CN120927770A - Electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device and method for target in serum and application - Google Patents

Abstract

This application provides a photo-induced photoelectrochemical integrated device, achieving for the first time simultaneous output of electrochemiluminescence and PEC signals in a single sensing system, and realizing dual-mode simultaneous quantification of biomarkers in serum. A wavelength-matched ECL/PEC material pair, luminol-Au/ _{Bi₂WO₆ ,} was prepared and screened. Using a layer-by-layer self-assembly method, _Bi₂WO₆ and luminol-Au-labeled probes were sequentially assembled onto an indium tin oxide electrode. Upon applying a voltage, luminol-Au emitted a strong ECL signal, triggering a significant photocurrent _{in Bi₂WO₆} . Using the biomarker miRNA- ₂₁ as a model, a recognition molecule with a hairpin structure was introduced into the system. Enzyme-assisted amplification was employed to achieve sensitive and accurate dual-mode analysis of miRNA-21. The detection method provided in this application not only enables simultaneous ECL-PEC dual-mode detection of target analytes but also possesses strong universality. By replacing the recognition molecule with a different sensor, it provides a new paradigm for precision bioanalysis.

Description

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

Technical Field

The application relates to the technical field of biology, in particular to an electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device and method for a target in serum and application thereof.

Background

Biosensors are attracting attention of researchers as a rapid device that can convert biological signals into measurable light or electrical signals for quantitative analysis. Currently, various biosensors for quantitative detection of a target substance in serum have been developed, such as Photoelectrochemical (PEC) biosensors, electrochemical (EC) biosensors, electrochemical luminescence (ECL) biosensors, surface Plasmon Resonance (SPR) biosensors, fluorescence (FL) biosensors, and enzyme-linked immunosorbent assay (ELISA), etc. They collect biological signals through the biosensing material and convert them into a single light or electrical signal for quantification of the target. However, although this single detection method provides rapid and convenient detection, the result may be interfered by the complex detection environment and similar characteristics of coexisting materials, and a dual-mode sensing platform with independent signal output becomes one of reliable and effective strategies for constructing sensitive and accurate biosensors.

The dual-mode biosensor, such as PEC-photo-thermal, PEC-EC, ECL-PEC, ECL-colorimetric method and FL-ECL, uses two independent signals to compare and verify each other, overcomes the defect of high single signal output false positive/false negative, and remarkably improves the reliability of test results. Among them, ECL-PEC dual-mode sensing detection methods are receiving attention because of their advantages of high sensitivity, good selectivity, strong anti-interference capability, fast response speed, convenient operation, etc. In particular, both PEC and ECL techniques use the same electrochemical instrumentation, which further improves the accuracy of the test results, while reducing costs and simplifying operational procedures.

Currently, a variety of PEC-ECL dual-mode sensing platforms have been reported for the sensitive detection of target substances, but each achieves separate quantification based on sequential output of PEC and ECL signals. The application relates to a novel method for realizing dual-mode simultaneous detection of a target substance ECL-PEC in serum, the method realizes the simultaneous output of ECL and PEC signals through a set of self-assembled devices, and has not been reported at home and abroad in the field of life analysis.

Disclosure of Invention

In order to solve the problems in the prior art, the application provides an electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection method for a target in serum, which comprises the following steps:

Synthesizing an ECL luminescent probe, preparing the ECL luminescent probe pDNA-lumineol-Au by HAuCl ₄, lumineol-NaOH and NaBH ₄, and preparing the ECL luminescent probe pDNA-lumineol-Au by HS-functionalized pDNA and lumineol-Au;

Dispersing Bi (NO) ₃·5H₂ O and Na ₂WO₄·2H₂ O into pure water to obtain Bi (NO ₃)₃ suspension, dripping Na ₂WO₄ aqueous solution into the Bi (NO ₃)₃ suspension to obtain a mixed solution, and reacting the mixed solution at high temperature to obtain the PEC photosensitive material Bi ₂WO₆;

Step 3, constructing an electrochemiluminescence-photoelectrochemistry dual-mode sensor, wherein the construction of the dual-mode sensor comprises the following steps:

step 3-1, modifying the PEC photosensitive material Bi ₂WO₆ prepared in the step 2 on the surface of an indium tin oxide electrode;

Step 3-2, after cleaning the indium tin oxide electrode modified by Bi ₂WO₆, dropwise adding a molecular beacon, incubating, and cleaning again to modify bovine serum albumin;

Step 3-3, cleaning the electrode surface of the modified bovine serum albumin, dropwise adding a DSN enzyme mixed solution containing miRNA-21 and Mg ²⁺ to the electrode surface, and after incubation, inactivating the DSN enzyme and stopping the reaction;

Step 3-4, adding the ECL luminescent probe pDNA-luminol-Au solution prepared in the step 1, and incubating for 1h at 37 ℃ to obtain an electrochemiluminescence-photoelectrochemistry dual-mode sensor for a target in serum;

