A Low-Voltage Driven Ratio-Modulated Bipolar Electrode Electrochemiluminescence Biosensor and Its Application

Abstract

This invention discloses a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor and its applications. It utilizes an HNCQDs / H2O2 system and Ru(bpy)3... 2+ The / TPrA system serves as a dual-anode signal source, enabling the construction of a novel ratiometric bipolar electrode electrochemiluminescence biosensor for miRNA detection and analysis. By capturing probes that specifically recognize target miRNAs and auxiliary probes modified with ALP-Au NPs, a "Y-shaped" nucleic acid hybridization configuration is formed. Based on enzyme-catalyzed signal amplification strategies and the working principle of bipolar electrodes, quantitative analysis of miRNA-222 can be achieved through signal intensity ratio analysis based on the changes in the intensity of the two ECL signals, reducing the probability of false positives. Furthermore, the selected dual-anode ECL luminescent probes have low excitation potentials, effectively reducing the driving voltage of the ratiometric bipolar electrode electrochemiluminescence biosensor and expanding the selectivity of ECL luminescent probes. This invention offers advantages such as good portability, low cost, and high sensitivity, and has broad application prospects in clinical diagnosis.

Description

Technical Field

[0001] This invention relates to a low-voltage driven ratio bipolar electrode electrochemiluminescence biosensor for miRNA-222 detection, belonging to the field of biosensing technology. This biosensor is based on the change in the intensity of the dual-anodine ECL signal, and realizes the quantitative detection and analysis of miRNA in serum through the signal ratio. Background Technology

[0002] MicroRNAs (miRNAs) play crucial roles in cell proliferation, migration, and apoptosis, and are typically composed of 18 to 23 nucleotides. In recent years, numerous studies have demonstrated that miRNAs can serve as important biomarkers for early cancer diagnosis. With ongoing research, researchers have discovered that miRNAs can participate in regulating the expression of target proteins and inducing drug resistance in cancer cells. Elevated miRNA-222 expression is a risk factor for many cancers, including thyroid cancer, breast cancer, and prostate cancer. Furthermore, abnormal miRNA-222 expression is highly correlated with the development of drug resistance in tumor cells. Therefore, accurate detection of miRNA-222 is of great significance for early clinical diagnosis of cancer and for guiding personalized medication.

[0003] Electrochemiluminescence (ECL) technology combines the advantages of electrochemistry and chemiluminescence. Because the input electrical signal and output optical signal do not interfere with each other and no external excitation light source is required, it features high sensitivity and low background signal, making it particularly suitable for the detection and analysis of low-abundance targets. With the continuous development of ECL technology, various methods are employed to improve the detection performance of ECL biosensors, such as designing different ECL systems combined with diverse signal amplification strategies, developing novel and efficient luminescent probes, and modifying with nanomaterials. Ratio-coupled ECL biosensors based on dual signals use the ratio between the two ECL intensities for self-calibration, effectively avoiding external environmental interference and overcoming the limitations of single-signal analysis methods. They are particularly advantageous in the detection of low-abundance miRNAs that are easily degraded by RNases. However, in constructing ratio-coupled ECL biosensors, both ECL systems exist in the same reaction environment. Selecting a suitable ECL system is crucial, requiring consideration of factors such as the cross-reaction between the ECL luminescent probe and co-reactants, the regulation of the excitation potential, and the differentiation of ECL spectra. These factors, to some extent, limit the range of ECL system choices and significantly hinder the development and application of ratio-coupled ECL biosensors.

[0004] Bipolar electrodes (BPEs) have gained a foothold in the analytical field as a novel "wireless technology" due to their ease of control and the absence of direct voltage contact. Closed-loop BPEs effectively isolate the exchange of solutions between two electrolytic cells, avoiding complex analytical environments. Furthermore, based on the principle of electroneutrality, the entire closed-loop BPE follows the law of electron conservation, meaning the redox reactions in the two electrolytic cells are interconnected. This provides a unique advantage for constructing ratiometric ECL biosensors. Currently, ratiometric bipolar electrode electrochemiluminescence (BPE-ECL) biosensors have been reported in the field of bioanalysis. However, the cathode ECL system often requires a high driving voltage to generate a signal, which can easily damage the interface of the conductive material and generate side reactions, hindering stable signal output and accurate quantification of target analytes. Therefore, using ECL luminescent probes with low excitation potentials to construct ratiometric BPE-ECL biosensors will be beneficial for developing highly sensitive and stable point-of-care detection devices.

