DR1 detection kit based on electro-deposition AuNPs / PTAA and polymer PLGA dual signal amplification

Abstract

The invention discloses a DR1 detection kit based on electro-deposition AuNPs / PTAA and polymer PLGA dual signal amplification. The DR1 detection kit mainly comprises the following raw materials: a gold electrode, HAuCl4. 4H2O, 3-TAA, tetrabutylammonium perchlorate, EDC, NHS, Ab1, BSA, Ab2 and a PLGA solution. The AuNPs / PTAA is modified on the surface of the electrode through electro-deposition, so that the specific surface area of the electrode is increased, more target objects are loaded, and the sensitivity of a sensing system is improved. Due to the introduction of the polymer PLGA, a large number of electroactive tags are attached to the biosensing system, and the sensitivity of the sensing system is further improved. Meanwhile, the biosensing system constructed by the invention has excellent selectivity, stability, reproducibility and anti-interference capability. Besides, the sensing system disclosed by the invention has excellent reliability, can be used for detecting DR1 in a complex biological substrate, and has potential application prospects in clinical HT diagnosis.

Description

Technical Field

[0001] The invention relates to a kit for detecting DR1 with high sensitivity and without labeling electrochemical biosensing based on dual signals of electrodeposited AuNPs / PTAA and polymer PLGA and its application, belonging to the technical field of bioanalysis. Background Art

[0002] Hashimoto's thyroiditis (HT) is the most common autoimmune disease of the thyroid gland, characterized by thyroid enlargement, lymphocyte infiltration in the parenchyma, and hypothyroidism. In recent years, the incidence of HT combined with thyroid cancer and papillary thyroid cancer has been increasing, seriously threatening human life and health. Early detection of Hashimoto's thyroiditis can help with treatment, reduce the risk of death, and improve patient survival. Transcriptional down regulation factor 1 (DR1) is an inhibitor of RNA polymerase (polII) transcription and is considered to be a specific marker for HT. Therefore, the development of a sensitive and rapid DR1 detection method can be used to prevent the occurrence or development of HT.

[0003] At present, researchers have developed a variety of disease marker detection methods, including enzyme-linked immunosorbent assay, fluorescence, electrochemiluminescence, electrochemical sensing technology, etc. Among them, electrochemical biosensors are widely used in disease-related detection due to their high sensitivity, selectivity and portability. Label-free electrochemical biosensors, especially electrochemical biosensors based on electrochemical impedance analysis (EIS), can efficiently, quickly and label-free detect proteins, DNA and low molecular weight biomolecules, and have been proven to be reliable tools for detecting disease markers. However, the stability of existing electrochemical sensors still needs to be improved, and the sensitivity cannot meet the detection requirements of trace targets. And common polymerization methods such as ATRP, ROP, etc. involve expensive catalysts or heavy metal catalysts, which are not green and non-toxic. Therefore, the purpose of the present invention is to use electrodeposited AuNPs / PTAA and polymer PLGA as a signal amplification strategy to improve the sensitivity of biosensors and construct a label-free electrochemical biosensing method to achieve early diagnosis of diseases. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a DR1 detection kit and application based on dual signal amplification of electrodeposited AuNPs / PTAA and polymer PLGA, which has the advantages of high sensitivity, selectivity, repeatability, good stability, strong anti-interference ability, etc.

[0005] In order to achieve the above-mentioned object, the technical scheme of the present invention is to provide a DR1 detection kit based on dual signal amplification of electrodeposited AuNPs / PTAA and polymer PLGA, comprising the following raw materials: gold electrode, HAuCl4·4H2O, 3-TAA, tetrabutylammonium perchlorate, EDC, NHS, Ab1, BSA, Ab2, PLGA, anhydrous acetonitrile, [Fe(CN)6] 3- / 4- Electrolyte solution.

[0006] Furthermore, some raw materials need to be prepared into solutions when used, among which the mass concentration of HAuCl4·4H2O solution is 1%, and the concentration of 3-TAA solution is 1 mg·mL -1 , the concentration of tetrabutylammonium perchlorate solution was 50 mM, the concentration of EDC solution was 40 mM, the concentration of NHS solution was 10 mM, and the concentration of Ab1 solution was 1 μg·mL -1 , the concentration of BSA solution is 1%, v / v, and the concentration of Ab2 solution is 100 ng·mL -1 The concentration of PLGA solution was 5 mg mL -1 .

