CN113866235B - Electrochemiluminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrode and construction method and application thereof - Google Patents

Electrochemiluminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrode and construction method and application thereof Download PDF

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CN113866235B
CN113866235B CN202110936924.2A CN202110936924A CN113866235B CN 113866235 B CN113866235 B CN 113866235B CN 202110936924 A CN202110936924 A CN 202110936924A CN 113866235 B CN113866235 B CN 113866235B
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李迎春
胡悦
朱亮
梅学翠
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Shenzhen Graduate School Harbin Institute of Technology
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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, and can be used for detecting one target object or two target objects simultaneously, so that the device has a wide market application prospect.

Description

Electrochemiluminescence-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 increasing detection demands, there has been a great deal of attention to constructing a new type of sensing device by combining different sensing technologies together. Currently, various detection methods such as electrochemical-colorimetry, electrochemical-Electrochemiluminescence (ECL), electrochemical-photoelectrochemistry (electrochemical-photoelectrochemistry) and fluorescence-colorimetry are integrated. Such dual mode sensing devices not only retain the unique advantages of each mode sensing device, but generally result in a wider dynamic response range. The dual mode sensing detection device is more complementary, versatile and reliable than the single mode sensing detection device.
However, most of the current dual-mode sensing detection devices are constructed by mixing all reagents in a reaction system, so that a simple hybrid system is formed, and the design of the system lacks novelty and flexibility. In addition, due to the mashup of the reaction environment, the detection system inevitably faces the compromise of working conditions among different detection methods, and further influences the sensing performance of the respective detection modes, so that the sensitivity and the accuracy of detection are adversely affected. In addition, the dual mode signal output by such a detection system is subject to interference between two different detection schemes. Therefore, how to construct a better dual-mode sensing detection device becomes a research direction for analysis and detection technicians.
CN 112540073A teaches a method for preparing a dual output mode sensor based on Fc-apt amplified electrochemiluminescence signal and its application, by first preparing self-enhancing electrochemiluminescence material SiO 2 @Ru-NGQDs, spherical SiO with Nafion film 2 The @ Ru-NGQDs material is fixed on the surface of the clean glassy carbon electrode; then assembling AuNPs on the surface of the modified glassy carbon electrode, and fixing the complementary strand of the aptamer through Au-S covalent interaction; introducing an aptamer marked by ferrocene, and assembling by base complementation to obtain an electrochemical signal of ferrocene and an enhanced electrochemical luminescence signal; finally, a novel dual-mode electrochemical-electrochemiluminescence biosensor is obtained. Although the sensor can realize sensitive and rapid analysis on actual samples, the sensor is complex to prepare, has high cost and is not beneficial to large-scale use.
CN 110426438A teaches a wireless photoelectrochemical analysis detection device based on a closed bipolar electrode and a method for manufacturing the same, wherein a first control electrode is positioned in a first electrolyte in a first electrolytic cell, a second control electrode is positioned 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 positioned in the first electrolytic cell and the second electrolytic cell, and the first electrolytic cell and the second electrolytic cell are connected through the conductor; the light source is fixed above the photoelectric pole; 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, and performs potential control on the photoelectrodes. Although the device realizes photoelectrochemical and biological sensing which are not directly connected through an external wire for the first time, the device can only measure a single analysis target in a targeted way.
In summary, how to rapidly and sensitively realize the dual-mode detection of the target object becomes a research focus in the technical field of analysis.
Disclosure of Invention
In view of the above problems, the invention provides a novel electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode for an existing dual-mode sensing detection device, which is simple, low in cost and strong in plasticity, and can be used for detecting one target or two targets at the same time, 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 device based on a closed bipolar electrode, which comprises a detection cell, a detection cell separator, a first working electrode, a second working electrode and a conductor, wherein the detection cell separator divides the detection cell into two non-communicated areas, namely, a first detection cell and a second detection cell, the first working electrode is positioned in the first detection cell, the second working electrode is positioned in the second detection cell, 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 insulating materials are coated on the non-reaction area of the second working electrode and the surface of the conductor.
Unlike conventional electrodes, the closed bipolar electrode (closed bipolar electrode, c-BPE) has physically separate anode and cathode compartments, wherein oxidation and reduction reactions occur separately and simultaneously in the anode and cathode compartments of the c-BPE, which simplifies the complexity of the analysis device as well as reduces the cost of the equipment. Furthermore, it is worth mentioning that the physically separated anode and cathode compartments are not only suitable for single-target assays, but also for dual-target analysis. Briefly, we find that c-BPE is an ideal candidate for constructing a dual-mode sensor, and the present invention designs an electrochemiluminescence-colorimetric dual-mode sensing detection device based on c-BPE. Wherein, the first working electrode of the c-BPE is modified with Prussian Blue (PB) for colorimetric determination, and the second working electrode is used as ECL detection, which can take ibrinox 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 electrochemiluminescence-colorimetry dual-mode sensing detection device based on the c-BPE, H related to a large number of physiological processes is selected 2 O 2 And H 2 O 2 The relevant analyte served as the model target. Due to H 2 O 2 Is not only a coreactant of luminol, but also a strong oxidant for converting PW into PB, so that the platform can realize H 2 O 2 Dual output detection of ECL and colorimetry. 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 related experiments are also carried out 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-described electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, the detection cell and the detection cell separator are made of a transparent material, in particular a colorless transparent material. The detection cell and the detection cell partition plate made of colorless transparent materials prevent the influence of the detection cell and the detection cell partition plate on signal output when colorimetric tests are carried out.
More preferably, in the above-described electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, the colorless transparent material includes 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 detection cell and the detection cell separator are made of inorganic glass based on the consideration of production cost and transparency.
Preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed 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 arranged, a plurality of parallel signals can be simultaneously output, so that the stability of the detection device can be evaluated, and the parallel signals output simultaneously are averaged in actual testing, which is equivalent to one sensing array. In addition, when the number of the first working electrode and the second working electrode is plural, the number of the conductors is plural, so that the conductors connect the working electrodes.
Preferably, in the 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, platinized 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 electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the first working electrode and the second working electrode have the dimensions of 1-3cm in length, 1-3cm in width and 0.5-1.5mm in thickness; the sheet resistance is 8-15ohm/sq, and most preferably, the dimensions of the first working electrode and the second working electrode are 2cm in length, 2cm in width, 1.1mm in thickness and 12ohm/sq.
Preferably, the above seal-basedIn the electrochemical luminescence-colorimetric dual-mode sensing detection device of the closed bipolar electrode, prussian blue is modified on the surface of the first working electrode in an electrodeposition mode, wherein electrolyte is 0.05-0.15M hydrochloric acid solution, and the hydrochloric acid solution contains 2-8.0mM K 3 [Fe(CN) 6 ]、2-8.0mM FeCl 3 And 0.05-0.2M KCl, with an applied potential of 0.2-0.6V, and a deposition time of 120-360s. Prussian blue deposited by means of electrodeposition is distributed relatively uniformly.
More preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, the condition for electrodepositing prussian blue on the surface of the first working electrode is as follows: the electrolyte is 0.1M hydrochloric acid solution containing 5.0mM K 3 [Fe(CN) 6 ]、5.0mM FeCl 3 And 0.1M KCl, applied potential 0.4V, deposition time 240s. Prussian blue deposited under the electrodeposition condition is relatively uniform in deposition and is very suitable for colorimetric detection.
Preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the reaction area of the second working electrode is circular, and the diameter is 0.4-1.0cm. The reaction area arranged on the second working electrode can be in other shapes, is not strictly limited, and only needs to be consistent in the related test process of the same substance or the object to be tested, and the reaction area is required to correspond to the center position of the detection port of the ECL device.
More preferably, in the above-described electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, the conductor is made of a metal including, but not limited to, a metal such as copper, silver, aluminum, iron, etc., an alloy such as copper-aluminum alloy, etc.
Preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the conductor is a strip, so that the conductor is fully contacted with the working electrode, thereby facilitating smooth transmission of electrons, and facilitating reaction in the whole detection device.
More preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode, the dimension of the strip-shaped conductor is 5-10cm in length, 1-2cm in width and 0.1-0.3mm in thickness. The most dimension is 8cm in length, 1.5cm in width and 0.2mm in thickness. In a strict sense, the dimensions of the conductor are sufficient to meet the requirements of use.
More preferably, in the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the enclosed bipolar electrode, the insulating material is insulating paint or insulating tape, and more preferably, the insulating tape is an insulating tape, because the insulating tape can be detachably coated on the surfaces of the conductor and the second working electrode, and when not in use, the working electrode and the conductor can be discarded and remain, so that the device is easy to disassemble, assemble and clean.
Furthermore, according to a second aspect of the present invention, there is provided a method for constructing the above-mentioned electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, comprising the steps of:
(1) Modification of the surface of the first working electrode: prussian blue is modified on the surface of the first working electrode;
(2) Setting a second working electrode reaction zone: a reaction area is arranged on the surface of the second working electrode, and other non-reaction areas are covered with insulating materials;
(3) Construction of a closed bipolar electrode: the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode is prepared by connecting a first working electrode and a second working electrode through two ends of a conductor, coating an insulating material on the surface of the conductor, placing the first working electrode in a first detection cell, and placing the second working electrode in a second detection cell.
The construction method provided by the invention has the advantages that the sequence of the steps (1) and (2) can be changed, the construction is simple and feasible, no complex process is caused, and the method can be used for analyzing the target object only by simple assembly when the method is used outdoors or in other occasions, thereby providing great possibility for large-scale application of the detection device.
Preferably, in the construction method, in order to make the conductor fully attached to the first working electrode and the second working electrode, thereby facilitating the transmission of electrons, the overlapping lengths of the conductor and the first working electrode and the second working electrode are respectively 0.5 cm to 0.9cm.
Preferably, in the construction method, the construction method further comprises assembling the detection cells, specifically, bonding the side walls and the bottom surface of the detection cells by glue, and then placing the detection cell partition board in the detection cells to divide the detection cells into two areas which are not communicated, namely, a first detection cell and a second detection cell.
Preferably, in the construction method, the specific process of step (3) is as follows: taking a strip conductor, taking a proper amount of insulating tape at two ends of the strip conductor to enable the strip conductor to be connected with a first working electrode and a second working electrode respectively, fully fixing the strip conductor to enable the strip conductor to be tightly attached to the first working electrode and the second working electrode, wrapping the strip conductor by the insulating tape, and then folding the strip conductor along the middle and placing the strip conductor in a detection tank, wherein the first working electrode is positioned in the first detection tank, and the second working electrode is positioned in the second detection tank.
