CN113866235A

CN113866235A - Electrochemical luminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrode and construction method and application thereof

Info

Publication number: CN113866235A
Application number: CN202110936924.2A
Authority: CN
Inventors: 李迎春; 胡悦; 朱亮; 梅学翠
Original assignee: Shenzhen Graduate School Harbin Institute of Technology
Current assignee: Shenzhen Graduate School Harbin Institute of Technology
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-12-31
Anticipated expiration: 2041-08-16
Also published as: CN113866235B

Abstract

The invention discloses an electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode and a construction method and application thereof. The device is simple, low in cost and strong in plasticity, can be used for detecting one target or two targets simultaneously, and has wide market application prospect.

Description

Electrochemical luminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrode and construction method and application thereof

Technical Field

The invention belongs to the technical field of chemical analysis, and particularly relates to an electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, a construction method of the dual-mode sensing detection device and application of the dual-mode sensing detection device.

Background

In recent years, with the increasing detection demand, a wide range of attention has been paid to the construction of a novel sensing device by combining different sensing technologies together. At present, various detection methods such as an electrochemical-colorimetric method, an electrochemical-Electrochemiluminescence (ECL) method, an electrochemical-photoelectrochemical method, a fluorescence-colorimetric method and the like are integrated. Such dual mode sensing devices not only retain the unique advantages of each mode sensing device, but also generally produce a wider dynamic response range. Compared with a single-mode sensing detection device, the dual-mode sensing detection device is more supplementary, diversified and reliable.

However, most of the current dual-mode sensing devices are constructed by mixing all reagents in a reaction system, so as to form a simple hybrid system, and the design of the system lacks novelty and flexibility. In addition, due to the mixed reaction environment, the detection system inevitably faces the compromise of working conditions among different detection methods, and further influences the sensing performance of each detection mode, thereby having adverse effects on the sensitivity and accuracy of detection. In addition, the dual-mode signal output by such a detection system is also subject to interference between two different detection regimes. Therefore, how to construct a better dual-mode sensing detection device becomes a research direction for analyzing the detection technicians.

CN 112540073A teaches a preparation method and application of a dual-output mode sensor based on Fc-apt amplified electrochemiluminescence signalsSelf-reinforced electrochemical luminescent material SiO₂@ Ru-NGQDs coating spherical SiO with Nafion film₂The material of @ Ru-NGQDs is fixed on the surface of a clean glassy carbon electrode; then, AuNPs are assembled on the surface of the modified glassy carbon electrode, and an aptamer complementary chain is fixed through Au-S covalent interaction; then introducing an aptamer marked by ferrocene, and obtaining an enhanced electrochemiluminescence signal while obtaining a ferrocene electrochemical signal through base complementary pairing assembly; finally, a novel dual-mode electrochemical-electrochemiluminescence biosensor is obtained. Although the sensor can realize sensitive and rapid analysis on an actual sample, the sensor is complex to prepare, high in cost and not beneficial to large-scale use.

CN 110426438A teaches a wireless photoelectrochemical analysis and detection device based on a closed bipolar electrode and a manufacturing method thereof, in which a first control electrode is located in a first electrolyte in a first electrolytic cell, a second control electrode is located in a second electrolyte in a second electrolytic cell, the closed bipolar electrode is composed of a conductor, a third control electrode and a photoelectrode loaded with a photoelectric semiconductor material, the third control electrode and the photoelectrode are respectively located in the first and second electrolytic cells, and the first and second electrolytic cells are connected through the conductor; the light source is fixed above the photoelectrode; the second electrolyte contains a substance to be detected; the electrochemical workstation is electrically connected with the first control electrode and the second control electrode through leads to control the potential of the photoelectrode. Although this device for the first time enables photoelectrochemical and biosensing without direct connection via external leads, it is only possible to specifically detect a single analytical object.

In summary, how to rapidly and sensitively realize the dual-mode detection of the target object becomes a research focus in the field of analysis technology.

Disclosure of Invention

In view of the above problems, the present invention provides a novel electrochemiluminescence-colorimetric dual-mode sensing and detecting device based on a closed bipolar electrode, which is simple, low in cost, and strong in plasticity, and can be used for detecting one target or two targets simultaneously, so that the device has a wide market application prospect.

In order to achieve the above object, a first aspect of the present invention provides an electrochemiluminescence-colorimetric dual-mode sensing detection apparatus based on a closed bipolar electrode, which includes a detection cell, a detection cell partition plate, a first working electrode, a second working electrode and a conductor, wherein the detection cell partition plate divides the detection cell into two non-communicated regions, namely, the first detection cell and the second detection cell, the first working electrode is located in the first detection cell, the second working electrode is located in the second detection cell, two ends of the conductor are respectively in contact with the first working electrode and the second working electrode, the surface of the first working electrode is modified with prussian blue, the second working electrode is provided with a reaction region, and the non-reaction region and the conductor surface of the second working electrode are covered with an insulating material.

Unlike conventional electrodes, closed bipolar electrode (c-BPE) has physically separated anode and cathode compartments, where oxidation and reduction reactions occur separately and simultaneously in the anode and cathode compartments of the c-BPE, which both simplifies the complexity of the analysis apparatus and reduces equipment costs. In addition, it is worth mentioning that the physically separated anode chamber and cathode chamber are not only suitable for single target measurement, but also for dual target analysis. In short, c-BPE is found to be an ideal candidate for constructing a dual-mode sensor, and the invention designs an electrochemiluminescence-colorimetric dual-mode sensing detection device based on the c-BPE. Wherein the first working electrode of the c-BPE is modified with Prussian Blue (PB) for colorimetric assay, and the second working electrode is used for ECL detection, and luminol can be used as a signal source. Wherein, while the second working electrode collects ECL signals, PB on the first working electrode will be reduced to Prussian White (PW) for subsequent colorimetric determination. Subsequently, in order to verify the feasibility of the electrochemical luminescence-colorimetric dual-mode sensing detection device based on c-BPE constructed by the invention, H related to a large number of physiological processes is selected₂O₂And H₂O₂The analyte of interest serves as a model target. Due to H₂O₂Not only is a co-reactant of luminol, but also is a strong oxidant for converting PW into PB, so that the platform can realize H₂O₂The ECL and colorimetric dual output detection. In addition, the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the c-BPE can be further expanded to be used for dual-target analysis, and relevant experiments are subsequently performed for verification.

Preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the size of the detection cell is 5-10cm in length, 3-7cm in width and 2-5cm in height. The detection pool is small in size, does not occupy large space, and is easy to carry.

Preferably, in the above-mentioned electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the detection cell and the detection cell separator are made of a transparent material, particularly a colorless transparent material. The detection pool and the detection pool clapboard made of colorless transparent materials prevent the detection pool and the detection pool clapboard from influencing signal output when the colorimetric test is carried out.

More preferably, in the above electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the colorless transparent material includes, but is not limited to, one or more of Polydimethylsiloxane (PDMS), polycarbonate, cyclic olefin copolymer, polymethyl methacrylate (PMMA), Polystyrene (PS), styrene-methyl methacrylate copolymer (MS), inorganic glass, and the like. In addition, the test cell and the test cell separator are made of inorganic glass in consideration of production cost and transparency.

Preferably, in the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode, the number of the first working electrode and the second working electrode is more than 1, when a plurality of first working electrodes and/or second working electrodes are provided, a plurality of parallel signals can be output simultaneously, and thus the stability of the detection device of the present invention can be evaluated, and an average value is taken for the parallel signals output simultaneously in an actual test, which corresponds to a sensing array. In addition, when the number of the first working electrode and the second working electrode is plural, the number of the conductor is also plural so that the conductor connects the working electrodes.