And 4, testing a target object, constructing a three-electrode system by taking the electrochemiluminescence-photoelectrochemistry dual-mode sensor obtained in the step 3 as a working electrode, taking Ag/AgCl as a reference electrode and taking a platinum wire as an auxiliary electrode, placing the three-electrode system into a glass detection pool containing 5mmol/L H ₂O₂ of phosphate buffer solution (0.01 mol/L PBS), and transferring the glass detection pool into an electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device, wherein the electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device consists of a LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, and displaying and collecting ECL signals and PEC signals by corresponding software of the LVIUM electrochemical workstation and the BPCL-1-TIC weak luminescence detector respectively, and performing dual-mode simultaneous detection analysis to obtain information of the target object in serum.

The application also provides a device for simultaneously detecting the electrochemiluminescence and photoelectrochemistry modes of the detection method, which is characterized in that the detection device comprises LVIUM electrochemical workstations, a BPCL-1-TIC weak luminescence detector, wherein the BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier and a signal analyzer host, ECL signals are collected by the weak luminescence detector and PEC signals are collected by the LVIUM electrochemical workstations, the BPCL-1-TIC weak luminescence detector is connected with a computer after being connected, an S-Connector port in the detector is connected with an S-Input port of a signal analyzer host, a V-Connector port is connected with a V-output port, an electrochemiluminescence detection Cell in the detector is connected with the LVIUM electrochemical workstations through the Cell Connector port, the S-output port is connected with the computer and is used for transmitting ECL signals detected by the photomultiplier, and the BPCL-1-TIC weak luminescence detector is used for measuring and analyzing real-time display in a system;

The LVIUM electrochemical workstation is used as a voltage supply system to be connected with a BPCL-1-TIC weak luminescence detector, a PERIPHERAL POT empty part used for connecting a light source in the LVIUM electrochemical workstation is firstly connected with an electrochemical luminescence detection pool in the BPCL-1-TIC detection system through a Cell Connector port of the LVIUM electrochemical workstation, luminescence voltage is applied to an electrode in a Chrono Amperanetry detection mode, PEC signals on the surface of the electrode are collected, and a USB jack is connected with a computer and used for transmitting the PEC signals obtained through real-time detection.

The application also provides application of the detection method in serum target detection.

Advantageous effects

According to the self-made light-induced PEC integrated sensor provided by the application, the dual-mode simultaneous detection of the ECL and the PEC of target substances in serum can be realized in one-time measurement. The electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection method for the target in the serum provided by the application is the case of realizing ECL and PEC signals simultaneously for the first time at present.

The electrochemical luminescence-photoelectrochemistry dual-mode simultaneous detection method for the target in the serum synthesizes and screens Bi ₂WO₆ (semiconductor nano material with high PEC performance) as a photoelectrode material, and lumineol-Au as an ECL luminophor, and Bi ₂WO₆, a molecular beacon MB, miRNA-21 and a pDNA-lumineol-Au probe are sequentially modified on the surface of an ITO electrode by a layer-by-layer modification method. When a voltage of 0.6V (greater than the ECL luminescence potential of luminol) is applied to the electrode, the luminol-Au probe generates blue light with a wavelength of 435nm (the wavelength of the blue light is in the light absorption wavelength range of Bi ₂WO₆), and after the blue light is absorbed by Bi ₂WO₆, carriers of the blue light are promoted to be separated, and photocurrent is generated. Through a homemade optoelectronic system, ECL signals generated by luminal-Au and PEC signals generated by Bi ₂WO₆ can be collected simultaneously.

The technical scheme of the application solves a long-term challenge, namely, simultaneous output of ECL and PEC dual-mode signals, which promotes development of sensitive and accurate analysis of target substances in serum.

In general, there are two ways to achieve ECL-PEC dual-mode detection of an object:

(1) Two sets of three-electrode devices are used for respectively constructing an ECL and PEC dual-sensor system, the two systems are physically isolated by glass with good transmittance, and the ECL and PEC devices are used for simultaneously collecting two signals. Although ECL-PEC dual-mode simultaneous detection can be realized by the method, the sensor is complex in construction, time consuming in material consumption, and the physical distance between two systems needs to be strictly controlled, so that the development of the sensor is limited.

(2) The ECL and PEC dual-sensor system is built in a set of three-electrode device, and the ECL and PEC devices are used for sequentially collecting two signals, so that real simultaneous detection is not realized. In the application, in order to realize the simultaneous collection of double signals, ECL and PEC devices are skillfully integrated together, and after a square wave voltage is applied, a set of devices can be utilized to realize the simultaneous collection of double signals.

The application greatly simplifies the device and the construction process, provides a more consistent detection environment for the dual-mode detection of the target substances in the serum, and realizes more accurate and sensitive detection. The method provided by the application is beneficial to accurate analysis and detection, and provides organic support and accurate basis for scientific research.