In addition, according to the third aspect of the present invention, there is also provided an application of the above-mentioned electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode, wherein the electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode is applied to 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 detectable substance in the electrochemiluminescence detection end, i.e., the second detection cell, is provided that the concentration of the co-reactant (H 2 O 2 ) And (3) obtaining the product. In other words, H can be monitored 2 O 2 And H 2 O 2 Related analytes of (i.e.H can be formed after a series of reactions 2 O 2 ) The method comprises the steps of carrying out a first treatment on the surface of the Or by modifying specific recognition substances such as MIP, specific antibodies, aptamers, etc. on the surface of the second working electrodeThe substance(s) and the specific recognition substance(s) are combined to influence the electron transmission rate of the electrode surface, so that the change of ECL signals can be detected by the device, and therefore, the detection device can detect a wide range of substances, such as molecules, ions, nucleic acid fragments, proteins and the like. The substance detectable in the colorimetric detection end, i.e., the first detection cell, can be selected from H as long as the substance to be detected has strong oxidizing property (i.e., prussian white can be oxidized into Prussian blue with color conversion) 2 O 2 The substance to be detected further comprises a strong oxidant and a substance which can generate the strong oxidant after a series of reaction processes. Obviously, the detection device has a wider application range and can be widely applied to different fields of medical treatment, environmental monitoring and the like.
More preferably, the application is for the detection of glucose, lactic acid, very low density lipoprotein (very low density lipoprotein, VLDL), acetylcholinesterase (AChE), reduced nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NADH) and H 2 O 2 。
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 reaction condition trade-off 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-described electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode for detecting glucose, comprising the steps of:
(1) Drawing an ECL standard curve and a colorimetric standard curve: adding a series of glucose standard solutions with different concentrations into phosphate buffer solution containing glucose oxidase, and reacting at room temperature to generate H 2 O 2 Obtaining an enzymolysis mixed solution; adding the carbonic acid buffer solution containing luminol into a second detection tank to generate H 2 O 2 The enzymolysis mixed solution of (2)Adding the solution into a second detection tank, adding a phosphate buffer solution containing halide into a first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, and drawing an ECL standard curve according to a linear relation between the collected ECL signal and the concentration of the standard solution; after each ECL signal of one standard solution is measured, the solution in the first detection cell is removed, and H is generated 2 O 2 Adding the enzymolysis mixed solution into 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 the linear relation between the RGB blue values and the concentration of a standard solution;
(2) Detecting a sample to be detected: adding glucose sample solution into phosphate buffer solution containing glucose oxidase, reacting at room temperature to generate H 2 O 2 Obtaining a sample enzymolysis mixed solution; adding the carbonic acid buffer solution containing luminol into a second detection tank to generate H 2 O 2 And (2) adding the sample enzymolysis mixed solution into a second detection tank, adding a phosphate buffer solution containing halide into a first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, detecting to obtain an ECL signal, and comparing the ECL signal with an ECL standard curve to calculate so as to obtain the concentration of glucose in the sample to be detected.
In the test process of the invention, when ECL signals are collected, prussian blue deposited by the first working electrode generates Prussian white due to electrochemical reaction, in other words, ECL signals are generated in the ECL test process, and preparation is provided for subsequent colorimetric determination.
The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode adopts a standard curve for measuring glucose in a dual mode, can examine the sensitivity and stability of the detection device, and is very suitable for field measurement.
Preferably, in the above use, the step (2) further includes measurement of colorimetric signals, specifically: and 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 RGB blue values after the same time as the drawing of the standard curve, 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. And meanwhile, the sensitivity and the stability of the detection device can be compared by obtaining the colorimetric signal and the ECL signal, and the average value of the colorimetric signal and the ECL signal is more accurate.
Preferably, in the above-mentioned use, the concentration of glucose in the glucose solution in the second detection cell, i.e., the concentration of glucose detected by the present device using an electrochemical luminescence signal, ranges from 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 present device using a colorimetric signal ranges from 500. Mu.M to 20mM.
Preferably, in the above use, the conditions for determining ECL signal are: the scanning potential range is 0-2.5V, and the scanning speed is 100mV/s.
Preferably, in the above-mentioned use, the H-formed is obtained during the measurement of the standard curve 2 O 2 The conditions in the enzymolysis mixed solution are as follows: the concentration of 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 at room temperature is 5-15min, and the volume ratio of the glucose standard solution to the phosphate buffer solution containing the glucose oxidase is 1: (8-50).
Preferably, in the above use, the pH of the carbonic acid buffer solution containing luminol is 10.5, which 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.
Preferably, in the above-mentioned uses, the halide includes, but is not limited to, one or more of potassium chloride, sodium chloride, etc., with potassium chloride and/or sodium chloride being more preferred for cost reasons.
Preferably, in the above use, the pH of the phosphate buffer solution containing the halide is 6.0, which consists of 0.18mM potassium dihydrogen phosphate, 0.02mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, wherein the concentration of the halide is 0.05-1.5M.
Preferably, in the above-mentioned use, the parameters involved in the step (2) are the same except for the glucose solution.
More preferably, the specific process of the step (1) is as follows: a series of 100. Mu.L glucose standard solutions with different concentrations are respectively added into 1.0mL phosphate buffer solution (pH 7.2) containing 1.0mg/mL glucose oxidase, and reacted at room temperature for 10min to generate H 2 O 2 Obtaining an enzymolysis mixed solution; after adding 10. Mu.M luminol in carbonic acid buffer solution (pH 10.5) to the second detection cell, 10. Mu.L of the buffer solution was subjected to H formation 2 O 2 Adding the enzymolysis mixed solution into a second detection tank, adding a phosphoric acid buffer solution (pH is 6.0) containing 0.1M potassium chloride into the first detection tank, applying a driving voltage to the system by adopting cyclic voltammetry, 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 ECL signal of one standard solution is measured, the solution in the first detection cell is removed, and H is generated 2 O 2 Adding the enzymolysis mixed solution into a first detection pool, identifying RGB blue values after 15s, obtaining colorimetric signals, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue values and the concentration of the standard solution.