Preferably, in the above electrochemical luminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode, the first working electrode and the second working electrode are made of platinum, platinum alloy, platinum-plated material, stainless steel, graphite or carbon, gold, ITO conductive glass. More preferably, the first working electrode and the second working electrode are ITO conductive glass.

Preferably, in the above electrochemical luminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode, the dimensions of the first working electrode and the second working electrode are 1-3cm in length, 1-3cm in width and 0.5-1.5mm in thickness; the square resistance is 8-15ohm/sq, most preferably, the sizes of the first working electrode and the second working electrode are 2cm in length, 2cm in width, 1.1mm in thickness and 12 ohm/sq.

Preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the Prussian blue is modified on the surface of the first working electrode through electrodeposition, wherein the electrolyte is 0.05-0.15M hydrochloric acid solution, and the hydrochloric acid solution contains 2-8.0mM K₃[Fe(CN)₆]、2-8.0mM FeCl₃And 0.05-0.2M KCl, the applied potential is 0.2-0.6V, and the deposition time is 120-360 s. The prussian blue deposited by electrodeposition has a relatively uniform distribution.

More preferably, in the above electrochemical luminescence-colorimetric dual-mode sensing and detecting device based on the enclosed bipolar electrode, the conditions for electrodepositing prussian blue on the surface of the first working electrode are as follows: the electrolyte was a 0.1M hydrochloric acid solution containing 5.0mM K₃[Fe(CN)₆]、5.0mM FeCl₃And 0.1M KCl, with an applied potential of 0.4V and a deposition time of 240 s. Under the condition of the electrodeposition, the Prussian blue deposited is relatively uniform in deposition and is very suitable for colorimetric detection.

Preferably, in the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing and detecting device based on the enclosed bipolar electrode, the second working electrode is provided with a reaction area having a circular shape and a diameter of 0.4-1.0 cm. The reaction area arranged on the second working electrode can be in other shapes without strict limitation, and only the reaction areas in the related test process of the same substance or the object to be tested need to be consistent, and the reaction areas need to correspond to the central position of the detection port of the ECL device.

More preferably, in the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing and detecting device based on the closed bipolar electrode, the conductor is made of, but not limited to, metals such as copper, silver, aluminum, iron, etc., alloys such as copper-aluminum alloy, etc.

Preferably, in the above-mentioned electrochemiluminescence-colorimetric dual-mode sensing and detecting device based on the enclosed bipolar electrode, the conductor is a ribbon, so that the conductor is in sufficient contact with the working electrode, thereby facilitating smooth transmission of electrons and thus facilitating the reaction in the whole detecting device.

More preferably, in the above electrochemical luminescence-colorimetric dual-mode sensing and detecting device based on the closed bipolar electrode, the size of the strip-shaped conductor is 5-10cm in length, 1-2cm in width and 0.1-0.3mm in thickness. The most important dimensions are length 8cm, width 1.5cm and thickness 0.2 mm. Strictly speaking, the size of the conductor is only required to meet the use requirement.

More preferably, in the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing device based on the closed bipolar electrode, the insulating material is an insulating paint or an insulating tape, and more preferably, the insulating tape is detachably attached to the surface of the conductor and the second working electrode, and when the device is not used, the insulating tape can be discarded and the working electrode and the conductor can be retained, so that the device can be easily disassembled and cleaned.

In addition, according to the second aspect of the present invention, there is also provided a method for constructing the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode, which comprises the following steps:

(1) modification of the surface of the first working electrode: modifying the surface of the first working electrode with Prussian blue;

(2) and (3) setting a second working electrode reaction area: arranging a reaction area on the surface of the second working electrode, and covering insulating materials on other non-reaction areas;

(3) construction of a closed bipolar electrode: and connecting the first working electrode and the second working electrode through two ends of a conductor, then coating an insulating material on the surface of the conductor, then placing the first working electrode in a first detection cell, and placing the second working electrode in a second detection cell to obtain the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode.

The sequence of the steps (1) and (2) of the construction method can be exchanged, and the method can show that the construction is simple and easy to implement, does not have complex processes, can be used for analyzing the target object only by simple assembly when used outdoors or in other occasions, and provides great possibility for large-scale application of the detection device.

Preferably, in the construction method, in order to make the conductor sufficiently attached to the first working electrode and the second working electrode, and further facilitate the transmission of electrons, the lengths of the overlapping of the conductor and the first working electrode and the second working electrode are respectively 0.5-0.9 cm.

Preferably, the construction method further comprises the step of assembling the detection cell, specifically, the side wall and the bottom surface of the detection cell are bonded by glue, and then the detection cell partition plate is placed in the detection cell to divide the detection cell into two unconnected areas, namely the first detection cell and the second detection cell.

Preferably, in the construction method, the specific process of step (3) is: and taking a strip conductor, taking a proper amount of insulating adhesive tape at two ends of the strip conductor to enable the strip conductor to be respectively connected with the first working electrode and the second working electrode, fully fixing the strip conductor to enable the conductor to be tightly attached to the first working electrode and the second working electrode, wrapping the strip conductor with the insulating adhesive tape, folding the conductor along the middle part and placing the conductor into a detection pool, wherein the first working electrode is positioned in the first detection pool, and the second working electrode is positioned in the second detection pool, so that the construction of the electrochemiluminescence-colorimetric dual-mode sensing device based on the closed bipolar electrode is completed.

In addition, according to the third aspect of the present invention, there is also provided an application of the above-mentioned sealed bipolar electrode-based electrochemiluminescence-colorimetric dual-mode sensing detection device, wherein the sealed bipolar electrode-based electrochemiluminescence-colorimetric dual-mode sensing detection device is applied to the detection fields of medical diagnosis, environmental monitoring, food and the like. The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can be used for detecting one target object and two target objects.

In the application of the present invention, the substance detectable in the electrochemiluminescence detection end, i.e. the second detection cell, is only required to influence the electron transport rate on the surface of the electrode or to change the concentration of the co-reactant (H) in the system₂O₂) And (4) finishing. In other words, H can be monitored₂O₂And H₂O₂Is determined (i.e., H is generated after a series of reactions)₂O₂) (ii) a Or by modifying some specific recognition substances, such as MIP, specific antibodies, aptamers and the like, on the surface of the second working electrode, when the substance to be detected is combined with the specific recognition substances, the electron transmission rate on the surface of the electrode is influenced, and further the change of an ECL signal can be detected by the detection device provided by the invention, so that the detection device provided by the invention can detect a wide range of substances, such as molecules, ions, nucleic acid fragments, proteins and the like. For the substance detectable in the colorimetric detection end, i.e. the first detection cell, only the substance to be detected has strong oxidizing property (which can oxidize Prussian white into Prussian blue with color conversion), such as H₂O₂And the substances to be detected comprise strong oxidants and substances which can generate the strong oxidants after a series of reaction processes. Obviously, the detection device has a wide application range, and can be widely applied to different fields such as medical treatment, environmental monitoring and the like.

More preferably, the application is for detecting glucose, lactate, Very Low Density Lipoprotein (VLDL), acetylcholinesterase (AChE), reduced Nicotinamide Adenine Dinucleotide (NADH), and H₂O₂。

The electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode has the advantages of unique physically separated reaction tanks, and different sensing modes have independent reaction interfaces, so that the problems of compromise of reaction conditions and mutual interference of different sensing signals in the traditional dual-mode sensing detection device are solved.

