Electrochemiluminescence-photoelectrochemical dual-mode simultaneous detection device and method for target object in serum and application

Abstract

According to the light-induced photoelectrochemical integrated device provided by the invention, electrochemical luminescence and PEC signal simultaneous output are realized in a single sensing system for the first time, and dual-mode simultaneous quantification of markers in serum is realized. The preparation method comprises the following steps: preparing and screening an ECL / PEC material pair with matched wavelength: luminol-Au / Bi2WO6, and successively assembling Bi2WO6 and luminol-Au labeled probes on an indium tin oxide electrode by utilizing a layer-by-layer self-assembly mode; when a voltage is applied, the luminol-Au emits a strong ECL signal and triggers the Bi2WO6 to generate an obvious light current. A marker miRNA-21 is taken as a model, recognition molecules with a hairpin structure are introduced into a system, and sensitive and accurate dual-mode analysis of the miRNA-21 is realized by adopting an enzyme-assisted amplification method. The detection method provided by the invention not only can realize ECL-PEC dual-mode simultaneous detection of the target object, but also has relatively high universality, and a new example can be provided for precise biological analysis by replacing and recognizing molecules through the sensor.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a device, method, and application for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target substances in serum. Background Technology

[0002] Biosensors, as rapid devices that convert biological signals into measurable light or electrical signals for quantitative analysis, have attracted considerable attention from researchers. Currently, various biosensors have been developed for the quantitative detection of target substances in serum, such as photoelectrochemical (PEC) biosensors, electrochemical (EC) biosensors, electrochemiluminescence (ECL) biosensors, surface plasmon resonance (SPR) biosensors, fluorescence (FL) biosensors, and enzyme-linked immunosorbent assays (ELISA). These methods collect biological signals using biosensitive materials and convert them into single light or electrical signals for the quantification of the target analyte. However, while this single detection method offers rapid and convenient detection, its results may be affected by complex detection environments and the similar properties of coexisting substances. Therefore, dual-mode sensing platforms with independent signal outputs have become a reliable and effective strategy for constructing sensitive and accurate biosensors.

[0003] Dual-mode biosensors, such as PEC-photothermal, PEC-EC, ECL-PEC, ECL-colorimetric, and FL-ECL, utilize two independent signals for comparison and verification, overcoming the high false positive / false negative rate of single-signal output and significantly improving the reliability of test results. Among these, the ECL-PEC dual-mode sensing method has attracted widespread attention due to its high sensitivity, good selectivity, strong anti-interference ability, fast response speed, and ease of operation. In particular, both the PEC method and ECL technology use the same electrochemical instrument, which further improves the accuracy of test results while reducing costs and simplifying procedures.

[0004] Currently, various PEC-ECL dual-mode sensing platforms have been reported for the sensitive detection of target substances, but all of them rely on the sequential output of PEC and ECL signals to achieve separate quantification. This application relates to a novel method for the simultaneous ECL-PEC dual-mode detection of target substances in serum. This method achieves the simultaneous output of ECL and PEC signals through a self-assembled device, and its application in the field of life analysis has not been reported domestically or internationally. Summary of the Invention

[0005] To address the problems in the prior art, this application provides a method for simultaneous detection of target analytes in serum using both electrochemiluminescence and photoelectrochemical modes, the method comprising the following steps:

[0006] Step 1: Synthesis of ECL luminescent probe; luminol-Au was prepared by HAuCl4, luminol-NaOH and NaBH4; ECL luminescent probe pDNA-luminol-Au was prepared by HS-functionalized pDNA and luminol-Au;

[0007] Step 2: Synthesis of PEC photosensitive material; Bi(NO)3·5H2O and Na2WO4·2H2O were dispersed in pure water to obtain a Bi(NO3)3 suspension, and Na2WO4 aqueous solution was added dropwise to the Bi(NO3)3 suspension to obtain a mixed solution. The mixed solution was reacted at high temperature to prepare the PEC photosensitive material Bi2WO6.

[0008] Step 3: Construction of an electrochemiluminescence-photoelectrochemical dual-mode sensor, wherein the construction of the dual-mode sensor includes:

[0009] Step 3-1: Modify the PEC photosensitive material Bi2WO6 obtained in step 2 onto the surface of the indium tin oxide electrode;

[0010] Step 3-2: After cleaning the Bi2WO6-modified indium tin oxide electrode, add a molecular beacon, incubate, clean again, and then modify with bovine serum albumin.

[0011] Step 3-3: Clean the electrode surface modified with bovine serum albumin, and add a mixture containing miRNA-21 and Mg to the electrode surface. 2+ The DSN enzyme mixture was incubated to inactivate the DSN enzyme and terminate the reaction.