[0007] On the other hand, the technical solution of the present invention is to provide a method for using the DR1 detection kit, comprising the following steps:

[0008] (1) Electrode pretreatment

[0009] The bare gold electrode is ground to obtain a polished mirror surface;

[0010] (2) Electrode modification

[0011] ① Soak the electrode in HAuCl4·4H2O solution (4-6mL) and perform constant potential electrodeposition;

[0012] ② Soaking the electrode of step ① in an anhydrous acetonitrile solution (4-6 mL) containing 3-TAA and tetrabutylammonium perchlorate for electrodeposition;

[0013] ③ Soak the electrode in step ② in an anhydrous acetonitrile solution (100-200 μL) containing EDC and NHS for reaction;

[0014] ④ Add Ab1 solution (5-10 μL) dropwise onto the electrode in step ③ to react;

[0015] ⑤ Soak the electrode in step ④ in BSA solution (100-200 μL) and react;

[0016] ⑥ Add the solution to be tested (5-10 μL) dropwise onto the electrode in step ⑤ for reaction;

[0017] ⑦ Add Ab2 solution (5-10 μL) dropwise onto the electrode in step ⑥ to react;

[0018] ⑧ Soak the electrode in step ⑦ in a polymer PLGA solution (100-200 μL) for reaction;

[0019] ⑨ Place the electrode from step ⑧ on [Fe(CN)6] 3- / 4- The impedance value of the electrode is measured in the electrolyte solution, and the concentration of DR1 in the solution to be tested is calculated based on the impedance value.

[0020] Furthermore, the temperature of the constant potential electrodeposition in step ① is 35-38°C, and the time is 90s; the temperature of the electrodeposition in step ② is 35-38°C; the temperature of the reaction in step ③ is 35-38°C, and the time is 0.5-1h; the temperature of the reaction in step ④ is 35-38°C, and the time is 1-2h; the temperature of the reaction in step ⑤ is 35-38°C, and the time is 0.5-1h; the temperature of the reaction in step ⑥ is 35-38°C, and the time is 1-2h; the temperature of the reaction in step ⑦ is 35-38°C, and the time is 1-2h; the temperature of the reaction in step ⑧ is 35-38°C, and the time is 1-2h.

[0021] Furthermore, the potential of the constant potential electrodeposition in step ① is -0.2 V; the voltage of the electrodeposition in step ② is -0.55 to -1.9 V, the scanning rate is 50 mV / s, and the cycle is repeated 7 times.

[0022] On the other hand, the technical solution of the present invention is to provide an application of the DR1 detection kit in the preparation of a product for detecting Hashimoto's thyroiditis.

[0023] The detection method of the present invention is as follows Figure 1 shown.

[0024] The present invention uses electro-deposition of HAuCl4 to uniformly deposit AuNPs on the surface of a bare electrode, and then electro-deposit TAA to form PTAA on the surface of the electrode, and activate its terminal carboxyl group with EDC / NHS. The carboxyl group reacts with EDC / NHS to form an intermediate (PTAA*), and the intermediate reacts with the amino group of antibody Ab1 to connect Ab1 to the electrode surface. In the next step, BSA is used to block the unbound sites on the electrode surface to prevent subsequent non-specific binding on the electrode surface. DR1 is connected to the electrode surface through specific recognition with Ab1. Then Ab2 and DR1 are specifically recognized on the electrode surface to form an Ab1-DR1-Ab2 sandwich structure to improve the specificity of the sensing system. Subsequently, the carboxyl-terminated copolymer PLGA is activated and connected to Ab2 through an amide bond to amplify the signal and improve the detection sensitivity of the sensing system. Finally, an electrochemical workstation is used to convert the biological signal into a measurable impedance signal value to quantitatively detect DR1. By electro-depositing nanogold and introducing polymer PLGA, the detection range of the biosensor system is effectively expanded.

[0025] Beneficial effects of the present invention

[0026] 1. The dual signal amplification strategy based on electrodeposition of AuNPs / PTAA and polymer PLGA greatly improves the detection sensitivity.

[0027] 2. The present invention belongs to label-free detection, and the detection platform has the advantages of easy operation and environmental friendliness.

[0028] 3. The present invention modifies the electrode surface with AuNPs / PTAA by electrodeposition to increase the specific surface area of ​​the electrode and load more targets, thereby improving the sensitivity of the sensing system. In addition, the introduction of the polymer PLGA enables a large number of electroactive tags to be attached to the biosensor system, further improving the sensitivity of the sensing system.