In addition, according to a fourth aspect of the present invention, there is also provided a use of the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrodes for detecting glucose, comprising the steps of:

(1) drawing an ECL standard curve and a colorimetric standard curve: respectively adding a series of glucose standard solutions with different concentrations into a phosphoric acid buffer solution containing glucose oxidase, and reacting at room temperature to generate H₂O₂Obtaining enzymolysis mixed liquor; adding carbonic acid buffer solution containing luminol into a second detection pool to generate H₂O₂Adding the enzymolysis mixed solution into a second detection tank, adding a phosphate buffer solution containing halide into the first detection tank, applying a driving voltage to a system by adopting a cyclic voltammetry method, and drawing an ECL standard curve according to a linear relation between the collected ECL signal and the concentration of a standard solution; after each measurement of the ECL signal of one standard solution, the solution in the first detection cell is removed and H will be generated₂O₂Adding the enzymolysis mixed solution to the surface of a first working electrode in a first detection pool, identifying RGB blue values after a period of time, obtaining colorimetric signals, and drawing a colorimetric standard curve by obtaining a linear relation between the RGB blue values and the concentration of a standard solution;

(2) and (3) detection of a sample to be detected: adding the glucose sample solution into a phosphate buffer solution containing glucose oxidase, and reacting at room temperature to generate H₂O₂Obtaining a sample enzymolysis mixed solution; adding carbonic acid buffer solution containing luminol into a second detection pool to generate H₂O₂And adding the sample enzymolysis mixed solution into a second detection tank, adding a phosphate buffer solution containing halide into the first detection tank, applying a driving voltage to a system by adopting a cyclic voltammetry method, detecting to obtain an ECL signal, comparing the ECL signal with an ECL standard curve, and calculating to obtain the concentration of glucose in the sample to be detected.

In the test process of the invention, when the ECL signal is collected, the Prussian blue deposited by the first working electrode generates Prussian white due to electrochemical reaction, in other words, the ECL signal is generated in the ECL test process, and preparation is also made for subsequent colorimetric determination.

The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode adopts a standard curve for determining glucose in dual modes, can investigate the sensitivity and stability of the detection device, and is very suitable for field determination.

Preferably, in the above application, the step (2) further comprises measuring a colorimetric signal, specifically: removing the solution in the first detection pool, adding the sample enzymolysis mixed solution to the surface of the first working electrode in the first detection pool, identifying the RGB blue value after the same time as that when the standard curve is drawn, obtaining a colorimetric signal, and comparing the colorimetric signal with the colorimetric standard curve to obtain the concentration of glucose in the sample to be detected. Meanwhile, the sensitivity and the stability of the detection device can be compared by obtaining a colorimetric signal and an ECL signal, and the average value of the colorimetric signal and the ECL signal is more accurate.

Preferably, in the above-mentioned application, the concentration of glucose in the glucose solution in the second detection cell, i.e., the concentration of glucose detected by the device of the present invention using an electrochemiluminescence signal, is in the range of 10. mu.M to 2mM, and the concentration of glucose in the glucose solution in the first detection cell, i.e., the concentration of glucose detected by the device of the present invention using a colorimetric signal, is in the range of 500. mu.M to 20 mM.

Preferably, in the above use, the conditions for measuring ECL signal are: the scanning potential range is 0-2.5V, and the scanning speed is 100 mV/s.

Preferably, in the above-mentioned use, the formation of H is obtained in the course of measuring the standard curve₂O₂The conditions in the enzymolysis mixed solution are as follows: the concentration of the glucose oxidase in the phosphate buffer solution of the glucose oxidase is 0.5-1.5mg/ml, the pH of the phosphate buffer solution of the glucose oxidase is 7.2, the phosphate buffer solution of the glucose oxidase consists of 0.06mM potassium dihydrogen phosphate, 0.14mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, the reaction time is 5-15min at room temperature, and the glucose standardThe volume ratio of the solution to the phosphate buffer solution containing glucose oxidase is 1: (8-50).

Preferably, in the above use, the carbonic acid buffer solution containing luminol has a pH of 10.5 and consists of 0.01mM potassium dihydrogen phosphate, 0.19mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, wherein the concentration of the luminol is 8-15. mu.M.

Preferably, in the above uses, the halide includes, but is not limited to, one or more of potassium chloride, sodium chloride, and the like, with potassium chloride and/or sodium chloride being more preferred based on cost considerations.

Preferably, in the above use, the phosphate buffer solution containing a halide has a pH of 6.0 and consists of 0.18mM potassium dihydrogenphosphate, 0.02mM dipotassium hydrogenphosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, wherein the halide is at a concentration of 0.05-1.5M.

Preferably, in the above-mentioned use, the parameters involved in said step (2) are the same except that the glucose solution is different.

More preferably, the specific process of step (1) is: a series of 100. mu.L glucose standard solutions of different concentrations were added to 1.0mL phosphate buffer solution (pH 7.2) containing 1.0mg/mL glucose oxidase, and reacted at room temperature for 10min to produce H₂O₂Obtaining enzymolysis mixed liquor; adding carbonic acid buffer solution (pH 10.5) containing 10 μ M luminol into the second detection cell, and generating H into 10 μ L₂O₂Adding the enzymolysis mixed solution into a second detection pool, adding a phosphoric acid buffer solution (with the pH value of 6.0) containing 0.1M potassium chloride into the first detection pool, applying a driving voltage to the system by adopting a cyclic voltammetry method, and drawing an ECL standard curve according to the linear relation between the collected ECL signal and the concentration of the standard solution; after each measurement of the ECL signal of one standard solution, the solution in the first detection cell is removed and H will be generated₂O₂The enzymolysis mixed solution is added into a first detection pool, the RGB blue value is identified after 15s, a colorimetric signal is obtained, and a colorimetric standard curve is drawn by obtaining the linear relation between the RGB blue value and the concentration of the standard solution.

The electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode realizes output signals in different modes according to different detection principles, has higher sensitivity and stability, keeps the respective unique advantages of each mode detection device, and also generates a wider dynamic response range. Compared with a single-mode sensing detection device, the detection device has the advantages of more complementarity, diversity and reliability.

In addition, according to a fifth aspect of the present invention, there is also provided a use of the above-mentioned electrochemiluminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode for detecting Very Low Density Lipoprotein (VLDL) and acetylcholinesterase (AChE), which includes the following steps:

(1) modification of the second working electrode: modifying a Molecularly Imprinted Polymer (MIP) in a reaction region of a second working electrode, soaking the second working electrode in ethanol containing (3-aminopropyl) trimethoxysilane, drying the surface of the second working electrode through air flow, immersing the second working electrode in a VLDL solution with the concentration of 0.05-2g/dL for incubation, then blocking the second working electrode in an acrylamide solution, polymerizing the second working electrode in a phosphate buffer solution containing acrylamide, N' -methylene bisacrylamide and ammonium persulfate, and then immersing the second working electrode in an oxalic acid solution to remove the VLDL embedded in the polymer membrane;

(2) plotting of ECL standard curve for VLDL: soaking the second working electrode in a series of VLDL standard solutions with different concentrations, and soaking the VLDL standard solutions containing luminol and H₂O₂After the carbonic acid buffer solution is added into the second detection cell, the second working electrode is placed into the second detection cell, and then the phosphorus containing the halide is addedAfter the acid buffer solution is added into the first detection cell, a driving voltage is applied to the system by adopting a cyclic voltammetry method, and an ECL standard curve is drawn according to the linear relation between the collected ECL signal and the concentration of the standard solution;

(3) drawing a colorimetric standard curve of AChE: respectively adding a series of AChE standard solutions with different concentrations into a phosphoric acid buffer solution containing choline oxidase and acetylcholine, and reacting at room temperature to obtain an enzymolysis mixed solution; removing the solution in the first detection pool after measuring the ECL signal of each standard solution, adding the enzymolysis mixed solution into the first detection pool, identifying the RGB blue value after a period of time, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue value and the concentration of the standard solution;

(4) detection of VLDL-containing test sample: soaking the modified second working electrode in a VLDL-containing sample solution, and adding luminol and H₂O₂After the carbonic acid buffer solution is added into a second detection pool, a second working electrode is placed into the second detection pool, then the phosphoric acid buffer solution containing halide is added into the first detection pool, electrochemical detection is carried out by adopting an electrochemical alternating current impedance method to obtain an ECL signal, the ECL signal is compared with an ECL standard curve for calculation, and the concentration of VLDL in the sample to be detected is obtained;

(5) detection of AChE-containing test samples: adding a sample to be detected containing AChE into a phosphate buffer solution containing choline oxidase and acetylcholine, and reacting at room temperature to obtain an enzymolysis mixed solution; and then removing the solution in the first detection pool after the detection of the VLDL-containing solution is completed, adding the enzymolysis mixed solution to the surface of the first working electrode in the first detection pool, identifying the RGB blue value after the same time as that when the standard curve is drawn, obtaining a colorimetric signal, and comparing the colorimetric signal with the colorimetric standard curve to obtain the concentration of AChE in the sample to be detected.