[0005] Carbon quantum dots (CQDs) have become an emerging carbon-based nanomaterial for electrochemical liquid chromatography (ECL) due to their advantages such as tunable size, easy surface modification, low preparation cost, and good biocompatibility. However, the application of CQDs in BPE-ECL sensors has been rarely reported, mainly due to two factors: (1) the generation of excited states by CQDs in electrochemical reactions requires the participation of strongly oxidizing co-reactants, resulting in a high excitation potential. Due to the voltage division effect of BPE, the driving voltage is too high to achieve effective driving; (2) the interaction between CQDs and co-reactants is weak, resulting in low luminescence efficiency and weak ECL signal, which limits the construction of high-sensitivity detection methods. These problems still need to be solved.

[0006] This invention discloses a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor for miRNA-222 detection. Using citric acid as the carbon source, hydrazine hydrate as the modifier, and N,N-dimethylformamide as the solvent, nitrogen-doped carbon quantum dots (HNCQDs) with an acylhydrazine-like structure are rapidly and efficiently prepared as ECL luminescent probes via microwave irradiation. 2+Using a dual-anodic ECL signal source, a novel ratiometric BPE-ECL biosensor was constructed to detect miRNA-222, employing a / TPrA system and an HNCQDs / H2O2 system. Gold nanoparticles modified the BPE cathode region, enabling the capture probe DNA to be immobilized via Au-S bonds. In the presence of miRNA-222, the capture probe recognized both miRNA-222 and an ALP-Au NP-labeled auxiliary probe; these three components formed a Y-shaped nucleic acid hybridization conformation through base complementarity. The modified ALP catalytic precursor... p -APP undergoes hydrolytic ester bond breakage, generating p -AP is prone to oxidation at the anode of the driving electrode, which accelerates the electron transfer rate, increases the total current in the BPE chip, and leads to an increase in the signal (ECL) of the HNCQDs / H2O2 system. HNCQDs ) increases. At the same time, p -AP and oxidation products p -QI versus Ru(bpy)3 2+ / TPrA system signal (ECL) Ru It has a quenching effect, leading to ECL. Ru Signal decreases. (via ECL) HNCQDs With ECL Ru The intensity ratio was used to achieve quantitative analysis of miRNA-222. The constructed ratiometric BPE-ECL biosensor can achieve highly sensitive detection of miRNA-222 under low voltage drive, and exhibits excellent specificity and signal stability, showing broad application prospects in the early diagnosis of clinical diseases. Summary of the Invention

[0007] 1. The purpose of this invention is to provide a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor for the detection of miRNA-222.

[0008] 2. The low-voltage driven ratio type BPE-ECL biosensor of the present invention uses Ru(bpy)3. 2+ Using the / TPrA system and HNCQDs / H2O2 system as dual-anodine ECL signal sources, and combining them with a "Y"-type nucleic acid hybridization structure and using ALP-Au NPs-labeled auxiliary probes as signal probes in the enzyme catalytic signal amplification strategy, a ratiometric BPE-ECL biosensor capable of operating under low voltage drive was constructed to achieve quantitative analysis of miRNA-222.

[0009] 3. A method for preparing a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor according to the present invention comprises the following steps in sequence:

[0010] (1) Synthesis of HNCQDs

[0011] 2.1 g of citric acid was dissolved in 15 mL of DMF, and then 1.25 mL of hydrazine hydrate was added dropwise to form a gel-like mixed precursor. The gel-like precursor was placed in a microwave reactor (800 W) and reacted for 300 s. A large porous solid was obtained, and 15 mL of water was added to dissolve the reaction product. The mixture was centrifuged (10,000 rpm, 10 min) to remove large black precipitates. The precipitate was allowed to stand, and the supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm filters, and dialyzed in deionized water for 24 h using a dialysis bag with a molecular weight cutoff (MWCO) of 100-500 Dalton. After dialysis, the mixture was dried at 60 °C to obtain HNCQDs solid, which was stored in a sealed container protected from light.

[0012] (2) Synthesis of ALP-Au NPs-labeled auxiliary probes

[0013] Synthesis of Au NPs: 10 mL of HAuCl4 solution (1.2 mM) was added to 85 mL of boiling water, and after heating to boiling, 5 mL of sodium citrate solution (38.7 mM) was added. The reaction system was kept at a gentle boil for 30 min to obtain Au NPs solution.

[0014] Synthesis of ALP-Au NPs-labeled auxiliary probe: 80 µL of ALP dilution buffer (0.8 mg / mL) and 40 µL of auxiliary probe DNA (1 µM) were added to 4 mL of Au NPs solution (pH = 8.2) and reacted for 2 h. 80 µL of MCH solution (10 mM) was added, and the mixture was stirred for 30 min. The mixture was then centrifuged at 12,000 rpm for 20 min, and the precipitate was washed three times to obtain the ALP-Au NPs-labeled auxiliary probe, which was then reconstituted in 400 µL of ultrapure water for later use.