[0029] 4. Under the optimal experimental conditions, the analytical performance of the present invention within a certain range of DR1 concentration was studied. Through calculation, it was found that there was a good linear relationship between the logarithm of DR1 concentration and the impedance value signal intensity within a certain range. Under the optimal conditions, the biosensor system has a 50fg·mL -1 To 50ng·mL -1 Wide linear range, detection limit as low as 19.4 fg·mL -1 In addition, the constructed biosensor system has excellent selectivity, stability, reproducibility and anti-interference ability. It is worth noting that the sensing system has excellent reliability and can be used for the detection of DR1 in complex biological substrates, and has potential application prospects in clinical HT diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0031] Figure 2 EIS graphs (A) and CV graphs (B) of different modified electrodes: (a) bare electrode; (b) AuNPs / Au; (c) PTAA / AuNPs / Au; (d) PTAA* / AuNPs / Au; (e) Ab1 / PTAA* / AuNPs / Au; (f) BSA / Ab1 / PTAA* / AuNPs / Au; (g) DR1 / BSA / Ab1 / PTAA* / AuNPs / Au; (h) Ab2 / DR1 / BSA / Ab1 / PTAA* / AuNPs / Au; (i) PLGA / Ab2 / DR1 / BSA / Ab1 / PTAA* / AuNPs / Au modified electrode. (The concentration of DR1 is 1 ng·mL -1 )

[0032] Figure 3 The water contact angle spectra of the electrode surfaces at different modification steps: (A) bare electrode; (B) AuNPs modified electrode; (C) PTAA / AuNPs modified electrode; (D) PTAA* / AuNPs modified electrode; (E) Ab1 / PTAA* / AuNPs modified electrode; (F) BSA / Ab1 / PTAA* / AuNPs modified electrode; (G) DR1 / BSA / Ab1 / PTAA* / AuNPs modified electrode; (H) Ab2 / DR1 / BSA / Ab1 / PTAA* / AuNPs modified electrode; (I) PLGA / Ab2 / DR1 / BSA / Ab1 / PTAA* / AuNPs modified electrode.

[0033] Figure 4 Optimization of reaction conditions: (A) optimization of AuNPs electrodeposition time; (B) optimization of polymer PLGA concentration; (C) optimization of polymer PLGA reaction time. (Error bars are standard deviations of three sets of parallel experiments)

[0034] Figure 5 (A) is the EIS test result diagram; (B) is the linear relationship diagram between impedance value and different concentrations of DR1. (The curves ah in Figure A correspond to 50fg·mL -1 , 100fg·mL -1 , 1pg·mL -1 , 10pg·mL -1 , 100pg·mL -1 , 1ng·mL -1 , 10ng·mL -1 , 50ng·mL -1DR1) (the inner graph in Figure B is a linear curve, and the error bars are the standard deviation of three sets of parallel experiments).

[0035] Figure 6 The concentration is 1 ng·mL -1 EIS reaction histograms of DR1, BSA, cTnI, CEA, CY21-1 and HER-2 (error bars are the standard deviation of three sets of parallel experiments).

[0036] Figure 7 The impedance response of DR1 in PBS buffer, 1% (v / v) normal human serum and 5% (v / v) normal human serum (the concentration of DR1 is 100 fg·mL -1 , 10pg·mL -1 and 1 ng·mL -1 ; Error bars represent the standard deviation of 3 sets of parallel experiments). DETAILED DESCRIPTION

[0037] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0038] Ab1 is a rabbit anti-DR1 polyclonal antibody (Cat. No.: D163620), purchased from Sangon Biotech (Shanghai) Co., Ltd. Ab2 is a DR1 mouse anti-polyclonal antibody (Cat. No.: ab88597), purchased from Abcam (Shanghai) Trading Co., Ltd. The detection target DR1 used in the present invention is DR1 antigen, which is a mouse monoclonal antibody (Cat. No.: sc-515083) generated against amino acids 1-176 of full-length DR1 representing human origin, purchased from Guangzhou Aikenman Biotechnology Co., Ltd.