Drawings

FIG. 1A schematic construction of ECL-PEC dual-mode integrated sensor for miRNA-21 in serum.

FIG. 2 shows (A) XRD, (B) ultraviolet-visible absorption spectrum and (C) SEM characterization of Bi ₂WO₆ photosensitive material, (D) ultraviolet-visible absorption spectrum of luminol (curve a) and luminol-Au (curve B) and (E, F) TEM of luminol-Au, and F and its insets are high resolution TEM of luminol-Au.

FIG. 3, wherein (A) Bi ₂WO₆ ultraviolet-visible absorption spectrum (black curve) and ECL emission spectrum of luminol-Au (red curve), dual mode integrated sensor construction process, (B) EIS characterization, (C) ECL characterization and (D) PEC characterization.

FIG. 4, wherein (A) ECL signal of biosensors incubated with different concentrations of miRNA-21 and (B) ECL intensity versus miRNA-21 concentration are plotted, the interpolated graph shows the logarithmic relationship of ECL intensity versus miRNA-21 concentration, and (C) PEC signal of biosensors incubated with different concentrations of miRNA-21 and (D) PEC intensity versus miRNA-21 concentration are plotted, the interpolated graph shows the logarithmic relationship of PEC intensity versus miRNA-21 concentration.

FIG. 5 shows a selectivity plot (A, B) and a stability plot (C, D) for ECL-PEC dual-mode integrated sensors detecting miRNA-21, wherein A and C are ECL modes and B and D are PEC modes.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

An embodiment of the present application provides a method for simultaneously detecting electrochemiluminescence and photoelectrochemistry modes of a target in serum, which is characterized in that the method comprises the following steps:

Synthesizing an ECL luminescent probe, preparing the ECL luminescent probe pDNA-lumineol-Au by HAuCl ₄, lumineol-NaOH and NaBH4, and preparing the ECL luminescent probe pDNA-lumineol-Au by HS-functionalized pDNA and lumineol-Au;

Dispersing Bi (NO) ₃·5H₂ O and Na ₂WO₄·2H₂ O into pure water to obtain Bi (NO ₃)₃ suspension, dripping Na ₂WO₄ aqueous solution into the Bi (NO ₃)₃ suspension to obtain a mixed solution, and reacting the mixed solution at high temperature to obtain the PEC photosensitive material Bi ₂WO₆;

Step 3, constructing an electrochemiluminescence-photoelectrochemistry dual-mode sensor, wherein the construction of the dual-mode sensor comprises the following steps:

step 3-1, modifying the PEC photosensitive material Bi ₂WO₆ prepared in the step 2 on the surface of an indium tin oxide electrode;

Step 3-2, after cleaning the indium tin oxide electrode modified by Bi ₂WO₆, dropwise adding a molecular beacon, incubating, and cleaning again to modify bovine serum albumin;

Step 3-3, cleaning the electrode surface of the modified bovine serum albumin, dropwise adding a DSN enzyme mixed solution containing miRNA-21 and Mg ²⁺ to the electrode surface, and after incubation, inactivating the DSN enzyme and stopping the reaction;

Step 3-4, adding the ECL luminescent probe pDNA-luminol-Au solution prepared in the step 1, and incubating for 1h at 37 ℃ to obtain an electrochemiluminescence-photoelectrochemistry dual-mode sensor for a target in serum;

And 4, testing a target object, constructing a three-electrode system by taking the electrochemiluminescence-photoelectrochemistry dual-mode sensor obtained in the step 3 as a working electrode, taking Ag/AgCl as a reference electrode and taking a platinum wire as an auxiliary electrode, placing the three-electrode system into a glass detection pool containing 5mmol/L H ₂O₂ of phosphate buffer solution (0.01 mol/L PBS), and transferring the glass detection pool into an electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device, wherein the electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device consists of a LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, and displaying and collecting ECL signals and PEC signals by corresponding software of the LVIUM electrochemical workstation and the BPCL-1-TIC weak luminescence detector respectively, and performing dual-mode simultaneous detection analysis to obtain information of the target object in serum.

In the embodiment, in the step 1, the preparation of the luminol-Au by HAuCl ₄, luminol-NaOH and NaBH ₄ is that HAuCl ₄ is added into a beaker under the condition of ice bath stirring, then the luminol-NaOH solution is added dropwise, after the stirring is uniform, naBH ₄ solution is added dropwise, the ice bath is removed after the reaction, stirring is continued for 6 hours at room temperature, and the luminol-Au is obtained by dialysis in a dialysis bag.

In one embodiment, in step 1, pDNA-luminel-Au is prepared by mixing HS-functionalized pDNA with luminel-Au, reacting in a shaker, then centrifuging to remove supernatant, and dispersing the centrifuged product in 500. Mu.L PBS to obtain ECL luminescent probe pDNA-luminel-Au.