[0012] Steps 3-4: Add the ECL luminescent probe pDNA-luminol-Au solution prepared in step 1, and incubate at 37°C for 1 h to obtain an electrochemiluminescence-photoelectrochemical dual-mode sensor for the target analytes in serum;

[0013] Step 4: Target analyte testing was performed. A three-electrode system was constructed using the electrochemiluminescence-photoelectrochemical dual-mode sensor obtained in Step 3 as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the auxiliary electrode. This three-electrode system was placed in a glass detection cell containing 5 mmol / L H2O2 in phosphate buffer solution (0.01 mol / L PBS). The glass detection cell was then transferred to an electrochemiluminescence-photoelectrochemical dual-mode simultaneous detection device, which consisted of an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector. The ECL and PEC signals were collected and displayed by the corresponding software of the LVIUM electrochemical workstation and the BPCL-1-TIC weak luminescence detector, respectively. Dual-mode simultaneous detection analysis was performed to obtain information about the target analyte in the serum.

[0014] This application also provides a device for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of the aforementioned detection method, characterized in that the detection device includes an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, wherein the BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier tube and a signal analyzer host. The ECL signal is collected by the weak luminescence detector and the PEC signal is collected by the LVIUM electrochemical workstation. After connection, the BPCL-1-TIC weak luminescence detector is connected to a computer: the S-Connector port in the detector is connected to the S-Input port of the signal analyzer host, the V-Connector port is connected to the V-output port, the electrochemiluminescence detection cell in the detector is connected to the LVIUM electrochemical workstation through the Cell Connector port, and the S-output port is connected to the computer for transmitting the ECL signal detected by the photomultiplier tube and displaying it in real time in the BPCL-1-TIC weak luminescence detector measurement and analysis system;

[0015] The LVIUM electrochemical workstation is connected to the BPCL-1-TIC weak luminescence detector as a voltage supply system: First, leave the Peripheral pot in the LVIUM electrochemical workstation, which is used to connect the light source, empty. Then, connect the Cell Connector port of the LVIUM electrochemical workstation to the electrochemiluminescence detection cell in the BPCL-1-TIC detection system to apply the luminescence voltage to the electrode in ChronoAmperanetry detection mode and collect the PEC signal on the electrode surface. The USB port is connected to the computer to transmit the PEC signal obtained in real time.

[0016] This application also provides the application of the detection method in the detection of serum targets.

[0017] Beneficial effects

[0018] This application provides a self-made photoinduced PEC integrated sensor that enables simultaneous detection of target substances in serum using both ECL and PEC modes in a single measurement. The electrochemiluminescence-photoelectrochemical dual-mode simultaneous detection method for target substances in serum provided in this application is the first case to achieve simultaneous output of ECL and PEC signals.

[0019] This application provides a method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum. Bi2WO6 (a semiconductor nanomaterial with high PEC performance) was synthesized and screened as the photoelectrode material, and luminol-Au as the ECL luminescent emitter. Using a layer-by-layer modification method, Bi2WO6, molecular beacon MB, miRNA-21, and pDNA-luminol-Au probe were sequentially modified onto the surface of an ITO electrode. When a voltage of 0.6V (greater than the ECL emission potential of luminol) is applied to the electrode, the luminol-Au probe generates blue light at approximately 435nm (the wavelength of which falls within the absorption wavelength range of Bi2WO6). After absorption by Bi2WO6, this light causes carrier separation, generating a photocurrent. Using a self-made photoelectric system, the ECL signal generated by luminol-Au and the PEC signal generated by Bi2WO6 can be collected simultaneously.

[0020] The technical solution of this application solves a long-standing challenge: the simultaneous output of ECL and PEC dual-mode signals, which will promote the development of sensitive and accurate analysis of target substances in serum.

[0021] Generally, there are two ways to achieve ECL-PEC dual-mode detection of a target object:

[0022] (1) Two sets of three-electrode devices were used to construct ECL and PEC dual-sensor systems respectively, and the two systems were physically isolated by glass with good light transmittance. The two signals were collected simultaneously by ECL and PEC devices respectively. Although this method can realize simultaneous detection of ECL-PEC dual modes, the sensor construction is complex, material consumption is time-consuming, and the physical distance between the two systems needs to be strictly controlled, which limits its development.

[0023] (2) The ECL and PEC dual-sensor system is built in a three-electrode device, and the two signals are collected sequentially by the ECL and PEC devices, which does not achieve true simultaneous detection. In this application, in order to achieve simultaneous collection of two signals, the ECL and PEC devices are cleverly integrated together. After applying a square wave voltage, the two signals can be collected simultaneously by a single device.

[0024] This application significantly simplifies the apparatus and construction process, providing a more consistent detection environment for dual-mode detection of target substances in serum, and achieving more accurate and sensitive detection. The method provided in this application facilitates precise analysis and detection, offering organic support and an accurate foundation for scientific research. Attached Figure Description

[0025] Figure 1 A schematic diagram of the construction of the ECL-PEC dual-mode integrated sensor for miRNA-21 in serum.