[0039] Example 1: Kit

[0040] A dual signal amplification detection kit for DR1 based on electrodeposition AuNPs and polymer PLGA includes the following raw materials: gold electrode, tetrachloroauric acid tetrahydrate (HAuCl4·4H2O), 3-thiopheneacetic acid (3-TAA), tetrabutylammonium perchlorate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), Ab1, bovine serum albumin (BSA), Ab2, poly(lactide-glycolide) (PLGA), anhydrous acetonitrile and [Fe(CN)6] 3- / 4- Electrolyte solution.

[0041] Some raw materials need to be prepared into solutions when used. The mass concentration of HAuCl4·4H2O solution is 1%, and the concentration of 3-TAA solution is 1 mg·mL -1, the concentration of tetrabutylammonium perchlorate solution was 50 mM, the concentration of EDC solution was 40 mM, the concentration of NHS solution was 10 mM, and the concentration of Ab1 solution was 1 μg·mL -1 , the concentration of BSA solution is 1%, v / v, and the concentration of Ab2 solution is 100 ng·mL -1 The concentration of PLGA solution was 5 mg mL -1 .

[0042] Example 2: Method of using the kit

[0043] (1) Electrode pretreatment

[0044] The bare gold electrode is ground to obtain a polished mirror surface;

[0045] (2) Electrode modification

[0046] ① Soak the electrode in 5mL 1% HAuCl4·4H2O solution and perform constant potential electrodeposition;

[0047] ② Soak the electrode from step ① in 5 mL of 1 mg·mL -1 3-TAA and 50 mM tetrabutylammonium perchlorate in anhydrous acetonitrile were electrodeposited;

[0048] ③ Soak the electrode in step ② in 200 μL of anhydrous acetonitrile solution containing 40 mM EDC and 10 mM NHS for reaction;

[0049] ④ Add 10 μL of 1 μg mL -1 Ab1 solution is added dropwise onto the electrode in step ③ to react;

[0050] ⑤ Soak the electrode in step ④ in 200 μL 1% BSA solution and react;

[0051] ⑥ Add 10 μL of the solution to be tested onto the electrode in step ⑤ for reaction;

[0052] ⑦ Add 10 μL of 100 ng mL -1 Ab2 solution is added dropwise onto the electrode in step ⑥ to react;

[0053] ⑧ Soak the electrode from step ⑦ in 200 μL 5 mg mL -1 In the polymer PLGA solution, the reaction;

[0054] ⑨ Place the electrode from step ⑧ on [Fe(CN)6] 3- / 4- The impedance value of the electrode is measured in the electrolyte solution, and the concentration of DR1 in the solution to be tested is calculated based on the impedance value.

[0055] Among them, the potential of constant potential electrodeposition in step ① is -0.2V, the temperature is 35°C, and the time is 90s; the voltage of electrodeposition in step ② is -0.55~-1.9V, the scanning rate is 50mV / s, the cycle is 7 times, and the temperature is 35°C; the reaction temperature of step ③ is 35°C, and the time is 0.5h; the reaction temperature of step ④ is 35°C, and the time is 1h; the reaction temperature of step ⑤ is 35°C, and the time is 0.5h; the reaction conditions of step ⑥ are 35°C, and the time is 1h; the reaction temperature of step ⑦ is 35°C, and the time is 1h; the reaction temperature of step ⑧ is 35°C, and the time is 1h.

[0056] Example 3: Method of using the kit

[0057] The method for using the kit of this embodiment is basically the same as that of Example 2, except that: the temperature of constant potential electrodeposition in step ① is 38°C; the temperature of electrodeposition in step ② is 38°C; the reaction temperature in step ③ is 38°C, and the time is 1h; the reaction temperature in step ④ is 38°C, and the time is 2h; the reaction temperature in step ⑤ is 38°C, and the time is 1h; the reaction conditions in step ⑥ are 38°C, and the time is 2h; the reaction temperature in step ⑦ is 38°C, and the time is 2h; the reaction temperature in step ⑧ is 38°C, and the time is 2h.

[0058] Example 4: Method of using the kit

[0059] The method for using the kit of this embodiment is basically the same as that of Example 2, except that: the temperature of constant potential electrodeposition in step ① is 36.5°C; the temperature of electrodeposition in step ② is 36.5°C; the reaction temperature in step ③ is 36.5°C, and the time is 0.75h; the reaction temperature in step ④ is 36.5°C, and the time is 1.5h; the reaction temperature in step ⑤ is 36.5°C, and the time is 0.75h; the reaction conditions in step ⑥ are 36.5°C, and the time is 1.5h; the reaction temperature in step ⑦ is 36.5°C, and the time is 1.5h; the reaction temperature in step ⑧ is 36.5°C, and the time is 1.5h.