In step 2, the high temperature reaction is that, after stirring, the Na ₂WO₄ aqueous solution is dripped into Bi (NO ₃)₃ suspension to obtain a mixed solution, the mixed solution is transferred into an autoclave, the high temperature reaction is carried out, the reaction product is centrifugally washed by ethanol and water, and the PEC photosensitive material Bi ₂WO₆ is obtained after drying.

In one embodiment, in step 3-1, bi ₂WO₆ of the PEC photosensitive material prepared in step 2 is modified on the surface of an indium tin oxide electrode, bi ₂WO₆ is dissolved in 0.05mg/mL to 0.1mg/mL chitosan solution, the modified surface of the indium tin oxide electrode is dried, glutaraldehyde is added dropwise on the surface of the Bi ₂WO₆ modified indium tin oxide electrode, and the mixture is placed in a dark place at room temperature.

In one embodiment, in step 3-2, after washing the indium tin oxide electrode modified by Bi ₂WO₆, dropwise adding a molecular beacon, incubating, and washing again, the modified bovine serum albumin is obtained by washing the indium tin oxide electrode modified by Bi ₂WO₆, dropwise adding a molecular beacon, incubating for 1h at 37 ℃, washing again with PBS, and modifying the bovine serum albumin in the same environment.

In one embodiment, in step 3-3, the electrode surface of the modified bovine serum albumin is cleaned, a DSN enzyme mixture containing miRNA-21 and Mg ²⁺ is dripped into the electrode surface, after incubation, the DSN enzyme is deactivated, the electrode surface is cleaned by a PBS solution containing Mg ²⁺, a DSN enzyme mixture containing miRNA-21 and Mg ²⁺ is dripped into the electrode surface, after incubation for 1h at 37 ℃, the mixture is left for 5min at 60 ℃ to deactivate the DSN enzyme, and the reaction is terminated.

In one embodiment, in step 4, a dual mode simultaneous detection analysis is performed as follows:

Step S4-1, exciting ECL (electro-chemical engineering) by using a chronoamperometry in LVIUM electrochemical workstation by adopting a potential step method, wherein the excitation potential is 0.6V (lasting 10S), the excitation state is 0V (lasting 10S), and the sampling interval is 0.5S;

And S4-2, setting the high voltage of a photomultiplier tube in the BPCL weak luminescence detector software to be-800V, and after the setting is finished, clicking the LVIUM electrochemical workstation software and the BPCL weak luminescence detector software to start, and carrying out dual-mode simultaneous detection.

The embodiment of the application provides a device for simultaneously detecting electrochemiluminescence and photoelectrochemistry modes for running the detection method, which is characterized in that the detection device comprises LVIUM electrochemical workstations, a BPCL-1-TIC weak luminescence detector, wherein the BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier and a signal analyzer host, ECL signals are collected by the weak luminescence detector and PEC signals are collected by the LVIUM electrochemical workstations, the BPCL-1-TIC weak luminescence detector is connected with a computer after being connected, an S-Connector port in the detector is connected with an S-Input port of a signal analyzer host, a V-Connector port is connected with a V-output port, an electrochemiluminescence detection Cell in the detector is connected with the LVIUM electrochemical workstations through a Cell Connector port, the S-output port is connected with the computer and is used for transmitting ECL signals detected by the photomultiplier, and the BPCL-1-TIC weak luminescence detector is used for measuring and analyzing real-time display in a system;

The LVIUM electrochemical workstation is used as a voltage supply system to be connected with a BPCL-1-TIC weak luminescence detector, a PERIPHERAL POT empty part used for connecting a light source in the LVIUM electrochemical workstation is firstly connected with an electrochemical luminescence detection pool in the BPCL-1-TIC detection system through a Cell Connector port of the LVIUM electrochemical workstation, luminescence voltage is applied to an electrode in a Chrono Amperanetry detection mode, PEC signals on the surface of the electrode are collected, and a USB jack is connected with a computer and used for transmitting the PEC signals obtained through real-time detection.

In one embodiment, the electrochemical luminescence-photoelectrochemistry dual-mode simultaneous detection device is composed of a LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, and ECL signals and PEC signals are respectively displayed and collected by software corresponding to the two parts.

In one embodiment, the weak luminescence detector is connected with a computer after the weak luminescence detector is connected with the computer, an S-Connector port in the detector is connected with an S-Input port of the signal analysis system, a V-Connector port is connected with a V-output port, an electrochemiluminescence detection pool in the detector is connected with a LVIUM electrochemistry workstation through a Cell Connector port, and the S-output port is connected with the computer for transmitting ECL signals detected by a photomultiplier and is displayed in real time in the weak luminescence detector measurement analysis system. And then connecting LVIUM an electrochemical workstation as a voltage supply system with the BPCL-1-TIC weak luminescence detector, namely firstly, connecting a Peripheralpot empty part of the LVIUM electrochemical workstation, which is used for connecting a light source, a Cell Connector port of the LVIUM electrochemical workstation with an electrochemical luminescence detection pool of the BPCL-1-TIC detection system, applying luminescence voltage to an electrode in a Chrono Amperanetry detection mode, collecting PEC signals on the surface of the electrode, and connecting a USB jack with a computer for transmitting the PEC signals obtained by real-time detection.