[0026] Figure 2 Among them, the (A) XRD, (B) UV-Vis absorption spectra and (C) SEM characterization of Bi2WO6 photosensitive material; (D) UV-Vis absorption spectra of luminol (curve a) and luminol-Au (curve b) and (E, F) TEM images of luminol-Au, with F and its inset being high-resolution TEM images of luminol-Au.

[0027] Figure 3 Among them, (A) the UV-Vis absorption spectrum of Bi2WO6 (black curve) and the ECL emission spectrum of luminol-Au (red curve); (B) EIS characterization, (C) ECL characterization and (D) PEC characterization of the dual-mode integrated sensor construction process.

[0028] Figure 4 (A) ECL signals of biosensors incubated with different concentrations of miRNA-21 and (B) the relationship between ECL intensity and miRNA-21 concentration, with insets showing the logarithmic relationship between ECL intensity and miRNA-21 concentration; (C) PEC signals of biosensors incubated with different concentrations of miRNA-21 and (D) the relationship between PEC intensity and miRNA-21 concentration, with insets showing the logarithmic relationship between PEC intensity and miRNA-21 concentration.

[0029] Figure 5 The experiment diagrams show the selectivity (A, B) and stability (C, D) of the ECL-PEC dual-mode integrated sensor for detecting miRNA-21. (A and C are ECL modes, and B and D are PEC modes.) Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] One embodiment of this application provides a method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of a target analyte in serum, characterized in that the method includes the following steps:

[0035] Step 1: Synthesis of ECL luminescent probe; luminol-Au was prepared by HAuCl4, luminol-NaOH and NaBH4; ECL luminescent probe pDNA-luminol-Au was prepared by HS-functionalized pDNA and luminol-Au;

[0036] Step 2: Synthesis of PEC photosensitive material; Bi(NO)3·5H2O and Na2WO4·2H2O were dispersed in pure water to obtain a Bi(NO3)3 suspension, and Na2WO4 aqueous solution was added dropwise to the Bi(NO3)3 suspension to obtain a mixed solution. The mixed solution was reacted at high temperature to prepare the PEC photosensitive material Bi2WO6.

[0037] Step 3: Construction of an electrochemiluminescence-photoelectrochemical dual-mode sensor, wherein the construction of the dual-mode sensor includes:

[0038] Step 3-1: Modify the PEC photosensitive material Bi2WO6 obtained in step 2 onto the surface of the indium tin oxide electrode;

[0039] Step 3-2: After cleaning the Bi2WO6-modified indium tin oxide electrode, add a molecular beacon, incubate, clean again, and then modify with bovine serum albumin.

[0040] Step 3-3: Clean the electrode surface modified with bovine serum albumin, and add a mixture containing miRNA-21 and Mg to the electrode surface. 2+ The DSN enzyme mixture was incubated to inactivate the DSN enzyme and terminate the reaction.

[0041] Steps 3-4: Add the ECL luminescent probe pDNA-luminol-Au solution prepared in step 1, incubate at 37°C for 1 h to obtain an electrochemiluminescence-photoelectrochemical dual-mode sensor for the target analytes in serum;

[0042] Step 4: Target analyte testing was performed. A three-electrode system was constructed using the electrochemiluminescence-photoelectrochemical dual-mode sensor obtained in Step 3 as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the auxiliary electrode. This three-electrode system was placed in a glass detection cell containing 5 mmol / L H2O2 in phosphate buffer solution (0.01 mol / L PBS). The glass detection cell was then transferred to an electrochemiluminescence-photoelectrochemical dual-mode simultaneous detection device, which consisted of an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector. The ECL and PEC signals were collected and displayed by the corresponding software of the LVIUM electrochemical workstation and the BPCL-1-TIC weak luminescence detector, respectively. Dual-mode simultaneous detection analysis was performed to obtain information about the target analyte in the serum.

[0043] In one embodiment, in step 1, the preparation of luminol-Au using HAuCl4, luminol-NaOH, and NaBH4 involves adding HAuCl4 to a beaker under ice bath stirring conditions, followed by dropwise addition of luminol-NaOH solution. After stirring until homogeneous, NaBH4 solution is added dropwise. After the reaction, the ice bath is removed, and stirring continues at room temperature for 6 hours. The luminol-Au is then obtained by dialysis in a dialysis bag.

[0044] In one embodiment, in step 1, pDNA-luminol-Au is prepared by mixing HS-functionalized pDNA with Luminol-Au. The HS-functionalized pDNA and Luminol-Au are mixed and reacted in a shaker. Then, the supernatant is removed by centrifugation. The centrifuged product is dispersed in 500 μL PBS to obtain the ECL luminescent probe pDNA-luminol-Au.