[0060] Example 5: Feasibility Verification

[0061] In order to prove the feasibility of the detection method of the present invention, electrochemical characterization was carried out by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), and the relative changes of the electrode impedance value and the corresponding current were tested. Figure 2It can be seen that the impedance signal value of the bare electrode (curve a) is the smallest, ~260Ω, and its corresponding CV current signal is the largest. This is because the charge transfer rate on the surface of the bare gold electrode is relatively fast. When nanogold is electro-deposited on the electrode (curve b), a uniform layer of AuNPs is formed on the surface of the gold electrode, which reduces the impedance value to 217Ω and slightly increases the current signal value. This is because the modification of nanogold accelerates the power of charge transfer on the electrode surface. It is worth noting that after the electrode surface is electro-deposited with TAA (curve c), the electron transfer efficiency and rate on the gold electrode surface are reduced, causing the impedance value to increase to 462Ω, and the corresponding current is reduced. After that, after the terminal carboxyl group of PTAA is activated by EDC and NHS (curve d), the measured impedance value is reduced and the current value is increased. This may be because the activation of the terminal carboxyl group of PTAA reduces the charge transfer resistance on the electrode surface. When Ab1 (curve e), BSA (curve f), DR1 (curve g) and Ab2 (curve h) were introduced into the electrode in sequence, the electron transfer process was blocked after these protein molecules were fixed on the gold electrode surface, resulting in an increase in the electron transfer impedance on the gold electrode surface and a continuous decrease in the current signal value. Finally, after modification with PLGA (curve i), the impedance signal value increased significantly and the current signal value decreased significantly, mainly because the larger molecular weight of PLGA increased the spatial resistance. The experimental results provide a reliable basis for the successful construction of the sensing system.

[0062] Example 6: Characterization Analysis

[0063] Water contact angle (WCA) can be used to illustrate the change in hydrophilicity and hydrophobicity of the electrode surface after modification with groups. The stronger the hydrophilicity of the group attached to the electrode, the smaller the water contact angle obtained. On the contrary, the stronger the hydrophobicity of the group, the larger the water contact angle obtained. Figure 3 As shown in Figure 2, due to the hydrophobicity of the cleaned gold electrode surface, the WCA value of the bare gold electrode is 91.8° ( Figure 3 A). When the electrode was modified with gold nanoparticles, the WCA decreased to 87.2° ( Figure 3 B). After electrodeposition of TAA, the WCA value decreased to 84.8° ( Figure 3 C), this is because the PTAA formed by electrodeposition has a carboxyl group at the end, which increases the hydrophilicity of the modified electrode surface. After the carboxyl group is activated with EDC / NHS, the contact angle increases to 86.4° ( Figure 3 D), because the terminal carboxyl group of PTAA was activated into a hydrophobic ester bond by EDC / NHS. Then, Ab1, BSA, DR1 and Ab2 were modified on the electrode surface in sequence, and the measured WCA values ​​were 79.6° ( Figure 3 E), 76.0°( Figure 3 F), 72.7°( Figure 3 G) and 68.2°( Figure 3H). Because these protein molecules contain hydrophilic amino and carboxyl groups, the WCA value continues to decrease. Finally, after the copolymer PLGA reaction, the WCA increased sharply to 92.1° ( Figure 3 I), this is because after the modification of PLGA, a large amount of hydrophobic polymers are formed on the electrode surface. The above results prove the successful construction of the sensing system.

[0064] Example 7: Condition Optimization

[0065] In order to achieve the best detection performance of the kit, under the reaction conditions of Example 2, the present invention optimizes the electrodeposition time, the concentration of PLGA and the reaction time, and uses the impedance signal value to determine the optimal reaction conditions of the sensing system.

[0066] 1. Time optimization of AuNPs electrodeposition

[0067] The time of electrodeposition of AuNPs is crucial to the performance of the electrochemical sensing system. Figure 4 As shown in Figure A, when other conditions remain unchanged, within the range of 60 to 90 seconds, the impedance signal value increases with time, and then decreases. Therefore, 90 seconds is selected as the optimal reaction time for electrodeposition of AuNPs.