An embodiment of the present application provides an application of any one of the detection methods in serum target detection.

Example 1 Synthesis of wavelength-adapted ECL/PEC Material pairs

(1) Synthesis of ECL luminescence probe, namely adding 20mL of 2.5X10 ^-4 mol/L HAuCl ₄ into a small beaker under the conditions of ice bath and stirring, then dropwise adding 4mL of 0.01mol/L luminol-NaOH solution, dropwise adding 0.6mL of 0.1mol/L NaBH ₄ solution after stirring uniformly, removing the ice bath after reacting for 10min, continuing stirring at room temperature for 6h, and finally continuing dialysis in a dialysis bag with D=14000 for 6h to obtain luminol-Au. Finally, 10. Mu.L of 100. Mu.M HS-functionalized pDNA (purchased from Zheng Biotechnology Co., ltd.) was mixed with 2000. Mu.L of luminol-Au, and properly reacted in a shaker at 20℃and 1000rpm for 16 hours, and then centrifuged at 10000rpm for 10min and the supernatant was removed. Finally, the centrifugal product is dispersed in 500 mu L PBS to obtain the pDNA-luminal-Au probe with ECL performance.

(2) Synthesis of PEC photosensitive Material 4mmol of Bi (NO) ₃·5H₂ O and 2mmolNa ₂WO₄·2H₂ O were dispersed in 30mL of pure water under vigorous stirring, then Na ₂WO₄ aqueous solution was slowly dropped into Bi (NO ₃)₃ suspension, stirred for 30min, then the mixed solution was transferred into an autoclave, reacted at 180℃for 12h, the obtained product was washed by centrifugation with ethanol and water several times, and dried overnight at 80℃to obtain Bi ₂WO₆ material with PEC response.

The results of XRD and SEM characterization, UV-visible absorption spectrum and TEM characterization of the Bi ₂WO₆ material, respectively, are shown in FIG. 2, wherein the XRD of the Bi ₂WO₆ photosensitive material is shown in FIG. 2, the UV-visible absorption spectrum of the Bi ₂WO₆ photosensitive material is shown in FIG. 2, and the SEM characterization of the Bi ₂WO₆ photosensitive material is shown in FIG. 2, the UV-visible absorption spectrum of the Bi (D) photosensitive material is shown in FIG. 2, the UV-visible absorption spectrum of the Bi (B) photosensitive material is shown in FIG. 2, and the TEM image of the Bi (E, F) photosensitive material is shown in FIG. 2. Experimental results prove that the two materials are successfully synthesized.

Example 2 construction and feasibility test of Dual-mode Integrated sensor

By studying the ultraviolet-visible absorption spectrum of the Bi ₂WO₆ material and the ECL emission spectrum of the luminol-Au NPs in example 1, the ECL emission wavelength range of the luminol-Au NPs was found to have a larger overlap with the ultraviolet-visible absorption wavelength range of the Bi ₂WO₆ material (FIG. 3A), which demonstrates the feasibility of the ECL signal of the luminol-Au NPs to trigger the PEC behavior of the Bi ₂WO₆ material.

The construction of the dual-mode integrated sensor is further carried out by modifying 20 mu L of Bi ₂WO₆ (dissolved in 0.1Mg/mL chitosan) on the surface of an Indium Tin Oxide (ITO) electrode, drying, dripping 20 mu L of 2.5% (v/v) glutaraldehyde on the surface, standing for 50min at room temperature in the dark, washing with PBS, dripping 20 mu L of 0.5 mu mol/L Molecular Beacon (MB) and incubating for 1h at 37 ℃, washing with PBS again, modifying 0.8% Bovine Serum Albumin (BSA) in the same environment, washing the surface of the electrode with a PBS solution containing 5mmol/L of Mg ²⁺, dripping a mixed solution containing miRNA-21 and DSN enzyme (containing Mg ²⁺) on the surface of the electrode, incubating for 1h at 37 ℃, standing for 5min at 60 ℃ to inactivate the DSN enzyme, terminating the reaction, and finally, adding a pDNA-lumine-Au probe solution and incubating for 1h at 37 ℃ to obtain the dual-mode sensing miRNA for detecting the dual-mode integrated sensor.

The electrode modified layer by layer was characterized using Electrochemical Impedance Spectroscopy (EIS) and dual mode signal output behavior, the results of which are shown in fig. 3. It was found that with the layer-by-layer modification of the above steps, the electron transfer resistance of the electrode surface became larger (B in fig. 3), which is caused by the smaller conductivity and larger steric hindrance of chitosan, glutaraldehyde, MB, BSA, miRNA-21 and pDNA-luminal-Au probes, it could be preliminarily demonstrated that the sensor was successfully constructed.