[0015] (3) Fabrication of a low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor

[0016] ① In a BPE chip, 100 μL of chloroauric acid solution (1 wt%) was added to the cathode cell, and 0.1 M PBS solution (pH 7.4) was added to the anode cell. A constant voltage of 3.5 V was applied to both ends of the driving electrode for 300 s, thus modifying Au NPs on the BPE cathode (Au NPs / ITO). ② 10 μL of a 1 μM capture probe was dropped onto the Au NPs / ITO and incubated at room temperature for 2 h. The sample was washed three times with washing buffer and dried under nitrogen to obtain Capture probe DNA / Au NPs / ITO. ③ 20 μL of 1 mM MCH solution was completely applied to the surface of the Capture probe DNA / Au NPs / ITO and incubated at room temperature in the dark for 1 h to avoid non-specific adsorption. The sample was washed three times and dried under nitrogen to obtain MCH / Capture probe DNA / Au NPs / ITO. ④ Take 10 μL of the mixture of LALP-Au NPs-labeled helper probe and miRNA-222 and drop it onto the surface of MCH / Capture probe DNA / Au NPs / ITO. Incubate at 37 °C for 50 min, then wash three times with washing buffer to obtain Y-shaped DNA / Au NPs / ITO. ⑤ Take 10 μL... p -APP solution (10 mM) was dropped onto the surface of Y-shaped DNA / Au NPs / ITO and reacted at 37 °C in the dark for 30 min.

[0017] (4) Signal detection of a low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor

[0018] The HNCQDs / H2O2 system was added to the anode cell; Ru(bpy)3 was added to the cathode cell. 2+ / TPrA system. Cyclic voltammetry was used to detect the ECL signal of the HNCQDs / H2O2 system in the range of 0 ~ 3.5 V, PMT = 600 V, and scan rate of 0.2 V / s. Cyclic voltammetry was also used to detect Ru(bpy)3 in the range of 0 ~ 2.75 V, PMT = 600 V, and scan rate of 0.2 V / s. 2+ ECL signal of the / TPrA system.

[0019] Specifically, the low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor of the present invention is characterized in that the biosensor includes a dual-anode ECL luminescent probe and a co-reactant, a bipolar electrode chip constructed based on ITO and PDMS, and nucleic acid probes and reagents for detecting miRNA-222 contained in the cathode cell. The biosensor can be used for the specific and sensitive detection of miRNA-222.

[0020] Furthermore, the dual-anode ECL luminescent probe and co-reactant are the HNCQDs / H2O2 system and the Ru(bpy)32+ / TPrA system.

[0021] Further, the synthesis of HNCQDs in the HNCQDs / H2O2 system is as follows: 1-5 g of citric acid is dissolved in 5-15 mL of DMF, and then 0.5-2.5 mL of hydrazine hydrate is added dropwise to form a gel-like mixed precursor. The mixture is placed in a microwave reactor with a power of 800 W and reacted for 100-500 s. A large porous solid is obtained. The reaction product is dissolved in water, allowed to stand to precipitate, and the supernatant is taken. After dialyzing for 24 h, it is dried at 60 °C to obtain HNCQDs. The concentration of Ru(bpy)32+ in the Ru(bpy)32+ / TPrA system is 0.25 mM-1 mM, and the concentration of TPrA is 2.5 mM-10 mM. The concentration of HNCQDs in the HNCQDs / H2O2 system is 0.1 mg / mL-1.25 mg / mL, and the concentration of H2O2 is 50 mM-800 mM.

[0022] Furthermore, the bipolar electrode driving voltage is 0 ~ 3.5 V.

[0023] Furthermore, the nucleic acid probe used to detect miRNA-222 is a capture probe and an ALP-Au NPs-labeled auxiliary probe, and its preparation steps include:

[0024] (1) Capture probe DNA, sequence: 5'-SH-(CH2)6-GGCTGTTTTTTGAGACCCACGATAGATTTTTTTCAGCC-(CH2)6-SH-3';

[0025] (2) Assistant probe DNA, sequence: 5'-TCTATCGGTAGCCAGATGTAGCT-(CH2)6-SH-3';

[0026] (3) The modification process of the ALP-Au NPs-labeled auxiliary probe is as follows:

[0027] Add 10 mL of 1.2 mM HAuCl4 solution to 85 mL of boiling water, heat to boiling, then add 5 mL of 38.7 mM sodium citrate solution, and maintain the reaction system at a gentle boil for 30 min to obtain Au NPs solution; add 80 µL of 0.8 mg / mL ALP diluent and 40 µL of 1 µM Assistant probe DNA to 4 mL of pH = 8.2 Au NPs solution and react for 2 h; add 80 µL of 10 mM MCH solution, mix for 30 min, centrifuge at 12,000 rpm for 20 min, wash the precipitate 3 times to obtain ALP-Au NPs labeled assistant probe, and redissolve in 400 µL of ultrapure water for later use.