[0045] In one embodiment, in step 2, the high-temperature reaction is as follows: after stirring, an aqueous solution of Na2WO4 is added dropwise to a Bi(NO3)3 suspension to obtain a mixed solution, which is then transferred to an autoclave for high-temperature reaction. The reaction product is washed with ethanol and water by centrifugation and dried to obtain the PEC photosensitive material Bi2WO6.

[0046] In one embodiment, in step 3-1, the PEC photosensitive material Bi2WO6 prepared in step 2 is modified on the surface of the indium tin oxide electrode by dissolving Bi2WO6 in a 0.05 mg / mL-0.1 mg / mL chitosan solution, modifying it on the surface of the indium tin oxide electrode, drying it, and then adding glutaraldehyde dropwise to the surface of the Bi2WO6-modified indium tin oxide electrode, and placing it at room temperature in the dark.

[0047] In one embodiment, in step 3-2, after cleaning the Bi2WO6-modified indium tin oxide electrode, a molecular beacon is added, and the electrode is incubated. After cleaning again, bovine serum albumin is modified as follows: after cleaning the Bi2WO6-modified indium tin oxide electrode, a molecular beacon is added, and the electrode is incubated at 37°C for 1 hour. After cleaning again with PBS, bovine serum albumin is modified under the same conditions.

[0048] In one embodiment, in step 3-3, the electrode surface modified with bovine serum albumin is cleaned, and then a mixture containing miRNA-21 and Mg is added to the electrode surface. 2+ The DSN enzyme mixture was incubated to inactivate the DSN enzyme. The reaction was terminated by adding a Mg-containing solution. 2+ The electrode surface was cleaned with PBS solution, and then a solution containing miRNA-21 and Mg was added to the electrode surface. 2+The DSN enzyme mixture was incubated at 37°C for 1 hour, and then placed at 60°C for 5 minutes to inactivate the DSN enzyme and terminate the reaction.

[0049] In one embodiment, in step 4, the dual-mode simultaneous detection analysis is performed as follows:

[0050] Step S4-1: Using the chronoamperometry method in the LVIUM electrochemical workstation, the potential step method is used to excite ECL--excitation potential 0.6V (lasting 10s), no-excitation state 0V (lasting 10s), sampling interval is 0.5s;

[0051] Step S4-2: Set the photomultiplier tube high voltage to -800V in the BPCL weak luminescence detector software. After setting, click "Start" in both the LVIUM electrochemical workstation software and the BPCL weak luminescence detector software to perform simultaneous detection in dual modes.

[0052] One embodiment of this application provides an apparatus for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of the detection method, characterized in that the detection apparatus includes an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, wherein the BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier tube and a signal analyzer host. The ECL signal is collected by the weak luminescence detector and the PEC signal is collected by the LVIUM electrochemical workstation. The BPCL-1-TIC weak luminescence detector is connected to a computer after self-connection: the S-Connector port in the detector is connected to the S-Input port of the signal analyzer host, the V-Connector port is connected to the V-output port, the electrochemiluminescence detection cell in the detector is connected to the LVIUM electrochemical workstation through the Cell Connector port, and the S-output port is connected to the computer for transmitting the ECL signal detected by the photomultiplier tube and displaying it in real time in the BPCL-1-TIC weak luminescence detector measurement and analysis system;

[0053] The LVIUM electrochemical workstation is connected to the BPCL-1-TIC weak luminescence detector as a voltage supply system: First, leave the Peripheral pot in the LVIUM electrochemical workstation, which is used to connect the light source, empty. Then, connect the Cell Connector port of the LVIUM electrochemical workstation to the electrochemiluminescence detection cell in the BPCL-1-TIC detection system to apply the luminescence voltage to the electrode in ChronoAmperanetry detection mode and collect the PEC signal on the electrode surface. The USB port is connected to the computer to transmit the PEC signal obtained in real time.

[0054] In one embodiment, the device for simultaneous detection in both electrochemiluminescence and photoelectrochemical modes consists of an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector. The ECL signal and PEC signal are collected and displayed by the corresponding software of the two components, respectively.

[0055] In one embodiment, the weak luminescence detector is first self-connected and then connected to the computer: the S-Connector port of the detector is connected to the S-Input port of the signal analysis system, the V-Connector port is connected to the V-output port, and the electrochemiluminescence detection cell in the detector is connected to the LVIUM electrochemical workstation via the Cell Connector port. The S-output port is connected to the computer to transmit the ECL signal detected by the photomultiplier tube and display it in real time in the weak luminescence detector measurement and analysis system. Then, the LVIUM electrochemical workstation is connected to the BPCL-1-TIC weak luminescence detector as a voltage supply system: the Peripheralpot in the LVIUM electrochemical workstation used for connecting the light source is left unused, and its Cell Connector port is connected to the electrochemiluminescence detection cell in the BPCL-1-TIC detection system to apply a luminescence voltage to the electrode in ChronoAmperanetry detection mode, while collecting the PEC signal on the electrode surface. The USB port is connected to the computer to transmit the PEC signal obtained in real time.