[0068] 2. Optimization of PLGA polymer concentration

[0069] Appropriate polymer concentration is a key factor in signal amplification. Therefore, the present invention studies 1-10 mg·mL -1 The influence of polymer concentration on the impedance signal value within the range. Figure 4 As shown in B, when other conditions remain unchanged, the signal value is between 1 and 5 mg·mL -1 There is a significant increase in the range. When the concentration increases to 6-10 mg·mL -1 The signal value decreased when the reaction was 5 mg mL -1 as the optimal concentration of polymer.

[0070] 3. Optimization of reaction time of polymer PLGA

[0071] In order to make PLGA fully react and maximize its benefits, the present invention studies the change trend of the impedance signal value within a reaction time of 30 to 80 minutes. Figure 4 As shown in Figure C, when other conditions remain unchanged, the impedance value increases with the extension of the reaction time, reaches the maximum value at 60 minutes, and then has no obvious change, indicating that the PLGA reaction reaches equilibrium at 60 minutes. Therefore, the present invention selects 60 minutes as the optimal time for the polymer reaction.

[0072] Example 8: Performance Analysis

[0073] Under the optimal conditions of Example 7, the analytical performance of the constructed biosensor system for DR1 was studied by comparing the responses of the constructed biosensor system to different concentrations of DR1. Figure 5 In A, the impedance response value gradually increases with the increase of DR1 concentration. Figure 5 As can be seen from B, R ct The logarithm of the antigen concentration is 50fg·mL -1 ~50ng·mL -1 There is a good linear relationship within the range. The linear regression equation is y = 533.85lgC DR1 +3424.89 (y represents the impedance signal value, R 2 =0.997), detection limit 19.4 fg·mL -1 . In addition, the present invention also compares this method with other protein marker detection sensors. As shown in Table 1, the impedance electrochemical sensing system constructed by the present invention has a wider detection range and a lower detection limit. The above results show that the electrochemical sensing system based on electrodeposited nanogold and polymer PLGA can be used for high-sensitivity detection of DR1.

[0074] Table 1 Comparison of this strategy with other protein marker detection sensors

[0075]

[0076] Example 9: Selectivity, reproducibility and stability analysis

[0077] To investigate the selectivity of the biosensing method, DR1, BSA, cardiac troponin I (cTnI), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CY21-1), and human epidermal growth factor receptor-2 (HER-2) (all at a concentration of 1 ng mL) were compared under the optimal experimental conditions. -1 ) of the impedance signal value. Figure 6 As shown in the figure, at the same concentration, the R ct DR1 R ct13.52%, 11.16%, 14.95%, 18.74% and 15.73%. These data, on the one hand, show that the specific binding rates of Ab1 and Ab2 to the target DR1 are very high, and on the other hand, they also prove that the electrochemical sensing system has good selectivity. In order to study the stability of the sensing system, the modified electrodes of the present invention are stored at 4°C for 14 days. Compared with the data measured before storage, the signal intensity of the electrode after 7 days was 95.61% of the original, and the signal intensity of the electrode after 14 days was 91.44% of the original. These results show that the proposed sensing system has good stability within a certain period of time. In addition, under the same conditions, the present invention carried out intra-group and inter-group experiments to evaluate the reproducibility of the sensing system. The results showed that the RSD values ​​of the inter-group and intra-group experiments were 4.57% and 2.66%, respectively, proving that the sensing system has good reproducibility.

[0078] Example 10: Anti-interference ability analysis and actual sample testing

[0079] In addition to selectivity, reproducibility and stability, anti-interference ability is also an important factor in evaluating the analytical performance of the sensor system. Therefore, the present invention uses the sensor system to perform multiple tests on samples diluted with 1% (v / v), 5% (v / v) normal human serum and samples diluted with PBS buffer solution, and compares the test results to evaluate the practical application ability of the analytical method.

[0080] The present invention determines the anti-interference ability of the electrochemical sensor system by calculating the ratio of the impedance signal value measured in serum environments of different concentrations to the impedance signal value measured in PBS buffer. Figure 7 As shown, the present invention measures the concentration of 100fg·mL in PBS buffer, 1% (v / v) and 5% (v / v) normal human serum. -1 , 10pg·mL -1 and 1 ng·mL -1 The impedance signal intensity of DR1. In 1% (v / v) and 5% (v / v) normal human serum, the concentration is 100fg·mL -1 DR1 R ct 96.83% and 94.99% in PBS buffer respectively; the concentration is 10 pg·mL -1 DR1 R ct 97.58% and 95.74% in PBS buffer respectively; the concentration is 1 ng·mL -1 DR1 R ct The above results show that the electrochemical impedance spectroscopy method still has high analytical performance in detecting DR1 in normal human serum.