[0028] Furthermore, the enzyme-catalyzed reaction of the ALP-Au NPs-labeled helper probe is preceded by aminophenyl phosphate (p-APP).

[0029] Furthermore, the method for fabricating a low-voltage driven ratio-type bipolar electrode electrochemiluminescence biosensor according to the present invention is characterized by comprising the following steps:

[0030] (1) In the BPE chip, 100 μL of chloroauric acid solution with a concentration of 1 wt% was added to the cathode cell, and 0.1 M PBS solution with a pH of 7.4 was added to the anode cell. A constant voltage of 3.5 V was applied to both ends of the driving electrode for 300 s, that is, Au NPs were modified to form a bipolar electrode cathode (Au NPs / ITO).

[0031] (2) Take 10 μL of the capture probe with a concentration of 1 μM and drop it onto the cathode of the bipolar electrode to modify Au NPs (Au NPs / ITO). Incubate at room temperature for 2 h, wash three times with washing solution, and blow dry with nitrogen to obtain Capture probe DNA / Au NPs / ITO.

[0032] (3) Take 20 μL of 1 mM MCH solution to completely cover the surface of Capture probe DNA / Au NPs / ITO, keep it at room temperature in the dark for 1 h to avoid non-specific adsorption, wash three times, and blow dry with nitrogen to obtain MCH / Capture probe DNA / Au NPs / ITO.

[0033] (4) Take 10 μL of the mixture of ALP-Au NPs labeled helper probe and miRNA-222 and drop it onto the surface of MCH / Captureprobe DNA / Au NPs / ITO. After incubating at 37 °C for 50 min, wash three times with washing solution to obtain Y-shaped DNA / Au NPs / ITO.

[0034] (5) Take 10 μL of 10 mM p-APP solution and drop it onto the surface of Y-shaped DNA / Au NPs / ITO. React at 37°C in the dark for 30 min to obtain a low voltage driven ratio bipolar electrode electrochemiluminescence biosensor.

[0035] Furthermore, an HNCQDs / H2O2 system was added to the anode cell; a Ru(bpy)32+ / TPrA system was added to the cathode cell; cyclic voltammetry was used to detect the ECL signal of the HNCQDs / H2O2 system in the range of 0 ~ 3.5 V, PMT = 600 V, and scan rate of 0.2 V / s; cyclic voltammetry was also used to detect the ECL signal of the Ru(bpy)32+ / TPrA system in the range of 0 ~ 2.75 V, PMT = 600 V, and scan rate of 0.2 V / s.

[0036] Furthermore, the low-voltage driven ratio bipolar electrode electrochemiluminescence biosensor of the present invention or the low-voltage driven ratio bipolar electrode electrochemiluminescence biosensor prepared by the above method is characterized in that it is used for the specific and sensitive detection of miRNA-222.

[0037] Advantages of this invention:

[0038] The present invention discloses a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor, which uses Ru(bpy)3. 2+ The / TPrA system and HNCQDs / H2O2 system serve as dual-anode ECL signal sources. Combined with a "Y"-shaped nucleic acid hybridization structure and an ALP-Au NPs-labeled auxiliary probe, this strategy amplifies the enzyme catalytic signal, balancing both specificity and sensitivity for target analyte detection. The constructed ratiometric BPE-ECL biosensor achieves sensitive detection of miRNA-222 under low-voltage operation, effectively reducing energy consumption and protecting the BPE chip from high-voltage damage. This sensor exhibits excellent specificity, a wide detection range, a low detection limit, good stability and reproducibility, and demonstrates good performance in detecting human serum samples. Attached Figure Description

[0039] Figure 1This is a schematic diagram illustrating the working principle of a low-voltage driven ratio bipolar electrode electrochemiluminescence biosensor for miRNA-222 detection according to the present invention.

[0040] Figure 2 In the low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor of the present invention: In the figure: A is the HRTEM image of HNCQDs, and B is the particle size distribution of HNCQDs.

[0041] Figure 3 In the low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor of the present invention: in the three-electrode system, A is the CV curve and B is the ECL intensity-voltage relationship curve: in the figure: (a) PBS, (b) PBS + H2O2, (c) PBS + HNCQDs, (d) PBS + H2O2 + HNCQDs.