The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode realizes different mode output signals through different detection principles, has higher sensitivity and stability, retains the unique advantages of each mode detection device, and also generates wider dynamic response range. Compared with a single-mode sensing detection device, the detection device has more supplement, diversity and reliability.
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 reaction condition trade-off and mutual interference of different sensing signals in the traditional dual-mode sensing detection device are solved.
In addition, according to a fifth aspect of the present invention, there is also provided the use of the above-described electrochemical luminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode for detecting very low density lipoproteins (very low density lipoprotein, VLDL) and acetylcholinesterase (AChE), comprising the steps of:
(1) Modification of the second working electrode: modifying a molecularly imprinted polymer (molecularly imprinted polymer, MIP) in a reaction zone of a second working electrode, firstly immersing the second working electrode in ethanol containing (3-aminopropyl) trimethoxysilane, subsequently drying the surface of the second working electrode by a gas flow, then immersing the second working electrode in a VLDL solution with a concentration of 0.05-2g/dL, incubating in an acrylamide solution, subsequently blocking, polymerizing in a phosphate buffer containing acrylamide, N' -methylenebisacrylamide and ammonium persulfate, and then immersing in an oxalic acid solution to remove embedded VLDL in the polymer film;
(2) Drawing an ECL standard curve of VLDL: respectively soaking the second working electrode in a series of VLDL standard solutions with different concentrations, and soaking the second working electrode in a solution containing luminol and H 2 O 2 After the carbonic acid buffer solution containing the halide is added into the first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, and drawing an ECL standard curve 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 phosphate 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 each ECL signal of one standard solution is detected, adding the enzymolysis mixed solution into the first detection pool, identifying RGB blue values after a period of time, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue values and the concentration of the standard solution;
(4) ComprisesDetection of VLDL test sample: immersing the modified second working electrode in a sample solution containing VLDL, and immersing the sample solution containing luminol and H 2 O 2 After the carbonic acid buffer solution containing the halide is added into a second detection tank, a second working electrode is placed into the second detection tank, then electrochemical alternating current impedance method is adopted to carry out electrochemical detection to obtain an ECL signal after the phosphoric acid buffer solution containing the halide is added into a first detection tank, and the ECL signal is compared with an ECL standard curve to calculate, so that the concentration of VLDL in a sample to be detected is obtained;
(5) Detection of test samples containing AChE: adding a sample to be tested 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 cell after the detection of the solution containing VLDL is completed, adding the enzymolysis mixed solution to the surface of the first working electrode in the first detection cell, identifying RGB blue values after the same time as the drawing of the standard curve, 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.
Preferably, in the above-mentioned use, the concentration of VLDL in the VLDL solution in the second detection cell, i.e. the concentration of VLDL detected by the device of the present invention using an electrochemiluminescence signal, is in the range of 2-100mg/dL.
Preferably, in the above application, the concentration of AChE in the AChE solution in the first detection cell, that is, the concentration range of AChE detected by the device of the present invention using colorimetric signal is 1.25-25U/mL.
Preferably, in the above use, the conditions for determining ECL signal are: the scanning potential range is 0-2.5V, and the scanning speed is 100mV/s.
Preferably, in the above use, the second working electrode in step (1) is washed, more preferably with acetone, ethanol and deionized water, one or more times before being immersed in the ethanol containing (3-aminopropyl) trimethoxysilane.
Preferably, in the above use, the concentration of (3-aminopropyl) trimethoxysilane in the ethanol containing (3-aminopropyl) trimethoxysilane in the step (1) is 5 to 15%.
Preferably, in the above-mentioned use, the second working electrode is immersed in ethanol containing (3-aminopropyl) trimethoxysilane for 60 to 120min in the step (1) to remove the amino groups not stably attached.
Preferably, in the above application, the gas flow used in the drying process of the surface of the second working electrode in the step (1) by the gas flow 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 electrode in step (1) by immersing it in VLDL solution respectively are: the concentration of VLDL in the VLDL solution was 1g/dL and the incubation time was 12-36h.
Preferably, in the above use, 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 as to effectively remove nonspecific adsorption on the ITO surface.
Preferably, in the above use, the phosphate buffer in the step (1) contains 5 to 15mM acrylamide, 50 to 100mM N, N' -methylenebisacrylamide and 40 to 100mM ammonium persulfate, and the pH of the phosphate buffer solution is 7.2, which consists of 0.06mM potassium dihydrogen phosphate, 0.14mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride. After the VLDL is captured, the electrode is immersed in the mixed solution, thereby achieving a polymer coating on the VLDL surface. The subsequent elution step, i.e. the step of removing the embedded VLDL, is aimed at washing off the VLDL entrapped therein, thereby forming specific recognition sites for VLDL, facilitating subsequent selective recombination.
Preferably, in the above-mentioned use, the polymerization time in the step (1) is 30 to 60 minutes so that there is enough time for the polymerization to be sufficient.
Preferably, in the above-mentioned uses, the halide includes, but is not limited to, one or more of potassium chloride, sodium chloride, etc., with potassium chloride and/or sodium chloride being more preferred for cost reasons.
Preferably, in the above use, the oxalic acid concentration in the oxalic acid solution in the step (1) is 0.5 to 2M, and the time for the second working electrode to enter oxalic acid is 6 to 18 hours, so that the VLDL embedded in the polymer film can be completely removed.
Preferably, in the above use, the time for immersing the second working electrode in the VLDL solution in the step (2) and the step (4) is 10 to 30min, and the immersing time may be selected in a wide range, but the immersing time in the step (2) and the step (4) is the same.
Preferably, in the above use, the composition contains luminol and H 2 O 2 The pH of the carbonic acid buffer solution of (2) is 10.5, which 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, H 2 O 2 The concentration of (2) is 0.05-2. Mu.M.