[0056] One embodiment of this application provides the application of any detection method in the detection of serum targets.

[0057] Example 1: Synthesis of wavelength-adapted ECL / PEC material pairs

[0058] (1) Synthesis of ECL luminescent probe: Under the conditions of ice bath and stirring at 4℃, 20mL of 2.5×10⁻⁶ ECL luminescent probe was first added to a small beaker. -4Add 4 mL of 0.01 mol / L luminol-NaOH solution dropwise to HAuCl4. After stirring until homogeneous, add 0.6 mL of 0.1 mol / L NaBH4 solution dropwise. After reacting for 10 min, remove the ice bath and continue stirring at room temperature for 6 h. Finally, dialyze for 6 h in a dialysis bag with D = 14000 to obtain luminol-Au. Then, mix 10 μL of 100 μM HS-functionalized pDNA (purchased from Zhengzhou Sangon Biotech Co., Ltd.) with 2000 μL of luminol-Au and react at 20℃ and 1000 rpm in a shaker for 16 h. Then, centrifuge at 10000 rpm for 10 min and remove the supernatant. Finally, disperse the centrifuged product in 500 μL PBS to obtain the pDNA-luminol-Au probe with ECL performance.

[0059] (2) Synthesis of PEC photosensitive material: Under vigorous stirring, 4 mmol of Bi(NO)3·5H2O and 2 mmol of Na2WO4·2H2O were dispersed in 30 mL of pure water. Then, the Na2WO4 aqueous solution was slowly added dropwise to the Bi(NO3)3 suspension. After stirring for 30 min, the mixed solution was transferred to an autoclave and reacted at 180 °C for 12 h. The product was washed by centrifugation with ethanol and water multiple times and dried overnight at 80 °C to obtain Bi2WO6 material with PEC response.

[0060] The Bi2WO6 material was characterized by XRD and SEM, and by luminol-Au UV-Vis absorption spectroscopy and TEM. The results are as follows: Figure 2 As shown, Bi2WO6 photosensitive material Figure 2 (A)XRD, Figure 2 (B) Ultraviolet-Vis absorption spectrum and Figure 2 (C) SEM characterization image; Figure 2 UV-Vis absorption spectra of (D)luminol (curve a) and luminol-Au (curve b) and Figure 2 TEM images of (E, F) luminol-Au. Figure 2 The middle (F) image and its inset are high-resolution TEM images of luminol-Au. The experimental results demonstrate the successful synthesis of both materials.

[0061] Example 2: Construction and Feasibility Testing of a Dual-Mode Integrated Sensor

[0062] By studying the UV-Vis absorption spectrum of Bi2WO6 material and the ECL emission spectrum of luminol-Au NPs in Example 1, it was found that the ECL emission wavelength range of luminol-Au NPs and the UV-Vis absorption wavelength range of Bi2WO6 material have a large overlap. Figure 3 (A) This demonstrates the feasibility of using the ECL signal of luminol-Au NPs to trigger the PEC behavior of Bi2WO6 materials.

[0063] Further construction of the dual-mode integrated sensor was carried out: 20 μL of 4 mg / mL Bi2WO6 (dissolved in 0.1 mg / mL chitosan) was modified on the surface of an indium tin oxide (ITO) electrode. After drying, 20 μL of 2.5% (v / v) glutaraldehyde was added and the electrode was incubated at room temperature in the dark for 50 min. After washing with PBS, 20 μL of 0.5 μmol / L molecular beacon (MB) was added and the electrode was incubated at 37 °C for 1 h. After washing with PBS again, 0.8% bovine serum albumin (BSA) was modified under the same conditions, followed by the addition of 5 mmol / L Mg... 2+ The electrode surface was cleaned with PBS solution, and then a solution containing miRNA-21 and DSN enzyme (containing Mg) was added to the electrode surface. 2+ The mixture of DSN enzyme and pDNA-luminol-Au probe solution was incubated at 37°C for 1 hour, and then placed at 60°C for 5 minutes to inactivate the DSN enzyme and terminate the reaction. Finally, pDNA-luminol-Au probe solution was added and incubated at 37°C for 1 hour to obtain a dual-mode sensing platform for miRNA-21 detection.