The dual mode output signals (i.e., ECL and PEC behavior) of the layer-by-layer modified electrodes were then tested, as shown in fig. 3C and 3D. When the photosensitive material Bi ₂WO₆ is modified on the surface of the electrode, the dual-mode integrated detection device does not collect a light signal (curve b, C in FIG. 3), but generates a photocurrent of about 400nA (curve b, D in FIG. 3) due to the application of a bias voltage of 0.6V, and the photocurrent gradually decreases with the modification layer by layer, and when the pDNA-luminol-Au probe with ECL luminescence property is incubated on the surface of the electrode, the dual-mode integrated detection device not only collects a light signal of about 10000a.u. (curve g, C in FIG. 3), but also increases the photocurrent of the PEC mode by about 500nA (interpolation between curves g and f, D in FIG. 3).

The above results are sufficient to demonstrate the successful construction of the sensor and the feasibility of dual mode detection for targets in serum.

Example 3 Dual mode detection of target miRNA-21 in solution

In order to perform dual-mode quantitative analysis on a target miRNA-21 in a solution by using a photoinduction PEC integrated device, ECL signals of biosensors with different concentrations of miRNA-21 and ECL intensity and miRNA-21 concentration graphs in FIG. 4 (A) are obtained by modifying the surfaces of working electrodes with different sensing interfaces respectively (0 fmol/L,50fmol/L,100fmol/L,500fmol/L,1pmol/L,10pmol/L,100pmol/L, 1nmol/L,10 nmol/L), and the like) according to the construction steps of an electrochemiluminescence-photoelectrochemistry dual-mode sensor, and then testing in a mode of step 5, wherein the ECL signals of the biosensors with different concentrations of miRNA-21 and the ECL intensity and the miRNA-21 concentration graphs in FIG. 4 (B) are shown by the interpolation graphs, and the PEC signals of the biosensors with different concentrations of miRNA-21 and the PEC intensity and the PEC intensity in FIG. 4 (D) show the logarithmic relations of the concentrations of miRNA-21 and the PEC intensity graphs in FIG. 4.

As a result of analysis in this example, when the concentration of miRNA-21 was in the range of 1.0X10 ^-16～1.0×10^-8 mol/L, the ECL signal intensity was positively correlated with the logarithmic value of the concentration, the linear equation was that Y _ECL＝17911.9+987.31g c(mol/L,R² =0.997, the detection limit was as low as 72.9amol/L (S/N=3), and when the concentration of miRNA-21 was in the range of 1.0X10 ^-14～1.0×10^-8 mol/L, the PEC signal intensity was positively correlated with the logarithmic value of the concentration, the linear equation was that I _PEC＝1605.5+102.9lg c(mol/L,R² =0.996, and the detection limit was as low as 6.46fmol/L (S/N=3).

The results show that the two developed analysis modes have wider linear range and lower detection limit, and the results measured by the two modes can be mutually complemented and mutually verified, so that the accuracy of the detection results is further improved, and a new method is provided for sensitive and accurate detection in complex samples.

Example 4 ECL-PEC Dual mode Integrated sensor to detect the Selectivity and stability of miRNA-21

To exclude false positive signals caused by miRNAs of other sequences in serum, selection experiments were performed using miRNAs of other sequences (including miRNA-141 and miRNA-451), single base mismatched miRNA-21 (MM 1) and three base mismatched miRNA-21 (MM 3) as interferents (at concentrations of 1.0 nmol/L). The ECL and PEC responses of the modified miRNA with other sequences are found to be lower, the ECL and PEC signals obtained by modifying the two interferents are only half of that of the modified target miRNA-21, and finally the ECL and PEC responses obtained by modifying the sensor of the substance mixture (miRNA-141+miRNA-451+MM1+MM3+miRNA-21) are similar to those obtained by modifying the target miRNA-21 only, see A and B in fig. 5.

In addition, stability of the dual mode sensor was also evaluated. As shown in fig. 5C and 5D, ECL pattern of the biosensor (containing 10nmol/L miRNA-21) showed stable photocurrent after 10 consecutive scans with a Relative Standard Deviation (RSD) of 2.3% and a PEC pattern of 1.1% current signal RSD, indicating high sensor stability.

Experimental results show that the method provided by the application has good selectivity and high stability, and can realize specific detection of the target substance in the complex sample.

Example 5 Dual mode analysis of target miRNA-21 in serum

MiRNA-21 is used as a potential tumor marker, is expressed in various cancers, and has lower miRNA-21 concentration in human serum and certain difficulty in actual sample analysis. In order to evaluate the applicability of the detection method, the developed dual-mode sensor is used for the labeling experimental analysis of miRNA-21 in serum. Different concentrations of miRNA-21 standard solutions (0.01, 0.1 and 1 pmol/L) were added to 2 human serum diluted 10-fold each, respectively, for dual mode detection, and the concentration was calculated from the calibration curve obtained in example 1.

