugation time is 5 - 15 min.

7. The construction method according to claim 1, characterized in that In step (3), the concentration of SH-TBA 1 solution is 2 - 3 μM, the stirring time is 10 - 12 h, the concentration of hemin solution is 1 mM, and the molar ratio of SH-TBA 1 to hemin is 2 - 3:1000.

8. A solid-state self-enhanced ECL aptamer thrombin sensor prepared by the preparation method according to any one of claims 1 - 7.

9. Application of the solid-state self-enhanced ECL aptamer thrombin sensor according to claim 8 in the electrochemical luminescence detection of thrombin. The specific steps are as follows: Incubate the working electrode with the test solution at room temperature. After the incubation ends, add hemin-G-quadruplex / AuNPs, dry and then dropwise add naphthol solution for blocking. Adopt a three-electrode system, use Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and a phosphate buffer solution with a pH of 8 - 9 as the electrolyte to carry out electrochemical luminescence signal detection. According to the linear relationship between the thrombin concentration and the electrochemical luminescence signal, calculate the thrombin concentration in the test solution.

10. The application according to claim 9, characterized in that The linear relationship between the thrombin concentration and the electrochemical luminescence signal is y = -4360.15 - 589.74x, where x = LgC, C is the thrombin concentration, and the electrochemical luminescence signal detection conditions are: the photomultiplier voltage is 600 V, the amplification factor is 2, and the scanning rate is 10 mV / s.

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