[0064] The layer-by-layer modified electrode was characterized using electrochemical impedance spectroscopy (EIS) and dual-mode signal output behavior, and the results are as follows: Figure 3 As shown, it was found that with each layer of modification described above, the electron transfer resistance on the electrode surface increased (…). Figure 3 (B) This is due to the low conductivity and large steric hindrance of the chitosan, glutaraldehyde, MB, BSA, miRNA-21 and pDNA-luminol-Au probes, which preliminarily proves the successful construction of the sensor.

[0065] Then the dual-mode output signal (i.e., ECL and PEC behavior) of the layer-by-layer modified electrode was tested, such as Figure 3 As shown in C and D of Figure 3. When the photosensitive material Bi2WO6 was modified on the electrode surface, the dual-mode integrated detection device did not collect any light signal (curve b). Figure 3 (C), but because a bias voltage of 0.6V was applied, a photocurrent of about 400nA was generated (curve b). Figure 3(D), and the photocurrent gradually decreases with each layer of modification; when a pDNA-luminol-Au probe with ECL luminescence properties is incubated on the electrode surface, the dual-mode integrated detection device not only collects a light signal of about 10000 a.u. (curve g, Figure 3 In the middle (C), the photocurrent in the PEC mode increased by approximately 500 nA (interpolation between curves g and f). Figure 3 (D).

[0066] The above results are sufficient to demonstrate the successful construction of the sensor and its feasibility for dual-mode detection of target substances in serum.

[0067] Example 3: Dual-mode detection of target miRNA-21 in solution

[0068] To perform dual-mode quantitative analysis of the target analyte miRNA-21 in solution using a photo-induced PEC integrated device, following the construction steps of an electrochemiluminescence-photoelectrochemical dual-mode sensor, different concentrations of miRNA-21 (0 fmol / L, 50 fmol / L, 100 fmol / L, 500 fmol / L, 1 pmol / L, 10 pmol / L, 100 pmol / L, 1 nmol / L, 10 nmol / L) were modified on the working electrode surface to construct different sensing interfaces. The analysis was then performed as described in step 5, simultaneously obtaining ECL and PEC signal curves for the modified analytes at different concentrations. (See [link to relevant documentation]). Figure 4 , Figure 4 (A) ECL signal of biosensors incubated with different concentrations of miRNA-21 and Figure 4 (B) Relationship between ECL intensity and miRNA-21 concentration, with insets showing the logarithmic relationship between ECL intensity and miRNA-21 concentration; Figure 4 PEC signals of biosensors incubated with different concentrations of miRNA-21 in the middle (C) and Figure 4 The relationship between PEC intensity and miRNA-21 concentration is shown in (D). The inset shows the logarithmic relationship between PEC intensity and miRNA-21 concentration.

[0069] Based on the results analysis of this embodiment, it can be seen that when the concentration of miRNA-21 is 1.0 × 10⁻⁶, -16 ~1.0×10 -8 Within the mol / L range, its ECL signal intensity is positively correlated with the logarithm of the concentration, and the linear equation is: Y ECL =17911.9+987.31gc(mol / L, R) 2 =0.997), with a detection limit as low as 72.9 amol / L (S / N = 3); simultaneously, when the miRNA-21 concentration is 1.0 × 10⁻⁶, the detection limit is as low as 72.9 amol / L (S / N = 3); -14~1.0×10 -8 Within the mol / L range, the PEC signal intensity is also positively correlated with the logarithm of the concentration, and the linear equation is: I PEC =1605.5+102.9lg c(mol / L, R) 2 =0.996), with a detection limit as low as 6.46 fmol / L (S / N=3).

[0070] The results show that both developed analytical modes have a wide linear range and a low detection limit, and the results obtained by the two modes can complement and corroborate each other, further improving the accuracy of the detection results and providing a new method for sensitive and accurate detection in complex samples.

[0071] Example 4: Selectivity and stability of miRNA-21 detection using the ECL-PEC dual-mode integrated sensor

[0072] To eliminate false positive signals from miRNAs with other sequences in serum, selector miRNAs with other sequences (including miRNA-141 and miRNA-451), single-base mismatched miRNA-21 (MM1), and triple-base mismatched miRNA-21 (MM3) were used as interfering agents (all at a concentration of 1.0 nmol / L) for selectivity experiments. It was found that the ECL and PEC responses of the modified miRNAs with other sequences were lower; while the ECL and PEC signals obtained by modifying the two interfering agents were only half that of the target miRNA-21 modified. Finally, the sensor modified with a mixture of these substances (miRNA-141 + miRNA-451 + MM1 + MM3 + miRNA-21) obtained ECL and PEC responses similar to those modified only with the target miRNA-21. Figure 5 A and B.

[0073] In addition, the stability of the dual-mode sensor was evaluated. For example... Figure 5 As shown in C and D, the biosensor (containing 10 nmol / L miRNA-21) exhibited a stable photocurrent in ECL mode after 10 consecutive scans, with a relative standard deviation (RSD) of 2.3%; the current signal RSD in PEC mode was 1.1%, indicating high sensor stability.