In the above-mentioned use, the concentration of VLDL in the VLDL solution in the second detection cell, that is, the concentration of VLDL detected by the device of the present invention using an electrochemiluminescence signal, is preferably in the range of 2 to 100 mg/dL.

Preferably, in the above application, the concentration of AChE in the AChE solution in the first detection pool, that is, the concentration of AChE detected by the device of the present invention using a colorimetric signal, is in the range of 1.25-25U/mL.

Preferably, in the above-mentioned use, the second working electrode needs to be washed in step (1) before the second working electrode is soaked in ethanol containing (3-aminopropyl) trimethoxysilane, and more preferably, the second working electrode is washed one or more times with acetone, ethanol and deionized water.

Preferably, in the above use, the concentration of (3-aminopropyl) trimethoxysilane in the ethanol containing (3-aminopropyl) trimethoxysilane in the step (1) is 5-15%.

Preferably, in the above use, the second working electrode is soaked in ethanol containing (3-aminopropyl) trimethoxysilane for 60-120min in the step (1) to remove the amino group which is not stably connected.

Preferably, in the above-mentioned use, the gas flow used in the drying of the surface of the second working electrode by the gas flow in the step (1) is an inert gas flow, such as a nitrogen gas flow, a carbon dioxide gas flow, or the like.

Preferably, in the above use, the conditions for incubating the second working electrodes immersed in the VLDL solution in step (1) are: the concentration of VLDL in the VLDL solution was 1g/dL and the incubation time was 12-36 h.

Preferably, in the above application, the conditions for blocking the second working electrode in the acrylamide solution in the step (1) are as follows: the concentration of acrylamide in the acrylamide solution is 5-15%, more preferably 10%, and the blocking time is 12-36h, so that the nonspecific adsorption on the ITO surface can be effectively removed.

Preferably, in the above use, the phosphate buffer solution in the step (1) contains 5 to 15mM of acrylamide, 50 to 100mM of N, N' -methylenebisacrylamide and 40 to 100mM of ammonium persulfate, and the phosphate buffer solution has a pH of 7.2 and consists of 0.06mM of potassium dihydrogenphosphate, 0.14mM of dipotassium hydrogenphosphate, 13.5mM of sodium chloride and 0.27mM of potassium chloride. After the VLDL is captured, an electrode is immersed in the mixed solution to achieve a layer of polymer encapsulation on the VLDL surface. The subsequent elution step, i.e.the step of removing intercalated VLDL, is aimed at washing away the VLDL entrapped therein, thereby forming specific recognition sites for VLDL, facilitating subsequent selective recombination.

Preferably, in the above use, the polymerization time in the step (1) is 30 to 60min, so that there is sufficient time for sufficient polymerization.

Preferably, in the above use, the concentration of oxalic acid in the oxalic acid solution in the step (1) is 0.5 to 2M, and the time for putting the second working electrode into oxalic acid is 6 to 18 hours, so that VLDL intercalated in the polymer film can be completely removed.

Preferably, in the above-mentioned use, the time for soaking the second working electrode in the VLDL solution in the step (2) and the step (4) is 10 to 30min, and the soaking time can be selected within a wide range, but the soaking time in the step (2) and the step (4) is the same.

Preferably, in the above use, the composition comprises luminol and H₂O₂Has a pH of 10.5 and consists of 0.01mM potassium dihydrogen phosphate, 0.19mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, wherein the concentration of luminol is 8-15. mu.M, H₂O₂The concentration of (B) is 0.05-2. mu.M.

In the above-mentioned applications, the parameters involved in the drawing of the ECL standard curve of VLDL and in the detection of a VLDL-containing test sample are the same except for the concentration of VLDL in the VLDL solution. The drawing of the colorimetric standard curve of the AChE is the same as the parameters involved in the detection process of the sample to be detected containing the AChE except for the AChE concentration in the AChE solution.

More preferably, the application to the detection of Very Low Density Lipoprotein (VLDL) and acetylcholinesterase (AChE) comprises the following steps:

(1) modification of the second working electrode: first immersing the second working electrode in ethanol containing 10% of (3-aminopropyl) trimethoxysilane for 90min, then immersing the second working electrode in a VLDL solution containing 1g/dL after drying the surface of the second working electrode by nitrogen gas flow, then incubating for 24h, then blocking in an acrylamide solution with an acrylamide concentration of 10%, and polymerizing for 40min in a phosphate buffer containing 10mM acrylamide, 70mM N, N' -methylenebisacrylamide and 60mM ammonium persulfate, and then immersing in a 1M oxalic acid solution for 12h to remove the embedded VLDL in the polymer film;

(2) plotting of ECL standard curve for VLDL: soaking the second working electrode in a series of VLDL standard solutions of different concentrations for 20min, and mixing the solution containing 10 μ M luminol and 1 μ M H₂O₂Adding a carbonic acid buffer solution (pH is 10.5) into a second detection cell, then placing a second working electrode into the second detection cell, adding a phosphoric acid buffer solution (pH is 6.0) containing 0.1M potassium chloride into a first detection cell, then applying a driving voltage to a system by adopting a cyclic voltammetry normal method, and drawing an ECL standard curve according to the linear relation between the collected ECL signal and the concentration of a standard solution;

(3) drawing a colorimetric standard curve of AChE: adding 10 μ L of a series of AChE standard solutions of different concentrations to 10mL of a phosphate buffer (pH 8.0) containing 10 μ g/mL choline oxidase and 100 μ M acetylcholine, respectively, and reacting at room temperature for 20min to obtain an enzymatic mixture; removing the solution in the first detection pool after measuring the ECL signal of one standard solution, adding the enzymolysis mixed solution into the first detection pool, identifying the RGB blue value after 5-20s, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue value and the concentration of the standard solution;

(4) detection of VLDL-containing test sample: the second working electrode after modification was immersed in the VLDL-containing sample solution for 20min, and then 10. mu.M luminol and 1. mu. M H were added₂O₂Adding a carbonic acid buffer solution (pH is 10.5) into a second detection cell, then placing a second working electrode into the second detection cell, adding a phosphoric acid buffer solution (pH is 6.0) containing 0.1M potassium chloride into a first detection cell, performing electrochemical detection by adopting an electrochemical alternating current impedance method to obtain an ECL signal, and comparing the ECL signal with an ECL standard curve for calculation to obtain the concentration of VLDL in a sample to be detected;

(5) detection of AChE-containing test samples: adding a test sample containing AChE into 10mL of a phosphate buffer (pH 8.0) containing 10 μ g/mL choline oxidase and 100 μ M acetylcholine, and reacting at room temperature for 20min to obtain an enzymatic mixture; and then removing the solution in the first detection pool after the detection of the VLDL-containing solution is completed, adding the enzymolysis mixed solution to the surface of the first working electrode in the first detection pool, identifying the RGB blue value after the same time as the standard curve is drawn, obtaining a colorimetric signal, and comparing the colorimetric signal with the colorimetric standard curve to obtain the concentration of AChE in the sample to be detected.