Preferably, in the above use, the pH of the phosphate buffer solution containing the halide is 6.0, which consists of 0.18mM potassium dihydrogen phosphate, 0.02mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride, wherein the concentration of the halide is 0.05-1.5M.
In the above-mentioned applications, the parameters involved in the drawing of the ECL standard curve of VLDL and the detection of the sample to be tested containing VLDL are the same except for the concentration of VLDL in the VLDL solution. The parameters involved in the drawing of the colorimetric standard curve of the AChE and the detection process of the sample to be detected containing the AChE are the same except for the AChE concentration in the AChE solution.
More preferably, the method is applied to the detection of very low density lipoproteins (very low density lipoprotein, VLDL) and acetylcholinesterase (AChE), and comprises the following steps:
(1) Modification of the second working electrode: the second working electrode was first immersed in ethanol containing 10% of (3-aminopropyl) trimethoxysilane for 90min, then dried by nitrogen flow, immersed in a VLDL solution containing 1g/dL for 24h, then blocked in an acrylamide solution having an acrylamide concentration of 10%, polymerized in a phosphate buffer 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 embedded in the polymer film;
(2) Drawing an ECL standard curve of VLDL: the second working electrode was immersed in a series of VLDL standard solutions of different concentrations for 20min, respectively, at a concentration of 10. Mu.M luminol and 1. Mu. M H 2 O 2 Adding a carbonic acid buffer solution (pH=10.5) into a second detection tank, then placing a second working electrode into the second detection tank, adding a phosphoric acid buffer solution (pH=6.0) containing 0.1M potassium chloride into a first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, and drawing an ECL standard curve 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: a series of AChE standard solutions of different concentrations were added to 10mL of phosphate buffer (ph=8.0) containing 10 μg/mL choline oxidase and 100 μΜ acetylcholine, respectively, and reacted at room temperature for 20min to obtain an enzymatic hydrolysis mixture; removing the solution in the first detection pool after each ECL signal of one standard solution is detected, adding the enzymolysis mixed solution into the first detection pool, identifying RGB blue values after 5-20s, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue values and the concentration of the standard solution;
(4) Detection of VLDL-containing test samples: immersing the modified second working electrode in a sample solution containing VLDL for 20min, and immersing the sample solution containing 10 mu M luminol and 1 mu M H 2 O 2 After adding the carbonic acid buffer solution (pH=10.5) into a second detection cell, placing a second working electrode into the second detection cell, adding a phosphoric acid buffer solution (pH=6.0) containing 0.1M potassium chloride into the 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 to calculate to obtain the concentration of VLDL in a sample to be detected;
(5) Detection of test samples containing AChE: the sample to be tested containing AChE was added to 10mL of phosphate buffer (ph=8.0) containing 10 μg/mL choline oxidase and 100 μm acetylcholine, and reacted at room temperature for 20min to obtain an enzymatic hydrolysis mixture; and then removing the solution in the first detection cell after the detection of the solution containing VLDL is completed, adding the enzymolysis mixed solution to the surface of the first working electrode in the first detection cell, identifying RGB blue values after the same time as the drawing of the standard curve, 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 targets by different detection principles, and has higher sensitivity and stability. The device avoids the inevitable compromise of reaction conditions and the mutual interference between signals, which are 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 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 application range of the detection device can be further widened by combining a molecular imprinting technology, and the detection object is not limited by the hydrogen peroxide related physiological index based on the 'gate effect' detection principle.
Compared with the prior art, the invention has the following beneficial effects:
(1) The electrochemical luminescence-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 electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode respectively places different sensing modes in different reaction spaces, so that the inevitable compromise of reaction conditions and the mutual interference between 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, but also serves for simultaneous detection of two targets, and is excellent in sensitivity and reliability;
(4) The detection device can realize array sensing by parallelly arranging the plurality of first working electrodes and the plurality of second working electrodes in the reaction tank, obtain a plurality of groups of parallel signals through 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, a plurality of different samples can be measured on the surface of the first working electrodes at the same time to obtain colorimetric signals, so that the measurement of a plurality of different sample targets is completed at the same time;
(5) The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode is easy to operate, does not need professional personnel and complex instruments and equipment, can realize field and family diagnosis by combining a small electrochemical workstation and a mobile phone, and has a particularly good application prospect.
Drawings
FIG. 1 is a schematic diagram of one embodiment of an electrochemiluminescence-colorimetry dual-mode sensing detection device based on a closed bipolar electrode of the present invention;
FIG. 2 is a physical photograph of the dual mode sensing device shown in FIG. 1;
FIG. 3 is a schematic diagram of another embodiment of the electrochemiluminescence-colorimetry dual-mode sensing detection apparatus based on a closed bipolar electrode according to the present invention;
FIG. 4 is a physical photograph of the dual mode sensing device of FIG. 3;
FIG. 5 is a schematic diagram of the dual mode sensing detection device of FIG. 2 in performing a glucose concentration determination in a glucose solution;
FIG. 6 is an ECL response curve of a series of concentration glucose solutions detected in a dual mode sensing detection apparatus as shown in FIG. 2 using example 1;
FIG. 7 is a linear fit of ECL's for a series of glucose concentration solutions using the dual mode sensing detection apparatus shown in FIG. 2 using example 1;
FIG. 8 is a graph showing the colorimetric response signals and a linear fit for detecting a series of glucose solutions at a dual mode sensing detection device as shown in FIG. 2 using example 1;
FIG. 9 is an ECL response curve of a series of concentration VLDL solutions detected using the dual-mode sensing detection device shown in FIG. 2 in application example 2;
FIG. 10 is an ECL linear fit of application example 2 to a dual mode sensing detection device of FIG. 2 for detecting a range of concentration VLDL solutions;
FIG. 11 is a graph showing the colorimetric response signals and a linear fit for detecting a series of concentration AChE solutions using the dual mode sensing detection apparatus shown in FIG. 2, applied example 2;
FIG. 12 is a graph showing the results of using the dual mode sensing device of FIG. 2 to observe the device selectivity in the presence of glucose, ascorbic acid, uric acid, urea and dopamine in application example 3;
FIG. 13 is a graph showing the result of colorimetric signals selectively obtained by using the dual-mode sensing device shown in FIG. 2 in application example 3 for two time periods of observation of the device 15s and 2min in the presence of glucose, ascorbic acid, uric acid, urea and dopamine;
FIG. 14 shows a method of detecting 50 μ M H in the dual mode sensor detection device of FIG. 2 according to application example 3 2 O 2 Observing a result graph of device repeatability;
FIG. 15 is a graph showing 50. Mu. M H of the same concentration detected in a dual mode sensor detection device for 13 consecutive days as shown in FIG. 2, which is applied to example 3 2 O 2 Results of (3) are shown.