[0074] Experimental results show that the method of this application has good selectivity and high stability, and can achieve specific detection of target substances in complex samples.

[0075] Example 5: Two-mode analysis of target miRNA-21 in serum

[0076] miRNA-21, a potential tumor marker, is expressed in various cancers. However, the concentration of miRNA-21 in human serum is low, posing a challenge for analysis in practical samples. To evaluate the applicability of the detection method in this application, the developed dual-mode sensor was used for spiked analysis of miRNA-21 in serum. Different concentrations of miRNA-21 standard solutions (0.01, 0.1, and 1 pmol / L) were added to two 10-fold diluted human serum samples for dual-mode detection, and the concentration was calculated based on the calibration curve obtained in Example 1.

[0077] As shown in Table 1, the ECL mode detection results showed that the recovery rate of miRNA-21 was 93.2%–117.7%, with an RSD between 1.8% and 3.6%; the PEC mode detection results showed that the recovery rate of miRNA-21 in serum was 87.1%–118.5%, with an RSD of 2.6%–5.1%. These results indicate that this invention has good application potential in the detection of target analytes in serum.

[0078] Table 1. Results of spiked recovery experiments of miRNA-21 in serum using the ECL-PEC dual-mode integrated sensor.

[0079]

[0080] In summary, this study constructed a novel ECL-PEC dual-mode integrated sensor, achieving sensitive and accurate detection of miRNA-21. This work has two innovations: First, by using ECL as a miniaturized internal light source, the use of an external light source is avoided, which shortens the distance required for PEC excitation, significantly improves photon utilization efficiency, and reduces background noise. Second, by utilizing different materials on the same electrode surface to achieve photon emission and Bi2WO6 photocurrent collection, simultaneously outputting ECL and PEC dual signals, the developed dual-mode biosensor achieves cross-validation detection of miRNA-21. Compared with single-mode sensors, it effectively reduces false positive / negative signals, making the detection results more reliable. This work not only makes an important contribution to the development of ECL-PEC detection technology and demonstrates its application potential in biochemical analysis and clinical diagnostics, but also provides new ideas for the construction of novel dual-mode biosensors.

[0081] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of a target analyte in serum, characterized in that, The method includes the following steps: Step 1: Synthesis of ECL luminescent probe; luminol-Au was prepared by HAuCl4, luminol-NaOH and NaBH4; ECL luminescent probe pDNA-luminol-Au was prepared by HS-functionalized pDNA and luminol-Au; Step 2: Synthesis of PEC photosensitive material; Bi(NO)3·5H2O and Na2WO4·2H2O were dispersed in pure water to obtain a Bi(NO3)3 suspension, and Na2WO4 aqueous solution was added dropwise to the Bi(NO3)3 suspension to obtain a mixed solution. The mixed solution was reacted at high temperature to prepare the PEC photosensitive material Bi2WO6. Step 3: Construction of an electrochemiluminescence-photoelectrochemical dual-mode sensor, wherein the construction of the dual-mode sensor includes: Step 3-1: Modify the PEC photosensitive material Bi2WO6 obtained in step 2 onto the surface of the indium tin oxide electrode; Step 3-2: After cleaning the Bi2WO6-modified indium tin oxide electrode, add a molecular beacon, incubate, clean again, and then modify with bovine serum albumin. Step 3-3: Clean the electrode surface modified with bovine serum albumin, and add a mixture containing miRNA-21 and Mg to the electrode surface. 2+ The DSN enzyme mixture was incubated to inactivate the DSN enzyme and terminate the reaction. Steps 3-4: Add the ECL luminescent probe pDNA-luminol-Au solution prepared in step 1, and incubate at 37°C for 1 h to obtain an electrochemiluminescence-photoelectrochemical dual-mode sensor for the target analytes in serum; Step 4: Target analyte testing was performed. A three-electrode system was constructed using the electrochemiluminescence-photoelectrochemical dual-mode sensor obtained in Step 3 as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the auxiliary electrode. This three-electrode system was placed in a glass detection cell containing 5 mmol / L H2O2 in phosphate buffer solution (0.01 mol / L PBS). The glass detection cell was then transferred to an electrochemiluminescence-photoelectrochemical dual-mode simultaneous detection device, which consisted of an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector. The ECL and PEC signals were collected and displayed by the corresponding software of the BPCL-1-TIC weak luminescence detector and the LVIUM electrochemical workstation, respectively. Dual-mode simultaneous detection analysis was performed to obtain information on the target analyte in the serum.

2. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum according to claim 1, characterized in that, In step 1, the preparation of luminol-Au using HAuCl4, luminol-NaOH, and NaBH4 involves adding HAuCl4 to a beaker under ice bath stirring conditions, followed by dropwise addition of luminol-NaOH solution. After stirring until homogeneous, NaBH4 solution is added dropwise. After the reaction, the ice bath is removed, and stirring continues at room temperature for 6 hours. The luminol-Au is then obtained by dialysis in a dialysis bag.

3. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target substances in serum according to claim 1, characterized in that, In step 1, pDNA-luminol-Au is prepared by mixing HS-functionalized pDNA with Luminol-Au. The HS-functionalized pDNA and Luminol-Au are mixed and reacted in a shaker; then, the supernatant is removed by centrifugation. The centrifuged product was dispersed in 500 μL PBS to obtain the ECL luminescent probe pDNA-luminol-Au.

4. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target substances in serum according to claim 1, characterized in that, In step 2, the high-temperature reaction is as follows: after stirring, Na2WO4 aqueous solution is added dropwise to Bi(NO3)3 suspension to obtain a mixed solution, which is then transferred to an autoclave for high-temperature reaction. The reaction product is washed with ethanol and water by centrifugation and dried to obtain PEC photosensitive material Bi2WO6.

5. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum according to claim 1, characterized in that, In step 3-1, the PEC photosensitive material Bi2WO6 prepared in step 2 is modified on the surface of the indium tin oxide electrode by dissolving Bi2WO6 in a 0.05 mg / mL to 0.1 mg / mL chitosan solution, modifying it on the surface of the indium tin oxide electrode, drying it, and then adding glutaraldehyde dropwise to the surface of the Bi2WO6-modified indium tin oxide electrode. The electrode is then placed at room temperature in the dark.

6. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum according to claim 1, characterized in that, In step 3-2, after cleaning the Bi2WO6-modified indium tin oxide electrode, a molecular beacon was added, and the electrode was incubated. After washing again, bovine serum albumin was modified. After cleaning the Bi2WO6-modified indium tin oxide electrode, a molecular beacon was added, and the electrode was incubated at 37°C for 1 hour. After washing again with PBS, bovine serum albumin was modified under the same conditions.

7. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum according to claim 1, characterized in that, In step 3-3, the electrode surface modified with bovine serum albumin is cleaned, and then a mixture containing miRNA-21 and Mg is added to the electrode surface. 2+ The DSN enzyme mixture was incubated to inactivate the DSN enzyme. The reaction was terminated by adding a Mg-containing solution. 2+ The electrode surface was cleaned with PBS solution, and then a solution containing miRNA-21 and Mg was added to the electrode surface. 2+ The DSN enzyme mixture was incubated at 37°C for 1 hour, and then placed at 60°C for 5 minutes to inactivate the DSN enzyme and terminate the reaction.

8. The method for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection of target analytes in serum according to claim 1, characterized in that, In step 4, the dual-mode simultaneous detection analysis is performed as follows: Step S4-1: Using the chronoamperometry method in the LVIUM electrochemical workstation, the potential step method is used to excite the ECL—excitation potential 0.6V (lasting 10s), no-excitation state 0V (lasting 10s), sampling interval is 0.5s; Step S4-2: Set the photomultiplier tube high voltage to -800V in the BPCL weak luminescence detector software. After setting, click "Start" in both the LVIUM electrochemical workstation software and the BPCL weak luminescence detector software to perform simultaneous detection in dual modes.

9. An apparatus for simultaneous electrochemiluminescence-photoelectrochemical dual-mode detection operating the detection method of any one of claims 1-8, characterized in that, The detection device includes an LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector. The BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier tube and a signal analyzer host. The ECL signal is collected by the weak luminescence detector and the PEC signal is collected by the LVIUM electrochemical workstation. After connection, the BPCL-1-TIC weak luminescence detector is connected to a computer: the S-Connector port in the detector is connected to the S-Input port of the signal analyzer host, the V-Connector port is connected to the V-output port, the electrochemiluminescence detection cell in the detector is connected to the LVIUM electrochemical workstation through the Cell Connector port, and the S-output port is connected to the computer to transmit the ECL signal detected by the photomultiplier tube and display it in real time in the BPCL-1-TIC weak luminescence detector measurement and analysis system. The LVIUM electrochemical workstation is connected to the BPCL-1-TIC weak luminescence detector as a voltage supply system: First, leave the Peripheral pot in the LVIUM electrochemical workstation, which is used to connect the light source, empty. Then, connect the Cell Connector port of the LVIUM electrochemical workstation to the electrochemiluminescence detection cell in the BPCL-1-TIC detection system to apply the luminescence voltage to the electrode in ChronoAmperanetry detection mode and collect the PEC signal on the electrode surface. The USB port is connected to the computer to transmit the PEC signal obtained in real time.

10. The application of any one of the detection methods of claims 1-8 in the detection of serum targets.

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