As shown in Table 1, ECL mode detection results show that the labeled recovery rate of miRNA-21 is 93.2-117.7%, the RSD is 1.8-3.6%, and PEC mode detection results show that the recovery rate of miRNA-21 in serum is 87.1-118.5%, and the RSD is 2.6-5.1%. The result shows that the invention has good application potential in the detection of serum target.

Table 1.ECL-PEC dual-mode integrated sensor is used for the labeling recovery experimental results of miRNA-21 in serum.

In conclusion, the research constructs a novel ECL-PEC dual-mode integrated sensor, and realizes sensitive and accurate detection of miRNA-21. The dual-mode biosensor has two innovation points, namely, firstly, ECL is used as a miniaturized inner light source, the use of an outer light source is avoided, the distance of the light source required by PEC excitation is shortened, the photon utilization efficiency is obviously improved, the background noise is reduced, secondly, photon emission and Bi ₂WO₆ photocurrent collection are realized on the surface of the same electrode by using different materials, meanwhile, ECL and PEC dual signals are output, the cross-validation detection of miRNA-21 is realized by the developed dual-mode biosensor, and compared with a single-mode sensor, false positive/negative signals are effectively reduced, so that the detection result is more reliable. The work not only makes an important contribution to the development of ECL-PEC detection technology, but also has application potential in the fields of biochemical analysis and clinical diagnosis, and provides a new idea for constructing a novel double-mode biosensor.

The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the invention.

Claims (10)

1. A method for simultaneous electrochemiluminescence-photoelectrochemistry dual-mode detection of a target in serum, the method comprising the steps of:

Synthesizing an ECL luminescent probe, preparing the ECL luminescent probe pDNA-lumineol-Au by HAuCl ₄, lumineol-NaOH and NaBH ₄, and preparing the ECL luminescent probe pDNA-lumineol-Au by HS-functionalized pDNA and lumineol-Au;

Dispersing Bi (NO) ₃·5H₂ O and Na ₂WO₄·2H₂ O into pure water to obtain Bi (NO ₃)₃ suspension, dripping Na ₂WO₄ aqueous solution into the Bi (NO ₃)₃ suspension to obtain a mixed solution, and reacting the mixed solution at high temperature to obtain the PEC photosensitive material Bi ₂WO₆;

Step 3, constructing an electrochemiluminescence-photoelectrochemistry dual-mode sensor, wherein the construction of the dual-mode sensor comprises the following steps:

step 3-1, modifying the PEC photosensitive material Bi ₂WO₆ prepared in the step 2 on the surface of an indium tin oxide electrode;

Step 3-2, after cleaning the indium tin oxide electrode modified by Bi ₂WO₆, dropwise adding a molecular beacon, incubating, and cleaning again to modify bovine serum albumin;

Step 3-3, cleaning the electrode surface of the modified bovine serum albumin, dropwise adding a DSN enzyme mixed solution containing miRNA-21 and Mg ²⁺ to the electrode surface, and after incubation, inactivating the DSN enzyme and stopping the reaction;

Step 3-4, adding the ECL luminescent probe pDNA-luminol-Au solution prepared in the step 1, and incubating for 1h at 37 ℃ to obtain an electrochemiluminescence-photoelectrochemistry dual-mode sensor for a target in serum;

And 4, testing a target object, constructing a three-electrode system by taking the electrochemiluminescence-photoelectrochemistry dual-mode sensor obtained in the step 3 as a working electrode, taking Ag/AgCl as a reference electrode and taking a platinum wire as an auxiliary electrode, placing the three-electrode system into a glass detection pool containing 5mmol/L H ₂O₂ of phosphate buffer solution (0.01 mol/L PBS), and transferring the glass detection pool into an electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device, wherein the electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device consists of a LVIUM electrochemical workstation and a BPCL-1-TIC weak luminescence detector, and displaying and collecting ECL signals and PEC signals by corresponding software of the BPCL-1-TIC weak luminescence detector and the LVIUM electrochemical workstation respectively, and performing dual-mode simultaneous detection analysis to obtain information of the target object in serum.

2. The method for simultaneous detection of electrochemiluminescence and photoelectrochemistry modes of a target in serum according to claim 1, wherein in the step1, the preparation of the luminol-Au by the HAuCl ₄, the luminol-NaOH and the NaBH ₄ is that under the condition of ice bath stirring, HAuCl ₄ is added into a beaker, then a luminol-NaOH solution is added dropwise, after stirring uniformly, naBH ₄ solution is added dropwise, after reaction, the ice bath is removed, stirring is continued for 6 hours at room temperature, and the luminol-Au is obtained by dialysis in a dialysis bag.

3. The method for simultaneous detection of electrochemiluminescence-photoelectrochemistry modes of a target in serum according to claim 1, wherein in step 1, pDNA-Luminol-Au is prepared by HS-functionalized pDNA and Luminol-Au, wherein HS-functionalized pDNA and Luminol-Au are mixed and reacted in a shaker, then supernatant is removed by centrifugation, and the centrifuged product is dispersed in 500. Mu.L PBS to obtain ECL luminescence probe pDNA-Luminol-Au.