[0042] Figure 4 This invention relates to the biosensor assembly process in a low-voltage driven ratio-type bipolar electrode electrochemiluminescence biosensor, specifically the ECL method. Ru (A) and ECL HNCQDs (B) Signal intensity-voltage relationship curves: In the figure: (a) bare ITO, (b) AuNPs / ITO, (c) Capture probe DNA / Au NPs / ITO, (d) Capture probe DNA / Au NPs / ITO without miRNA-222 and (e) Y-shaped DNA / Au NPs / ITO.

[0043] Figure 5 This is a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor of the present invention, showing ECL signal-potential curves (A and B) for different miRNA-222 concentrations. Different concentrations of miRNA-222 and ECL... HNCQDs Strength / ECL Ru The relationship curve between intensities (C in the figure) and the relationship between different concentrations of miRNA-222 and lg(ECL) HNCQDs Strength / ECL Ru Linear relationship graph of intensity (D in the figure). In the figure, A and B are (a) 1 fM, (b) 10 fM, (c) 100 fM, (d) 10 3 fM、(e)10 4 fM、(f)10 5 fM、(g)10 6 fM. Detailed Implementation

[0044] To make the technical problems, technical solutions and effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0045] like Figure 1 The diagram shown illustrates the working principle of a low-voltage driven ratio-type bipolar electrode electrochemiluminescence biosensor for miRNA-222 detection according to the present invention: It employs Ru(bpy)3... 2+ Using a dual-anodic ECL signal source, a novel ratiometric BPE-ECL biosensor was constructed to detect miRNA-222, employing a / TPrA system and an HNCQDs / H2O2 system. Gold nanoparticles modified the BPE cathode region, enabling the capture probe DNA to be immobilized via Au-S bonds. In the presence of miRNA-222, the capture probe recognized both miRNA-222 and an ALP-Au NP-labeled auxiliary probe; these three components formed a Y-shaped nucleic acid hybridization conformation through base complementarity. The modified ALP catalytic precursor... p -APP undergoes hydrolytic ester bond breakage, generating p -AP is prone to oxidation at the anode of the driving electrode, which accelerates the electron transfer rate, increases the total current in the BPE chip, and leads to an increase in the signal (ECL) of the HNCQDs / H2O2 system. HNCQDs ) increases. At the same time, p -AP and oxidation products p -QI versus Ru(bpy)3 2+ / TPrA system signal (ECL) Ru It has a quenching effect, leading to ECL. Ru Signal decreases. (via ECL) HNCQDs With ECL Ru The intensity ratio was used to achieve quantitative analysis of miRNA-222. Example 1

[0046] The fabrication of a BPE chip in a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor is as follows:

[0047] Based on the dimensions of the circuitry on the designed ITO glass, the corresponding screen printing template dimensions were designed using Adobe Illustrator 2021. Using screen printing technology, a squeegee was used to transfer the protective coating ink (model: Zhongyi GK-501, China Zhongyi Ink & Coating Co., Ltd.) onto the ITO glass through a screen printing mold, and the ink was dried at 120 °C for 12 h. After the ink formed a dense and smooth protective coating with a special pattern on the ITO layer, wet chemical etching was performed using an etching solution (containing 0.5 M FeCl3, 1 M HCl, and 1 M HNO3). After etching, the surface was cleaned with acetone and then boiled in an isopropanol solution containing 2 M KOH for 20 min to remove the ink from the ITO electrodes. The resulting ITO glass was cut to the appropriate dimensions, thus initially constructing the BPE chip substrate. The preparation of polydimethylsiloxane (PDMS) films involves mixing PDMS with a curing agent (Sylgard 184 including PDMS and curing agent, Dow Corning) (10:1, w / w), followed by vacuuming for 10 min to remove bubbles, molding, and crosslinking at 40 °C for 6 h. After cooling at room temperature, the films are demolded, and corresponding reservoirs are cut out. These reservoirs are then integrated with a BPE chip substrate prepared from ITO glass to form a BPE chip. Example 2

[0048] The preparation and characterization of HNCQDs in a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor are as follows:

[0049] Synthesis of HNCQDs: 2.1 g of citric acid was dissolved in 15 mL of DMF, and then 1.25 mL of hydrazine hydrate was added dropwise to form a gel-like mixed precursor. The gel-like precursor was placed in a microwave reactor (800 W) and reacted for 300 s. A large porous solid was obtained. 15 mL of water was added to dissolve the reaction product, and the mixture was centrifuged (10,000 rpm, 10 min) to remove large black precipitates. The precipitate was allowed to stand, and the supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm filters, and dialyzed in deionized water for 24 h using a dialysis bag with a molecular weight cutoff (MWCO) of 100-500 Daltons. The precipitate was then dried at 60 °C to obtain solid HNCQDs, which were stored in a sealed container protected from light. The morphology and structure were observed under a transmission electron microscope, and the results are as follows. Figure 2 As shown in A, the synthesized HNCQDs particles are uniform and have good dispersibility. Statistical analysis of particle size shows that the average particle size of the HNCQDs is approximately 2.11 nm. Figure 2 (B in the middle). Example 3