[0081] At the same time, the present invention also uses this method to detect clinical serum samples of multiple patients. 7 clinical samples were provided by the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine. The use of clinical samples in this study has been approved by the Ethics Committee of Henan Provincial Hospital of Traditional Chinese Medicine. The results are shown in Table 2. The results measured by the constructed strategy are consistent with the detection results of the ELISA method, with a relative error of -3.78% to 4.50%. The results show that the constructed electrochemical impedance analysis method for detecting DR1 has a high accuracy in actual sample detection.

[0082] Table 2 Comparison of the results of this method and ELISA

[0083]

Claims

1. A DR1 detection kit based on dual signal amplification of electrodeposited AuNPs / PTAA and polymer PLGA, characterized in that: The invention comprises the following raw materials: gold electrode, HAuCl4·4H2O, 3-TAA, tetrabutylammonium perchlorate, EDC, NHS, Ab1, BSA, Ab2 and PLGA.

2. The DR1 detection kit according to claim 1, characterized in that Also includes anhydrous acetonitrile, [Fe(CN)6] 3- / 4- Electrolyte solution.

3. The DR1 detection kit according to claim 1 or 2, characterized in that Some raw materials need to be prepared into solutions when used. The mass concentration of HAuCl4·4H2O solution is 1%, and the concentration of 3-TAA solution is 1 mg·mL -1 , the concentration of tetrabutylammonium perchlorate solution was 50 mM, the concentration of EDC solution was 40 mM, the concentration of NHS solution was 10 mM, and the concentration of Ab1 solution was 1 μg·mL -1 , the concentration of BSA solution is 1%, v / v, and the concentration of Ab2 solution is 100 ng·mL -1 The concentration of PLGA solution was 5 mg mL -1 .

4. A method for using the DR1 detection kit according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Electrode pretreatment The bare gold electrode is ground to obtain a polished mirror surface; (2) Electrode modification ① Soak the electrode in HAuCl4·4H2O solution and perform constant potential electrodeposition; ② Soaking the electrode of step ① in an anhydrous acetonitrile solution containing 3-TAA and tetrabutylammonium perchlorate for electrodeposition; ③ Soak the electrode in step ② in an anhydrous acetonitrile solution containing EDC and NHS for reaction; ④ Add Ab1 solution to the electrode in step ③ and react; ⑤ Soak the electrode in step ④ in BSA solution and react; ⑥ Add the solution to be tested to the electrode in step ⑤ and react; ⑦ Add Ab2 solution dropwise onto the electrode in step ⑥ to react; ⑧ Soaking the electrode in step ⑦ in a polymer PLGA solution for reaction; ⑨ Place the electrode from step ⑧ on [Fe(CN)6] 3- / 4- The impedance value of the electrode is measured in the electrolyte solution, and the concentration of DR1 in the solution to be tested is calculated based on the impedance value.

5. The method for using the DR1 detection kit according to claim 4, characterized in that: The temperature of the constant potential electrodeposition in step ① is 35-38° C., and the time is 90 s; the temperature of the electrodeposition in step ② is 35-38° C.; the temperature of the reaction in step ③ is 35-38° C., and the time is 0.5-1 h; the temperature of the reaction in step ④ is 35-38° C., and the time is 1-2 h; the temperature of the reaction in step ⑤ is 35-38° C., and the time is 0.5-1 h; the temperature of the reaction in step ⑥ is 35-38° C., and the time is 1-2 h; the temperature of the reaction in step ⑦ is 35-38° C., and the time is 1-2 h; the temperature of the reaction in step ⑧ is 35-38° C., and the time is 1-2 h.

6. The method for using the DR1 detection kit according to claim 4, characterized in that: The potential of the constant potential electrodeposition in step ① is -0.2 V; the voltage of the electrodeposition in step ② is -0.55 to -1.9 V, the scanning rate is 50 mV / s, and the cycle is repeated 7 times.

7. Use of the DR1 detection kit according to any one of claims 1 to 3 in preparing a product for detecting Hashimoto's thyroiditis.

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