Wherein, the technical characteristics that each reference sign indicates are as follows:
1. a detection pool; 11. a first detection cell; 12. a second detection cell; 2. detecting a pool 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 present invention more clear, the technical scheme of the present invention will be more clearly and completely described below with reference to the accompanying drawings and application examples. In the examples, the equipment used is the same as the production equipment used for conventional rock plates, unless otherwise specified.
Device embodiment
Device example 1
Referring to fig. 1 and 2, the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode of the present invention comprises a detection cell 1, a detection cell separator 2, a first working electrode 3, a second working electrode 4 and a conductor (not shown), wherein the detection cell separator 2 divides the detection cell 1 into two non-communicated areas, namely a first detection cell 11 and a second detection cell 12, the first working electrode 3 is positioned in the first detection cell 11, the second working electrode 4 is positioned 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, prussian blue is modified on the surface of the first working electrode 3, the second working electrode 4 is provided with a reaction area 41, and the non-reaction area of the second working electrode 4 and the conductor surface 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, and other nonconductive materials. Further, the surface of the first working electrode 3 modified with Prussian blue appears blue as shown in fig. 1 and 2, and the first working electrode 3 and the second working electrode 4 are easily distinguished.
The first working electrode 3 of the electrochemiluminescence-colorimetry dual-mode sensing detection device based on the c-BPE is modified by Prussian blue PB for colorimetry, and the second working electrode 4 is used for ECL detection by taking luminol as a signal source. Wherein, while the second working electrode 4 collects ECL signals, PB on the first working electrode 3 will be reduced to prussian white PW, which can be used for subsequent colorimetric determination. The feasibility of the electrochemiluminescence-colorimetric dual-mode sensing detection device based on the c-BPE constructed by the invention is verified in the following application example 1 and application example 2.
In this embodiment, the size of the detection cell 1 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. However, the size of the detection cell is not particularly limited and may be designed according to the specific application requirements.
The detection cell 1 and the detection cell separator 2 are made of glass, but of course the detection cell 1 and the detection cell separator 2 may 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, etc. This is because the detection cell 1 and the detection cell separator 2 made of colorless transparent materials can prevent the influence of the color of the detection cell 1 and the detection cell separator 2 on the signal output when performing a colorimetric test. In addition, the detection cell 1 and the detection 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 both made of ITO conductive glass, and the color of the ITO glass electrode is transparent, so that the subsequent color change is facilitated to be observed, the color change of the surface of the first electrode is used for providing a subsequent colorimetric signal, and therefore the color change is easier to observe, 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 made of polymers.
Further, regarding the dimensions of the first working electrode 3 and the second working electrode 4, the length may be 1 to 3cm, the width may be 1 to 3cm, and the thickness may be 0.5 to 1.5mm; the sheet resistance is 8-15ohm/sq, in this example 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. 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 requirements of the user 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 surface of the first working electrode 3 may be modified with the prussian blue by electrodeposition, but the surface of the first working electrode 3 may be modified with the prussian blue by other means. In the present embodiment, the surface of the first working electrode 3 is modified by electrodepositionPrussian blue, wherein the electrodeposition conditions are: the electrolyte is 0.1M hydrochloric acid solution containing 5.0mM K 3 [Fe(CN) 6 ]、5.0mM FeCl 3 And 0.1M KCl, applied potential 0.4V, deposition time 240s. Prussian blue deposited under the electrodeposition condition is relatively uniform in deposition and is very suitable for colorimetric detection. In addition, the electrodeposition conditions can be adjusted according to the practical situation so as to customize the first working electrode 3, for example, the electrolyte can be 0.05-0.15M hydrochloric acid solution, and the hydrochloric acid solution can contain 2-8.0mM K 3 [Fe(CN) 6 ]、2-8.0mM FeCl 3 And 0.05-0.2M KCl, the applied potential can be 0.2-6V, and the deposition time can be 120-360s. Specific electrodeposition conditions may be specifically defined in actual cases or as needed.
In an embodiment, the second working electrode 4 is provided with a reaction zone 41 of circular shape, with a diameter of 0.6cm. Of course, the diameter of the reaction area 41 may be 0.4 cm to 1.0cm, and in particular, the shape of the reaction area 41 may be other shapes, such as square, and the shape and size of the reaction area are not particularly limited, and only the same substance or the reaction area in the related test process of the object to be tested is required to be consistent, and the reaction area is required to correspond to the center position of the detection port of the ECL device.