[0050] The ECL characterization of the biosensor assembly process in a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor is as follows:

[0051] like Figure 3 As shown in Figure A, compared to the unmodified ITO (curve a), the ECL after Au NPs modification is significantly improved. Ru Significant improvement (curve b). The capture probe was modified onto Au NPs / ITO, ECL... Ru The signal decreases (curve c) because the nucleic acid probe hinders electron transfer at the electrode surface. In the absence of miRNA-222, ECL decreases after single incubation with the helper probe. Ru The signal remains almost unchanged (curve d) because the hybridization temperature between the auxiliary probe and the capture probe is low, resulting in low hybridization efficiency. In the absence of a target, the auxiliary probe cannot hybridize with the capture probe to introduce ALP-triggered enzyme catalysis. When miRNA-222 is present, a Y-shaped nucleic acid hybridization conformation forms on the electrode surface, and ECL... Ru A sharp drop (curve e) indicates the generation p -AP and p -QI to ECL Ru It produces a strong quenching effect.

[0052] Due to the conservation of electrons at the electrodes of BPE, ECL Ru With ECL HNCQDs The components are interconnected. Experiments showed that only after AuNPs and the capture probe were modified sequentially, under a voltage drive of 3.5 V, ECL... HNCQDs The changes were not obvious. Figure 3 In section B, curves a to c). It is noteworthy that when incubated with only the auxiliary probe and no target, the signal remains unchanged (curve d). However, after the Y-type nucleic acid hybridization conformation is generated, ECL... HNCQDs The intensity increased by an order of magnitude (curve e). In summary, the changes in ECL signal all indicate that this sensor specifically recognizes miRNA-222, leading to ECL. Ru With ECL HNCQDs The signal changes in reverse synchronization, thus the constructed ratiometric BPE-ECL biosensor can detect miRNA-222. Example 4

[0053] A low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor for the detection of miRNA-222

[0054] (1) Synthesis of ALP-Au NPs-labeled auxiliary probes

[0055] 1) Synthesis of Au NPs: Add 10 mL of HAuCl4 solution (1.2 mM) to 85 mL of boiling water, heat to boiling, then add 5 mL of sodium citrate solution (38.7 mM), and keep the reaction system at a gentle boil for 30 min to obtain Au NPs solution.

[0056] 2) Synthesis of ALP-Au NPs-labeled helper probes: 80 µL of alkaline phosphatase (ALP, Sigma-Aldrich) dilution buffer (0.8 mg / mL) and 40 µL of Assistantprobe DNA (1 µM, 5'-TCTATCGGTAGCCAGATGTAGCT-(CH2)6-SH-3', Sangon Biotech Shanghai Co., Ltd.) were added to 4 mL of the Au NPs solution (pH = 8.2) prepared in step 1) and reacted for 2 h. 80 µL of mercaptohexylhexanoate (MCH, Sigma-Aldrich) solution (10 mM) was added, and the mixture was stirred for 30 min. The mixture was then centrifuged at 12,000 rpm for 20 min, and the precipitate was washed three times to obtain the ALP-Au NPs-labeled helper probes, which were then reconstituted in 400 µL of ultrapure water for later use.

[0057] (2) Fabrication of a low-voltage driven ratio-type bipolar electrode electrochemiluminescence biosensor

[0058] In the BPE chip prepared in Example 1, 100 μL of chloroauric acid solution (1 wt%) was added to the cathode cell, and 0.1M PBS solution (pH 7.4) was added to the anode cell. A constant voltage of 3.5 V was applied to both ends of the driving electrode for 300 s, that is, Au NPs (referred to as Au NPs / ITO) were modified on the cathode of the BPE chip. (2) 10 μL of capture probe (1 μM, 5'-SH-(CH2)6-GGCTGTTTTTTGAGACCCACGATAGATTTTTTTCAGCC-(CH2)6-SH-3', referred to as Capture probe DNA, Sangon Biotech (Shanghai) Co., Ltd.) was dropped onto Au NPs / ITO, incubated at room temperature for 2 h, washed three times with washing solution, and dried with nitrogen to obtain Capture probe DNA / Au NPs / ITO. (3) Take 20 μL of MCH solution (1mM) to completely cover the surface of Capture probe DNA / Au NPs / ITO, incubate at room temperature in the dark for 1 h to avoid non-specific adsorption, wash three times, and dry with nitrogen to obtain MCH / Capture probe DNA / Au NPs / ITO. (4) Take 10 μL of the mixture of ALP-Au NPs-labeled auxiliary probe prepared in step 2) and different concentrations of miRNA-222 (5'-AGCUACAUCUGGCUACUGGGUCUC-3', Sangon Biotech (Shanghai) Co., Ltd.) and drop it onto the surface of MCH / Capture probe DNA / Au NPs / ITO, incubate at 37 °C for 50 min, and wash three times with washing solution to obtain Y-shaped DNA / Au NPs / ITO. (5) Take 10 μL of aminophenyl phosphate ( p -APP) solution (10 mM) was dropped onto the surface of Y-shaped DNA / Au NPs / ITO and reacted at 37 °C in the dark for 30 min.