The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can detect two target objects according to different detection principles, and has high sensitivity and stability. The device avoids the compromise of inevitable reaction conditions and the mutual interference among signals constructed by the traditional dual-mode sensing detection device, thereby being capable of accurately measuring the concentration of each target object.

In addition, the device of the invention can also carry out high specificity detection on the hydrogen peroxide and the detection object of the physiological index related to the hydrogen peroxide. Importantly, the detection device can further widen the application range by combining the molecular imprinting technology, and the detection object is not limited by the relevant physiological indexes of the hydrogen peroxide based on the gate effect detection principle.

Compared with the prior art, the invention has the following beneficial effects:

(1) the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode has the advantages of simple structure, no need of complex preparation process, easy assembly, low cost, suitability for outdoor or household use and extremely wide application prospect;

(2) the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode respectively places different sensing modes in different reaction spaces, so that the problems of compromise of inevitable reaction conditions and mutual interference among signals, which are constructed by the traditional dual-mode sensing detection device, are avoided;

(3) the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode is not only suitable for dual-mode detection of a single target object, but also serves for simultaneous detection of two target objects, and has excellent sensitivity and reliability;

(4) the detection device can realize array sensing by parallelly arranging a plurality of first working electrodes and second working electrodes in the reaction tank, obtain a plurality of groups of parallel signals by one-time detection, average the concentration of the detected samples to realize more accurate result, and importantly under the condition of the plurality of first working electrodes and the plurality of second working electrodes, can simultaneously detect a plurality of different samples on the surface of the first working electrode to obtain colorimetric signals so as to simultaneously complete the detection of a plurality of different sample targets;

(5) the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode is easy to operate, professional personnel and complex instruments and equipment are not needed, field and family diagnosis can be realized by combining a small-sized electrochemical workstation and a mobile phone, and therefore the detection device has a particularly good application prospect.

Drawings

FIG. 1 is a schematic structural diagram of one embodiment of the electrochemiluminescence-colorimetric dual-mode sensing device based on the enclosed bipolar electrode according to the present invention;

FIG. 2 is a pictorial photograph of the dual mode sensing apparatus of FIG. 1;

FIG. 3 is a schematic structural diagram of another embodiment of the electrochemiluminescence-colorimetric dual-mode sensing device based on the enclosed bipolar electrode according to the present invention;

FIG. 4 is a pictorial photograph of the dual mode sensing arrangement of FIG. 3;

FIG. 5 is a schematic diagram of the dual mode sensing device of FIG. 2 in performing glucose concentration determination in a glucose solution;

FIG. 6 is an ECL response curve of a series of concentrations of glucose solution measured using the dual-mode sensing device of FIG. 2 according to application example 1;

FIG. 7 is an ECL linear fit of a series of concentrations of glucose solution measured using the dual-mode sensor sensing device of FIG. 2 according to application example 1;

FIG. 8 is a graph showing a linear fit and a colorimetric response signal measured using the dual-mode sensor detection device of FIG. 2 for a series of concentrations of glucose solution according to application example 1;

FIG. 9 is an ECL response curve for a series of concentrations of VLDL solution using the dual mode sensor test device of FIG. 2, according to application example 2;

FIG. 10 is an ECL linear fit of a series of concentrations of VLDL solution measured using the dual mode sensor test device of FIG. 2, using application example 2;

FIG. 11 is a graph showing the colorimetric response signals and linear fit of the dual-mode sensor detection device of FIG. 2 for detecting AChE solutions at a range of concentrations;

FIG. 12 is a graph showing the results of observing the selectivity of the dual mode sensing device of FIG. 2 in the presence of glucose, ascorbic acid, uric acid, urea and dopamine in the device according to application example 3;

FIG. 13 is a graph showing results of colorimetric signals obtained by observing the dual mode sensing device shown in FIG. 2 in the presence of glucose, ascorbic acid, uric acid, urea and dopamine during two time periods of 15s and 2min in the dual mode sensing device according to application example 3;

FIG. 14 is a view showing the application of embodiment 3 to detect 50 μ M H using the dual mode sensing device shown in FIG. 2₂O₂Result chart for observing repeatability of device；

FIG. 15 is a graph showing the application of example 3 to the dual mode sensing device of FIG. 2 for detecting 50 μ M H at the same concentration for 13 consecutive days₂O₂The results are shown in the figure.

Wherein, the technical characteristics that each reference numeral refers to are as follows:

1. a detection cell; 11. a first detection cell; 12. a second detection cell; 2. a detection tank partition plate; 3. a first working electrode; 4. A second working electrode; 41. a reaction zone; 6. an insulating tape.

Detailed Description

In order to make the technical scheme and advantages of the invention clearer, the technical scheme of the invention is more clearly and completely described below with reference to the accompanying drawings and application examples. In the described embodiment, the equipment used is the same as the production equipment used for conventional rock plates, as it is not specifically described.

Device embodiment

Apparatus example 1

Referring to fig. 1 and 2, the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode of the present invention includes a detection cell 1, a detection cell partition plate 2, a first working electrode 3, a second working electrode 4 and a conductor (not shown), wherein the detection cell partition plate 2 divides the detection cell 1 into two unconnected areas, namely, a first detection cell 11 and a second detection cell 12, the first working electrode 3 is located in the first detection cell 11, the second working electrode 4 is located in the second detection cell 12, two ends of the conductor are respectively contacted with the first working electrode 3 and the second working electrode 4, the surface of the first working electrode 3 is modified with prussian blue, the second working electrode 4 is provided with a reaction zone 41, and the non-reaction zone and the surface of the second working electrode 4 are covered with an insulating tape 6.

In this embodiment, the insulating tape 6 may be replaced by other insulating materials, such as insulating paint, or other non-conductive materials. In addition, the surface of the first working electrode 3 modified with prussian blue appears blue as shown in fig. 1 and 2, and is easily distinguished from the first working electrode 3 and the second working electrode 4.

The first working electrode 3 of the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the c-BPE is modified by Prussian blue PB to carry out colorimetric determination, and the second working electrode 4 takes luminol as a signal source to be used for ECL detection. Wherein, while the second working electrode 4 collects the ECL signal, the PB on the first working electrode 3 will be reduced to prussian white PW, which can be used for subsequent colorimetric assays. The subsequent application example 1 and application example 2 both verify the feasibility of the electrochemical luminescence-colorimetric dual-mode sensing detection device based on c-BPE constructed by the invention.

In this embodiment, the detection cell 1 has a length of 5-10cm, a width of 3-7cm, and a height of 2-5 cm. The detection pool is small in size, does not occupy large space, and is easy to carry. However, the size of the detection cell is not particularly limited, and may be designed according to the requirements of a specific application.

The detection cell 1 and the detection cell partition plate 2 are made of glass, but the detection cell 1 and the detection cell partition plate 2 may also be made of one or more of other colorless transparent materials such as Polydimethylsiloxane (PDMS), polycarbonate, cyclic olefin copolymer, polymethyl methacrylate (PMMA), Polystyrene (PS), styrene-methyl methacrylate copolymer (MS), inorganic glass, and the like. This is because the detection cell 1 and the detection cell partition 2 made of a colorless transparent material can prevent the influence of the colors of the detection cell 1 and the detection cell partition 2 on the signal output when conducting a colorimetric test. In addition, the test cell 1 and the test cell separator 2 are made of inorganic glass in consideration of production cost and transparency.

The first working electrode 3 and the second working electrode 4 are made of ITO conductive glass, the color of the ITO glass electrodes is transparent, the change of subsequent colors can be observed conveniently, the color change of the surface of the first electrode is used for providing subsequent colorimetric signals, and therefore the color change can be observed more easily, and the measurement is more accurate. However, the first working electrode 3 and the second working electrode 4 may also be made of other materials such as platinum, platinum alloys, platinized materials, stainless steel, graphite or carbon, gold, or polymers.