The conductor is a copper strip, and the dimension of the conductor is 8cm in length, 1.5cm in width and 0.2mm in thickness. This is folded during placement in the test cell 1, specifically by selecting the positions 2cm,3.9cm,4.1cm and 6cm from one end of the copper strip to fold to fit the dimensions of the test cell 1. Specifically, in order to make the copper tape and the ITO sufficiently adhere, 0.8cm is selected as the connection overlap length of the copper tape and the ITO conductive glass.
In addition, the conductor may be made of other materials, such as metal (e.g., silver, aluminum, iron, etc.), alloy (e.g., copper-aluminum alloy), conductive polymer, etc., and may be in other shapes, not limited to a strip shape, such as a circular cross section, a triangle, a square, etc., and may be specifically set in practical situations.
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 reaction condition trade-off and mutual interference of different sensing signals in the traditional dual-mode sensing detection device are solved.
The process of constructing 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 bonding glass by adopting glass cement to obtain the glass detection cell 1. The device comprises a first detection cell 11 and a second detection cell 12 which are formed by physically separating a detection cell partition board 2, wherein the first detection cell 11 and the second detection cell are identical in size;
(2) Modification of the surface of the first working electrode 3: deposition of prussian was performed by electrodeposition on the surface of the first working electrode 3, wherein the electrodeposition conditions were: the electrolyte is 0.1M hydrochloric acid solution containing 5.0mM K 3 [Fe(CN) 6 ]、5.0mM FeCl 3 And 0.1M KCl, with an applied potential of 0.4V and a deposition time of 240s, flushing the obtained PB/ITO electrode with deionized water to serve as a first working electrode 3;
(3) Setting a second working electrode reaction zone: a circular reaction zone with the diameter of 0.6cm is arranged on the surface of the second working electrode 4, and the surfaces of other non-reaction zones are covered by insulating adhesive tapes;
(4) Construction of a closed bipolar electrode: cutting a piece of conductive copper strip into conductors with the length of 8cm, the width of 1.5cm and the thickness of 0.2mm, respectively contacting two ends of the conductors 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, wrapping the copper strip by using an insulating tape to fully fix the copper strip and the ITO, folding at the positions which are 2cm,3.9cm,4.1cm and 6cm away from one end of the copper strip, and placing the folded copper strip in a glass detection cell 1, wherein the first working electrode 3 is positioned in a first detection cell 11, and the second working electrode 4 is positioned in a second detection cell 12, so that the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can be obtained.
Device example 2
Referring to fig. 3 and 4, the structure in the electrochemical luminescence-colorimetric dual-mode sensing detection apparatus based on the closed bipolar electrode of the present embodiment is substantially the same as that of the apparatus embodiment 1, except that the number of the first working electrode 3, the second working electrode 4 and the conductors is 3, and accordingly, the size of the detection cell is also increased so that the first working electrode 3 and the second working electrode 4 can be placed. 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, so that the stability of the detection device can be evaluated, and the parallel signals output at the same time are averaged in actual test, which is equivalent to a sensing array.
The electrochemiluminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode can be used in the detection fields of medical diagnosis, environment monitoring, food and the like, and can be used for detecting one target object and two target objects. The detection objects are various, such as glucose, lactic acid, very low density lipoprotein and acetylcholinesterase, and have wide application prospects.
Application examples
Application example 1 measurement of glucose by using the detection device of device example 1
A series of different concentrations of 100. Mu.L glucose standard solution were added to 1.0mL of 1mg/mL glucose oxidase-containing phosphate buffer solution (pH 7.2, which consists of 0.06mM potassium dihydrogen phosphate, 0.14mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride), and reacted at room temperature for 10min to produce H 2 O 2 Obtaining an enzymolysis mixed solution; after adding 10. Mu.L of a carbonic acid buffer solution (pH 10.5, which consists of 0.01mM potassium dihydrogen phosphate, 0.19mM dipotassium hydrogen phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride) containing 10. Mu.M luminol to the second detection cell, 10. Mu.L of the solution was subjected to H-formation 2 O 2 Is also added into a second detection tank, and 25mL of phosphoric acid buffer solution (pH 6.0, which consists 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 is added into the first detection tank, and cyclic voltammetry scanning is applied to the device by using driving voltage The scanning potential range is 0-2.5V, the scanning speed is 100mV/s, the ECL response signal is recorded as shown in figure 6, and an ECL standard curve is drawn according to the linear relation between the ECL response signal and the concentration of the standard solution as shown in figure 7; after each ECL signal of one standard solution was measured, the solution in the first detection cell was removed, and 10. Mu.L was then subjected to H-formation 2 O 2 Adding the enzymolysis mixed solution into the surface of a first working electrode in a first detection pool, identifying RGB blue values after 15s, obtaining colorimetric signals, and drawing a colorimetric standard curve through obtaining the linear relation between the RGB blue values and the concentration of the standard solution, wherein the colorimetric standard curve 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 test device of the present embodiment, wherein first, a glucose sample is incubated with glucose oxidase to generate H 2 O 2 For subsequent determination. The treated sample was added to a second detection cell and cyclic voltammetric scans ranging from 0-2.5V were applied to the platform. Incubating H generated in a sample 2 O 2 As co-reactant for the luminol reaction, the excitation of luminol at the surface of the second electrode will be participated, such that the ECL signal is enhanced. At the same time, the first electrode surface will be accompanied by a reduction of the PB to PW transition. Subsequently, the electrolytes in the first and second detection cells are poured out, and the same treated glucose incubation liquid is added into the first reaction cell, so that H generated by PW on the surface of the first electrode can be observed 2 O 2 Again oxidized to PB, resulting in a color change, colorimetric quantification was performed by collecting 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 increases progressively with increasing concentration. As shown in FIG. 7, the ECL response signal shown in FIG. 6 has a logarithmic relationship with the concentration of the glucose solution, and thus the ECL signal (I) is linearly fitted to the concentration logarithm (lgC), and the regression equation is I=11822.9lgC+62172.2, R 2 =0.998, linear range is 10 -5 -2×10 -3 mol/L。
The invention detects the dressAs shown in FIG. 8, the colorimetric response to a series of glucose solutions of different concentrations is shown as a gradual decrease in the RGB Blue value of PW on the first working electrode with increasing concentration, the colorimetric signal is linearly related to the concentration of glucose solution, and the concentration (C) is linearly fitted to the RGB Blue value (RGB Blue) to give a regression equation of RGB blue= -22264.1C+195.4, R 2 =0.992, linear range is 5×10 -4 -2×10 -2 mol/L。
Each detection mode has its own advantages and disadvantages, ECL detection methods have the advantage of lower detection limits, while colorimetric methods are relatively less sensitive, but have the advantage of not requiring large-scale equipment. The detection ranges of the two detection methods are different due to the different sensitivities, so that repeated dilution work is needed to be carried out on the sample when the high concentration sample is detected by the sensitive detection methods, and measurement deviation can be caused. Whereas the insensitive detection method cannot achieve analysis of low concentration samples. Therefore, the two modes are combined to expand the detection range to a certain extent, and the mutual evidence of the two detection results can be realized for the area where the monitoring ranges of the two detection methods overlap.