[0059] (3) Signal detection of a low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor

[0060] The quantitative detection of miRNA-222 was performed as follows: The anolyte was filled with an HNCQDs / H2O2 system (100 μL of 0.1 M PBS buffer containing 0.5 mg / mL HNCQDs and 0.1 M H2O2, i.e., 100 μL of 0.1 M PBS buffer contained 0.5 mg / mL HNCQDs and 0.1 M H2O2); the cathode was filled with Ru(bpy)3. 2+ / Tripropylamine (TPrA) system (100 μL containing 0.5 mM Ru(bpy)3) 2+ 0.1 M PBS buffer containing 5 mM TPrA, i.e., 100 μL of 0.1 M PBS buffer containing 0.5 mM Ru(bpy)3. 2+ (and 5 mM TPrA). Cyclic voltammetry was used, range 0 ~ 3.5 V, PMT = 600 V, scan rate 0.2 V / s, to detect ECL. HNCQDs Cyclic voltammetry was used, with a range of 0 ~ 2.75 V, PMT = 600 V, and a scan rate of 0.2 V / s, to detect ECL. Ru .

[0061] ECL alone Ru and ECL HNCQDs The relationship with different concentrations of miRNA-222, such as Figure 5 As shown in A, when the concentration of miRNA-222 increases from 1 fM to 10... 4 fM, driving electrode anode ECL Ru The signal gradually decreased; as the concentration of miRNA-222 further increased (10... 4 fM to 10 6 fM), ECL Ru The downward trend is approaching a plateau. Furthermore, regarding BPE anode ECL... HNCQDs The relationship between intensity and the logarithm of miRNA-222 concentration was also investigated. Figure 5 (B in ECL) The two are directly proportional. HNCQDs / ECL Ru with lg C miRNA-222 The two showed an exponential growth trend. Figure 5 (C) in ECL. HNCQDs / ECL Ru After taking the logarithm, it is compared with lg C miRNA-222 Positive correlation ( Figure 5 D in the middle has a good correlation. R 2 = 0.994, the linear equation is: lg(ECL) HNCQDs / ECL Ru ) = 0.364lg C miRNA-222 -1.167, with a detection limit as low as 0.16 fM. These results demonstrate that, compared to single ECL signal detection methods, the miRNA-222 detection method based on the ratiometric BPE-ECL sensing strategy exhibits a wider linear range and better correlation.

[0062] Sequence list description:

[0063] Serial number 1 (ID): Assistant probe DNA

[0064] 5'-TCTATCGGTAGCCAGATGTAGCT-(CH2)6-SH-3';

[0065] Serial number 2 (ID): Capture probe DNA

[0066] 5'-SH-(CH2)6-GGCTGTTTTTTGAGACCCACGATAGATTTTTTTCAGCC-(CH2)6-SH-3';

[0067] Serial number 3 (ID): miRNA-222

[0068] 5'-AGCUACAUCUGGCUACUGGGUCUC-3'.