4. The method for simultaneous detection of electrochemiluminescence and photoelectrochemistry modes of a target in serum according to claim 1, wherein in the step 2, the high-temperature reaction is that after stirring, an aqueous solution of Na ₂WO₄ is dripped into Bi (NO ₃)₃ suspension to obtain a mixed solution, the mixed solution is transferred into an autoclave, the high-temperature reaction is carried out, and the reaction product is centrifugally washed by ethanol and water and dried to obtain the PEC photosensitive material Bi ₂WO₆.

5. The method for simultaneously detecting electrochemiluminescence and photoelectrochemistry modes of a target in serum according to claim 1, wherein in the step 3-1, bi ₂WO₆ of the PEC photosensitive material prepared in the step 2 is modified on the surface of an indium tin oxide electrode, bi ₂WO₆ is dissolved in 0.05mg/mL-0.1mg/mL chitosan solution, the modified surface of the indium tin oxide electrode is dried, glutaraldehyde is added dropwise on the surface of the indium tin oxide electrode modified by Bi ₂WO₆, and the modified surface is placed in a dark place at room temperature.

6. The method for simultaneous detection of electrochemical luminescence-photoelectrochemistry modes of a target in serum according to claim 1, wherein in step 3-2, after washing the indium tin oxide electrode modified with Bi ₂WO₆, a molecular beacon is added dropwise, incubation is performed, after washing again, the modified bovine serum albumin is obtained by washing the indium tin oxide electrode modified with Bi ₂WO₆, then a molecular beacon is added dropwise, and incubation is performed for 1 hour at 37 ℃, and after washing again with PBS, the modified bovine serum albumin is modified in the same environment.

7. The method for simultaneous detection of electrochemiluminescence and photoelectrochemistry modes of a target in serum according to claim 1, wherein in step 3-3, the electrode surface of the modified bovine serum albumin is cleaned, a DSN enzyme mixture containing miRNA-21 and Mg ²⁺ is dripped into the electrode surface, after incubation, the DSN enzyme is deactivated, the reaction is terminated by cleaning the electrode surface with a PBS solution containing Mg ²⁺, then a DSN enzyme mixture containing miRNA-21 and Mg ²⁺ is dripped into the electrode surface, after incubation for 1 hour at 37 ℃, the electrode surface is left for 5 minutes at 60 ℃ to deactivate the DSN enzyme, and the reaction is terminated.

8. The method for simultaneous detection of target substances in serum according to claim 1, wherein in step 4, the dual-mode simultaneous detection analysis is performed as follows:

Step S4-1, exciting ECL (electro-chemical engineering) by using a chronoamperometry in a LVIUM electrochemical workstation by adopting a potential step method, wherein the excitation potential is 0.6V (lasting 10S), the excitation state is 0V (lasting 10S), and the sampling interval is 0.5S;

And S4-2, setting the high voltage of a photomultiplier tube in the BPCL weak luminescence detector software to be-800V, and after the setting is finished, clicking the LVIUM electrochemical workstation software and the BPCL weak luminescence detector software to start, and carrying out dual-mode simultaneous detection.

9. An electrochemiluminescence-photoelectrochemistry dual-mode simultaneous detection device for running the detection method according to any one of claims 1-8, wherein the detection device comprises a LVIUM electrochemical workstation, a BPCL-1-TIC weak luminescence detector, wherein the BPCL-1-TIC weak luminescence detector consists of a detector containing a photomultiplier and a signal analyzer host, ECL signals are collected by the weak luminescence detector and PEC signals are collected by the LVIUM electrochemical workstation, the BPCL-1-TIC weak luminescence detector is connected with a computer after being connected, an S-Connector port in the detector is connected with an S-Input port of a signal analyzer host, a V-Connector port is connected with a V-output port, an electrochemiluminescence detection Cell in the detector is connected with the LVIUM electrochemical workstation through the Cell Connector port, and the S-output port is connected with the computer and is used for transmitting ECL signals detected by the photomultiplier, and the BPCL-1-TIC weak luminescence detector measures and displays real-time information in a weak luminescence detector measurement and analysis system;

The LVIUM electrochemical workstation is used as a voltage supply system to be connected with a BPCL-1-TIC weak luminescence detector, a PERIPHERAL POT empty part used for connecting a light source in the LVIUM electrochemical workstation is firstly connected with an electrochemical luminescence detection pool in the BPCL-1-TIC detection system through a Cell Connector port of the LVIUM electrochemical workstation, luminescence voltage is applied to an electrode in a Chrono Amperanetry detection mode, PEC signals on the surface of the electrode are collected, and a USB jack is connected with a computer and used for transmitting the PEC signals obtained through real-time detection.

10. Use of the detection method according to any one of claims 1-8 for the detection of serum targets.