Further, the dimensions for the first working electrode 3 and the second working electrode 4 may range from 1 to 3cm in length, from 1 to 3cm in width, and from 0.5 to 1.5mm in thickness; the square resistance is 8-15ohm/sq, and in the embodiment, the dimensions of the first working electrode 3 and the second working electrode 4 are 2cm in length, 2cm in width, 1.1mm in thickness and 12ohm/sq in square resistance. However, the dimensions of the first working electrode 3 and the second working electrode 4 are not particularly limited, and may be set according to the user's request, as long as they can be placed in the first detection cell 11 and the second detection cell 12, respectively.

In addition, regarding the prussian blue modified on the surface of the first working electrode 3, the prussian blue may be modified on the surface of the first working electrode 3 by electrodeposition, and of course, the prussian blue may be modified on the surface of the first working electrode 3 by other methods. In this embodiment, the surface of the first working electrode 3 is modified with prussian blue by electrodeposition, where the electrodeposition conditions are as follows: the electrolyte was a 0.1M hydrochloric acid solution containing 5.0mM K₃[Fe(CN)₆]、5.0mM FeCl₃And 0.1M KCl, with an applied potential of 0.4V and a deposition time of 240 s. Under the condition of the electrodeposition, the Prussian blue deposited is relatively uniform in deposition and is very suitable for colorimetric detection. In addition, the electrodeposition conditions may be adjusted to tailor the first working electrode 3, for example, the electrolyte may be a 0.05-0.15M hydrochloric acid solution, which may contain 2-8.0mM K₃[Fe(CN)₆]、2-8.0mM FeCl₃And 0.05-0.2M KCl, the applied potential can be 0.2-6V, and the deposition time can be 120-360 s. The specific electrodeposition conditions can be specifically defined in actual situations or needs.

In the embodiment, the second working electrode 4 is provided with the reaction region 41 having a circular shape with a diameter of 0.6 cm. Of course, the diameter of the reaction region 41 may also be 0.4-1.0cm, and in particular, the shape of the reaction region 41 may also be other shapes, such as a square, the shape and size of the reaction region are not particularly limited, and it is only required that the reaction regions are identical in the related test process of the same substance or the substance to be tested, and the reaction regions correspond to the central position of the detection port of the ECL apparatus.

The conductor is a copper strip, and the size of the conductor is 8cm in length, 1.5cm in width and 0.2mm in thickness. The copper strip is folded in the process of being placed into the detection cell 1, and specifically, positions 2cm, 3.9cm, 4.1cm and 6cm away from one end of the copper strip are selected to be folded so as to be more fit with the size of the detection cell 1. In particular, in order to make the copper strip and the ITO fully fit, 0.8cm is selected as the connection overlapping length of the copper strip and the ITO conductive glass.

In addition, the conductor can also be made of other materials, such as metal (such as silver, aluminum, iron, and the like), alloy (such as copper aluminum alloy), conductive polymer, and the like, and the conductor can also be in other shapes, not limited to a belt shape, such as a circular, triangular, square, and the like cross section, and can be arranged in a practical situation.

The construction process of the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode in the embodiment is as follows:

(1) assembling the detection cell 1: and (3) bonding the glass by using glass cement to obtain the glass detection cell 1. The device comprises a first detection cell 11 and a second detection cell 12 of two reaction cells with the same size, which are formed by physically separating the detection cells by a cell separation plate 2;

(2) modification of the surface of the first working electrode 3: prussian deposition was carried out by electrodeposition on the surface of the first working electrode 3 under the following electrodeposition conditions: the electrolyte was a 0.1M hydrochloric acid solution containing 5.0mM K₃[Fe(CN)₆]、5.0mM FeCl₃And 0.1M KCl, the applied potential is 0.4V, the deposition time is 240s, and the obtained PB/ITO electrode is washed by deionized water and serves as a first working electrode 3;

(3) and (3) setting a second working electrode reaction area: a circular reaction area with the diameter of 0.6cm is arranged on the surface of the second working electrode 4, and the surfaces of other non-reaction areas are covered by an insulating adhesive tape;

(4) construction of a closed bipolar electrode: a conductive copper strip is cut into a conductor with the size of 8cm in length, 1.5cm in width and 0.2mm in thickness, then two ends of the conductor are respectively contacted with a first working electrode 3 and a second working electrode 4, wherein the connection overlapping length of the copper strip and ITO conductive glass is 0.8cm, then the copper strip is wrapped by an insulating tape and fully fixed to enable the copper strip and the ITO to be tightly attached, then positions 2cm, 3.9cm, 4.1cm and 6cm away from one end of the copper strip are selected to be folded and placed in a glass detection pool 1, the first working electrode 3 is located in a first detection pool 11, and the second working electrode 4 is located in a second detection pool 12, so that the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can be obtained.

Apparatus example 2

Referring to fig. 3 and 4, the structure of the enclosed bipolar electrode-based electrochemiluminescence-colorimetric dual-mode sensing detection device of the present embodiment is substantially the same as that of device embodiment 1, except that the number of the first working electrode 3, the second working electrode 4 and the conductor is 3, and accordingly, the size of the detection cell is also increased to enable the placement of the first working electrode 3 and the second working electrode 4. When a plurality of first working electrodes and/or second working electrodes are arranged, a plurality of parallel signals can be output at the same time, and then the stability of the detection device can be evaluated.

The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can be used in the detection fields of medical diagnosis, environmental monitoring, food and the like, and can be used for detecting one target object and two target objects. The detection substances are various, such as glucose, lactic acid, very low density lipoprotein and acetylcholinesterase, and have wide application prospects.

Application examples

Application example 1 glucose was measured using the detection apparatus of example 1

Dissolving a series of 100 μ L glucose standards at different concentrationsThe solutions were added to 1.0mL of a phosphate buffer solution (pH 7.2 consisting of 0.06mM potassium dihydrogenphosphate, 0.14mM dipotassium hydrogenphosphate, 13.5mM sodium chloride and 0.27mM potassium chloride) containing 1mg/mL of glucose oxidase, respectively, and reacted at room temperature for 10min to produce H₂O₂Obtaining enzymolysis mixed liquor; mu.L of a carbonate buffer solution (pH 10.5 consisting of 0.01mM potassium dihydrogenphosphate, 0.19mM dipotassium hydrogenphosphate, 13.5mM sodium chloride, and 0.27mM potassium chloride) containing 10. mu.M luminol was added to the second detection cell, and 10. mu.L of a buffer solution having H formed therein was added to the second detection cell₂O₂The enzymatic mixture of (A) was also added to the second detection cell, 25mL of a phosphate buffer solution (pH 6.0 consisting of 0.18mM potassium dihydrogenphosphate, 0.02mM dipotassium hydrogenphosphate, 13.5mM sodium chloride and 0.27mM potassium chloride) containing 0.1M potassium chloride was added to the first detection cell, a cyclic voltammetric scan was applied to the apparatus using a driving voltage in a range of 0 to 2.5V at a scan rate of 100mV/s, ECL response signals were recorded as shown in FIG. 6, and an ECL standard curve was plotted according to its linear relationship with the concentration of the standard solution as shown in FIG. 7; after each measurement of ECL signal for one standard solution, the solution in the first test cell was removed and 10. mu.L was generated with H₂O₂The enzymolysis mixed solution is added to the surface of the first working electrode in the first detection pool, the RGB blue value is identified after 15s, a colorimetric signal is obtained, and a colorimetric standard curve is drawn by obtaining the linear relation between the RGB blue value and the concentration of the standard solution, and is shown in figure 8.