[0069] The DNA and RNA sequences in the sequence listing provided by this invention are known. If a residue contains "u", the "u", the initial "-" and the letters and numbers preceding it, and the final "-" and the letters and numbers following it are omitted. The above description is merely a preferred embodiment of this invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor, characterized in that, The biosensor comprises a dual-anode ECL luminescent probe and co-reactant, a bipolar electrode chip constructed based on ITO and PDMS, and nucleic acid probes and reagents for detecting miRNA-222 contained in the cathode cell; the dual-anode ECL luminescent probe and co-reactant are an HNCQDs / H2O2 system and Ru(bpy)3. 2+ / TPrA system; Synthesis of HNCQDs in the HNCQDs / H2O2 system: 1-5 g of citric acid was dissolved in 5-15 mL of DMF, and then 0.5-2.5 mL of hydrazine hydrate was added dropwise to form a gel-like mixed precursor. The mixture was placed in a microwave reactor with a power of 800 W and reacted for 100-500 s. A large porous solid was obtained. The reaction product was dissolved in water, allowed to stand to precipitate, and the supernatant was collected. After dialyzing for 24 h, the supernatant was dried at 60 °C to obtain HNCQDs. The Ru(bpy)3... 2+ Ru(bpy)3 in the / TPrA system 2+ The concentration of TPR is 0.25 mM ~ 1 mM, and the concentration of TPR is 2.5 mM ~ 10 mM; the concentration of HNCQDs in the HNCQDs / H2O2 system is 0.1 mg / mL ~ 1.25 mg / mL, and the concentration of H2O2 is 50 mM ~ 800 mM. The nucleic acid probe used to detect miRNA-222 is a capture probe and an ALP-Au NPs-labeled auxiliary probe, and its preparation steps include: (1) Capture probe DNA, sequence: 5'-SH-(CH2)6-GGCTGTTTTTTGAGACCCACGATAGATTTTTTTCAGCC-(CH2)6-SH-3'; (2) Assistant probe DNA, sequence: 5'-TCTATCGGTAGCCAGATGTAGCT-(CH2)6-SH-3'; (3) The modification process of the ALP-Au NPs-labeled auxiliary probe is as follows: Add 10 mL of 1.2 mM HAuCl4 solution to 85 mL of boiling water, heat to boiling, then add 5 mL of 38.7 mM sodium citrate solution, and maintain the reaction system at a gentle boil for 30 min to obtain Au NPs solution; add 80 µL of 0.8 mg / mL ALP diluent and 40 µL of 1 µM Assistant probe DNA to 4 mL of pH 8.2 Au NPs solution and react for 2 h; add 80 µL of 10 mM mercaptohexanol (MCH) solution, mix for 30 min, centrifuge at 12,000 rpm for 20 min, wash the precipitate 3 times to obtain ALP-Au NPs labeled assistant probe, and redissolve in 400 µL of ultrapure water for later use.

2. The low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor according to claim 1, characterized in that, The bipolar electrode driving voltage is 0 ~ 3.5 V.

3. A low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor according to claim 1, characterized in that, The enzyme-catalyzed reaction of the ALP-Au NPs-labeled helper probe is preceded by aminophenyl phosphate (APP). p -APP).

4. A method for preparing a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor according to any one of claims 1-3, characterized in that, Includes the following steps: (1) In the BPE chip, 100 μL of 1 wt% HAuCl4 solution was added to the cathode cell, and 0.1 M PBS solution with pH 7.4 was added to the anode cell. A constant voltage of 3.5 V was applied to both ends of the driving electrode for 300 s to form a bipolar electrode cathode modified Au NPs (Au NPs / ITO). (2) Take 10 μL of the capture probe with a concentration of 1 μM and drop it onto the cathode of the bipolar electrode to modify Au NPs (Au NPs / ITO). Incubate at room temperature for 2 h, wash three times with washing solution, and blow dry with nitrogen to obtain Capture probe DNA / Au NPs / ITO. (3) Take 20 μL of 1 mM MCH solution to completely cover the surface of Capture probe DNA / Au NPs / ITO, keep it at room temperature in the dark for 1 h to avoid non-specific adsorption, wash three times, blow dry with nitrogen to obtain MCH / Capture probe DNA / AuNPs / ITO; (4) Take 10 μL of the mixture of ALP-Au NPs labeled helper probe and miRNA-222 and drop it onto the surface of MCH / Captureprobe DNA / Au NPs / ITO. After incubating at 37 °C for 50 min, wash three times with washing solution to obtain Y-shaped DNA / Au NPs / ITO. (5) Take 10 μL of a 10 mM solution. p -APP solution was dropped onto the surface of Y-shaped DNA / Au NPs / ITO and reacted at 37 °C in the dark for 30 min to obtain a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor.

5. The method for fabricating a low-voltage driven ratiometric bipolar electrode electrochemiluminescence biosensor according to claim 4, characterized in that, The HNCQDs / H2O2 system was added to the anode cell; Ru(bpy)3 was added to the cathode cell. 2+ / TPrA system; cyclic voltammetry was used to detect the ECL signal of the HNCQDs / H2O2 system in the range of 0 ~ 3.5 V, PMT = 600 V, and scan rate of 0.2 V / s; cyclic voltammetry was also used to detect Ru(bpy)3 in the range of 0 ~ 2.75 V, PMT = 600 V, and scan rate of 0.2 V / s. 2+ ECL signal of the / TPrA system.

6. A low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor according to any one of claims 1-3, or a low-voltage driven ratio type bipolar electrode electrochemiluminescence biosensor prepared by the method of claim 4 or 5, characterized in that, For the specific and sensitive detection of miRNA-222.

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