FIG. 5 is a schematic diagram showing the measurement of the glucose concentration in a glucose solution by the dual-mode sensor detection device of the present embodiment, in which a glucose sample is first incubated in a glucose oxidase to generate H₂O₂For subsequent determination. The treated sample is added to a second detection cell and a cyclic voltammetric scan in the range of 0-2.5V is applied to the platform. Incubating H produced in the sample₂O₂As a co-reactant of the luminol ECL reaction, it will participate in the excitation of luminol at the surface of the second electrode, resulting in an enhanced ECL signal. At the same time, the first electrode surface will be accompanied by a reduction reaction of the PB to PW transition. Subsequently, the electrolyte in the first and second detection cells was poured off, and the same treated glucose incubation solution was added to the first and second detection cellsIn the first reaction cell, H generated from PW on the surface of the first electrode can be observed₂O₂Re-oxidation to PB caused a color change, which was calorimetrically quantified by collecting the RGB blue values of the first electrode surface.

The ECL response of the test device of the present invention to a series of glucose solutions of different concentrations is shown in fig. 6, and it can be seen that the ECL response signal generated by luminol in the second test cell gradually increases with increasing concentration. As shown in fig. 7, the ECL response signal shown in fig. 6 is logarithmic to the concentration of the glucose solution, and thus a linear fit is made between the logarithm of the concentration (lgC) and the ECL signal (I), resulting in a regression equation of I-11822.9 lgC +62172.2, R²Linear range of 10 at 0.998^-5-2×10^-3mol/L。

The colorimetric response of the detection device of the present invention to a series of glucose solutions with different concentrations is shown in fig. 8, and it can be seen that as the concentration increases, the RGB Blue value of PW on the first working electrode gradually decreases, the colorimetric signal and the concentration of the glucose solution form a linear relationship, and a linear fit is performed between the concentration (C) and the RGB Blue value (RGB Blue), and the obtained regression equation is that RGB Blue is-22264.1C +195.4, and R is²Linear range is 5 × 10 ═ 0.992^-4-2×10^-2mol/L。

Each detection mode has its own advantages and disadvantages, and the ECL detection method has the advantage of low detection limit, while the colorimetric method is relatively insensitive, but does not require large-scale equipment. The detection ranges of the two detection methods are different due to different sensitivities, and when the sensitive detection method is used for detecting a high-concentration sample, the sample needs to be repeatedly diluted for many times, so that the detection deviation can be caused. And the insensitive detection method cannot realize the analysis of low-concentration samples. Therefore, the two modes are combined to expand the detection range to a certain extent, and for the area with the overlapped monitoring ranges of the two detection methods, the mutual evidence of the two detection results can be realized.

Application example 2

The detection device of the embodiment 1 of the device is used for monitoring type IV hyperlipoproteinemia, specifically VLDL and AChE are two characteristic physiological indexes for detection.

Modifying MIP on the second working electrode, washing the ITO glass used as the second working electrode by acetone, ethanol and deionized water in sequence for one time, then the second working electrode was immersed in ethanol containing 10% of (3-aminopropyl) trimethoxysilane for 90min, followed by drying the surface of the second working electrode by a nitrogen gas flow, then the second working electrode was incubated in a VLDL solution at 1g/dL for 24h, followed by blocking in an acrylamide solution having an acrylamide concentration of 10% for 24h, and polymerized in a phosphate buffer (pH 7.2 consisting of 0.06mM potassium dihydrogenphosphate, 0.14mM dipotassium hydrogenphosphate, 13.5mM sodium chloride and 0.27mM potassium chloride) containing 10mM acrylamide, 70mM N, N' -methylenebisacrylamide and 60mM ammonium persulfate for 40min, and then immersed in a 1M oxalic acid solution for 12h to remove VLDL intercalated in the polymer film.

Soaking the second working electrode in a series of VLDL standard solutions of different concentrations for 20min, and mixing the solution containing 10 μ M luminol and 1 μ M H₂O₂After adding a carbonic acid buffer solution (pH 10.5, which is composed of 0.01mM potassium dihydrogen phosphate, 0.19mM dipotassium hydrogen phosphate, 13.5mM sodium chloride, and 0.27mM potassium chloride) to the second detection cell, the second working electrode was placed in the second detection cell, after adding a phosphoric acid buffer solution (pH 6.0, which is composed of 0.18mM potassium dihydrogen phosphate, 0.02mM dipotassium hydrogen phosphate, 13.5mM sodium chloride, and 0.27mM potassium chloride) containing 0.1M potassium chloride to the first detection cell, a cyclic voltammetric scan was applied to the device using a driving voltage, the scanning potential range was 0-2.5V, the scanning rate was 100mV/s, an ECL response signal was recorded as shown in fig. 9, and an ECL standard curve was plotted according to its linear relationship with the concentration of the standard solution as shown in fig. 10.

Adding 10 μ L of a series of AChE standard solutions of different concentrations to 10mL of a phosphate buffer (pH 8.0) containing 10 μ g/mL choline oxidase and 100 μ M acetylcholine, respectively, and reacting at room temperature for 20min to obtain an enzymatic mixture; and then removing the solution in the first detection pool after detecting the ECL signal of the VLDL standard solution each time, adding 50 mu L of enzymolysis mixed solution into the first detection pool, identifying the RGB blue value after 10s, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue value and the concentration of the standard solution as shown in figure 11.

In this process, we constructed a "signal-off" ECL assay format for VLDL, where the second working electrode of c-BPE was modified with MIP and the solution in the second assay cell was luminol-H₂O₂A light emitting system. The specific adsorption of VLDL by MIP will result in a strong inhibition of luminol luminescence intensity I, since the re-adsorption of VLDL prevents electron transfer of luminol at the electrode surface. In the first working electrode, AChE can catalyze acetylcholine to generate H in the presence of choline oxidase₂O₂. Therefore, acetylcholinesterase was quantitatively analyzed by a colorimetric method.

The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can provide response signals for two important indexes of IV-type hyperlipoproteinemia, namely VLDL and AChE. The ECL response for a series of VLDL solutions at different concentrations is shown in fig. 9, where it can be seen that the ECL response signal generated by luminol in the second detection cell gradually decreased with increasing concentration, where the ECL response signal was linearly fit as shown in fig. 10, and thus the ECL signal was linearly related to the VLDL concentration, and thus was linearly fit to the ECL signal (I) at a concentration (C) of-125.2C +19724.9, with R being the regression equation²Linear range of 2-100 mg/dL-0.998, which completely covers the physiological levels of AChE in normal human serum.

The colorimetric response of the device of the present invention to AChE series concentrations is shown in fig. 11, and it can be seen that as the concentration increases, the RGB Blue value of the cathode PW gradually decreases, wherein the colorimetric signal and AChE concentration have a linear relationship, so that a linear fit is performed between the concentration (C) and the RGB Blue value (RGB Blue), and the obtained regression equation is that RGB Blue is-2.857C +195.3, and R is²The linear range is 1.25-25U/mL, which is 0.992. The device can completely cover the physiological levels of VLDL (5-40 mg/dL) and AChE (5.4-13.2U/mL) in human serum, can meet the actual detection requirement, and further proves that the device has great potential in the field of medical diagnosis.

In addition, the appearance of the second working electrode during modification of the reaction zone of the second working electrode by the molecularly imprinted polymer was observed by a scanning electrode, and it was found that a large amount of VLDL was uniformly attached to the ITO electrode surface after immersion in VLDL for 24 hours. When the VLDL adsorbed ITO electrode is immersed in the polymerization solution, it is monomer wrapped to form a strong MIP film. From the VLDL removal, visible imprinted voids can be seen at the electrode surface, indicating successful removal of the template molecule.

Application example 3 evaluation of Selectivity, repeatability and stability of the device of the invention

ECL and colorimetry vs H were recorded in the presence of glucose, ascorbic acid AA, uric acid UA, urea and dopamine DA₂O₂To investigate the selectivity of the system. 1 μ M pure H according to ECL assay₂O₂And responses of mixed samples containing the same concentrations of the above interfering substances, respectively, and colorimetry on 50. mu.M pure H₂O₂And the responses of the mixed samples containing the same concentrations of the interfering substances, respectively, are shown in fig. 12. As can be seen from fig. 12, in the presence of an interference-free substance, the difference in the detection results was negligible at a significant level greater than 0.05, confirming its excellent selectivity. It is noteworthy that the colorimetric method was studied selectively in two periods of 15s and 2min, the color change increasing with the increase of the reaction time. The results are shown in FIG. 13. As can be seen from FIG. 13, pure H was obtained regardless of whether the reaction time was 15s or 2min₂O₂And the sample containing the interfering substance caused a uniform shift in the prussian white color, indicating that the reaction time did not affect the selectivity of the system. Therefore, analysis of a sample having a low concentration can be satisfied by appropriately extending the reaction time.

To verify the repeatability of the system, the device of the invention detects 50 mu M H for 9 consecutive times₂O₂It was found that the collected detection signals did not significantly decrease with increasing number of detections, and the relative standard deviations of the ECL and colorimetric detection results were 5.82% and 3.21%, respectively (fig. 14), indicating good reproducibility of the sensor. Subsequently, the test was carried out again for 13 days using the apparatus of the present inventionSame concentration 50 mu M H₂O₂Also, no significant deviation of the signal occurred, and the relative standard deviations of the ECL and colorimetric test results were 3.84% and 8.12%, respectively (fig. 15), demonstrating acceptable long-term stability of the device of the present invention.

Application example 4

To evaluate the feasibility of the device of the invention in the actual sample detection, glucose in the serum of newborn cattle and NADH and H in Hela cells were measured by a standard sample application method₂O₂And (4) content. The same samples were also tested with the quantification kit and the results are shown in table 1.

The glucose determination sample treatment process comprises the following steps: serum samples were centrifuged at 12000rpm for 5min and 1ml of supernatant was mixed with 9ml of PBS.

At H₂O₂And NADH detection, 5X 10⁶Hela cells were administered in an amount of 0.5mL and 0.6ng mL, respectively^-1Treating PMA and NADH extractive solution. The lysate was centrifuged at 12000rpm for 5min, and the final supernatant was collected and stored at 0 ℃. As shown in table 1, the results obtained with the device and kit of the invention are consistent, with the t-test showing negligible differences at the 95% confidence interval. The relative standard deviations of these independent tests were within satisfactory ranges. The result shows that the device has good reliability and effectiveness and can be used for practical application.

TABLE 1

Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. The electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode comprises a detection pool, a detection pool partition plate, a first working electrode, a second working electrode and a conductor, wherein the detection pool is divided into two unconnected areas by the detection pool partition plate, namely the first detection pool and the second detection pool, the first working electrode is positioned in the first detection pool, the second working electrode is positioned in the second detection pool, two ends of the conductor are respectively contacted with the first working electrode and the second working electrode, Prussian blue is modified on the surface of the first working electrode, a reaction area is arranged on the second working electrode, and an insulating material covers the non-reaction area of the second working electrode and the surface of the conductor.

2. Device according to claim 1, characterized in that the detection cells and the detection cell partitions are made of a transparent material, in particular a colorless transparent material.

3. The device of claim 1, wherein the number of first working electrodes and second working electrodes is 1 or more.

4. The device of claim 1, wherein the first and second working electrodes are made of platinum, platinum alloy, platinized material, stainless steel, graphite or carbon, gold, ITO conductive glass, more preferably the first and second working electrodes are ITO conductive glass.

5. The apparatus of claim 1, wherein the conditions for electrodepositing prussian on the surface of the first working electrode are: the electrolyte was a 0.1M hydrochloric acid solution containing 5.0mM K₃[Fe(CN)₆]、5.0mM FeCl₃And 0.1M KCl, with an applied potential of 0.4V and a deposition time of 240 s.

6. A construction method for preparing the closed bipolar electrode-based electrochemiluminescence-colorimetric dual-mode sensing detection device according to any one of claims 1 to 5, comprising the steps of:

7. The method of claim 6, wherein the conductors overlap the first working electrode and the second working electrode by a length of 0.5 cm to 0.9cm, respectively.

8. Use of the closed bipolar electrode based electrochemiluminescence-colorimetric dual-mode sensing detection device according to any one of claims 1 to 5, wherein the use is for detecting glucose (glucose), lactic acid, Very Low Density Lipoprotein (VLDL), acetylcholinesterase (AChE), reduced Nicotinamide Adenine Dinucleotide (NADH), and H₂O₂。

9. Use of the closed bipolar electrode based electrochemiluminescence-colorimetric dual-mode sensing device according to any of claims 1 to 5 for the detection of glucose, characterized in that it comprises the following steps:

(1) drawing an ECL standard curve and a colorimetric standard curve: respectively adding a series of glucose standard solutions with different concentrations into a phosphoric acid buffer solution containing glucose oxidase, and reacting at room temperature to generate H₂O₂Obtaining enzymolysis mixed liquor; adding carbonic acid buffer solution containing luminol into a second detection pool to generate H₂O₂Adding the enzymolysis mixed solution into a second detection tank, adding a phosphate buffer solution containing halide into the first detection tank, applying a driving voltage to a system by adopting a cyclic voltammetry method, and drawing an ECL standard curve according to a linear relation between the collected ECL signal and the concentration of a standard solution; removal of the solution from the first detection cell after each measurement of the ECL signal for one of the standard solutions will result in the formation of H₂O₂Adding the enzymolysis mixed solution to the surface of a first working electrode in a first detection pool, identifying RGB blue values after a period of time, obtaining colorimetric signals, and drawing a colorimetric standard curve by obtaining a linear relation between the RGB blue values and the concentration of a standard solution;

10. Use of the closed bipolar electrode based electrochemiluminescence-colorimetric dual mode sensing detection device for the detection of very low density lipoproteins and acetylcholinesterase according to any of claims 1-5, characterized in that it comprises the following steps:

(2) plotting of ECL standard curve for VLDL: soaking the second working electrode in a series of VLDL standard solutions with different concentrations, and soaking the VLDL standard solutions containing luminol and H₂O₂After the carbonic acid buffer solution is added into a second detection cell, a second working electrode is placed into the second detection cell, then the phosphoric acid buffer solution containing halide is added into the first detection cell, a driving voltage is applied to a system by adopting a cyclic voltammetry method, and an ECL standard curve is drawn according to the linear relation between the collected ECL signal and the concentration of a standard solution;

(4) detection of VLDL-containing test sample: soaking the modified second working electrode in a VLDL-containing sample solution, and adding luminol and H₂O₂Adding the carbonic acid buffer solution into a second detection cell, placing a second working electrode into the second detection cell, adding the phosphoric acid buffer solution containing halide into the first detection cell, applying a driving voltage to a system by adopting a cyclic voltammetry method, detecting to obtain an ECL signal, comparing the ECL signal with an ECL standard curve, and calculating to obtain the concentration of VLDL in the sample to be detected;

(5) detection of AChE-containing test samples: adding a sample to be detected containing AChE into a phosphate buffer solution containing choline oxidase and acetylcholine, and reacting at room temperature to obtain an enzymolysis mixed solution; and then, after removing the solution in the first detection pool after the detection of the VLDL-containing solution is completed, adding the enzymolysis mixed solution to the surface of the first working electrode in the first detection pool, identifying the RGB blue value after the same time as the standard curve is drawn, obtaining a colorimetric signal, and comparing the colorimetric signal with the colorimetric standard curve to obtain the concentration of AChE in the sample to be detected.