Application example 2
The detection device of device example 1 was used to monitor type iv hyperlipoproteinemia, specifically VLDL and AChE as two characteristic physiological indicators.
The MIP was modified at the second working electrode, the ITO glass as the second working electrode was washed once with acetone, ethanol and deionized water in this order, then the second working electrode was immersed in ethanol containing 10% of (3-aminopropyl) trimethoxysilane for 90min, then the surface of the second working electrode was dried by a nitrogen stream, then the second working electrode was immersed in a VLDL solution of 1g/dL for 24h, then the blocking was performed in an acrylamide solution of 10% concentration for 24h, and a phosphate buffer (ph=7.2 consisting of 0.06mM monobasic potassium phosphate, 0.14mM dibasic potassium phosphate, 13.5mM sodium chloride and 0.27mM potassium chloride) containing 10mM acrylamide, n' -methylenebisacrylamide and 60mM ammonium persulfate was polymerized in a 1M oxalic acid solution for 12h to remove the VLDL embedded in the polymer film.
The second working electrode was immersed in a series of VLDL standard solutions of different concentrations for 20min, respectively, at a concentration of 10. Mu.M luminol and 1. Mu. M H 2 O 2 After adding 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, and after adding a phosphate buffer solution (pH=6.0, which consists 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, cyclic voltammetry was applied to the device with a driving voltage in the range of 0 to 2.5V at a scanning rate of 100mV/s, ECL response signals were recorded as shown in FIG. 9, and an ECL standard curve was plotted as shown in FIG. 10 according to its linear relation to the standard solution concentration.
A series of AChE standard solutions of different concentrations were added to 10mL of phosphate buffer (ph=8.0) containing 10 μg/mL choline oxidase and 100 μΜ acetylcholine, respectively, and reacted at room temperature for 20min to obtain an enzymatic hydrolysis mixture; and then removing the solution in the first detection tank after each detection of ECL signals of the VLDL standard solution, adding 50 mu L of enzymolysis mixed solution into the first detection tank, identifying RGB blue values after 10s, obtaining colorimetric signals, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue values and the concentration of the standard solution, wherein the colorimetric standard curve is shown in figure 11.
In this procedure we constructed a "signal-off" ECL test mode for VLDL, wherein the second working electrode of the c-BPE was modified with MIP and the solution in the second test cell was luminol-H 2 O 2 A lighting system. The specific adsorption of the MIP to VLDL will have a strong inhibition effect on the luminous intensity I of the luminol, since the re-adsorption of VLDL prevents the electron transfer of the luminol at the electrode surface. Whereas AChE catalyzes the formation of H from acetylcholine in the presence of choline oxidase at the first working electrode 2 O 2 . The acetylcholinesterase was thus quantitatively analyzed by colorimetry.
The invention relates to an electric based on a closed bipolar electrode The chemiluminescent-colorimetric dual-mode sensing detection device can provide response signals for VLDL and AChE which are two important indicators of type IV hyperlipoproteinemia. As shown in FIG. 9, the ECL response of VLDL solutions of a series of different concentrations can be seen to decrease gradually with increasing concentration, wherein the ECL response signal generated by luminol in the second test cell is linearly fitted to the VLDL concentration as shown in FIG. 10, thus the ECL signal is linearly fitted to the ECL signal (I) at concentration (C), and the regression equation is I= -125.2C+19724.9, R 2 =0.998, the linear range is 2-100 mg/dL, which can completely cover physiological levels of AChE in human normal serum.
The colorimetric response of the device to AChE series concentration is shown in FIG. 11, and it can be seen that the RGB Blue value of the cathode PW gradually decreases with the increase of the concentration, wherein the colorimetric signal and the AChE concentration are in a linear relation, so that the concentration (C) and the RGB Blue value (RGB Blue) are subjected to linear fitting, and the regression equation is RGB blue= -2.857C+195.3, R 2 =0.992, the linear range is 1.25 to 25U/mL. 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 morphology of the second working electrode during the modification of the reaction zone of the second working electrode by the molecularly imprinted polymer was observed by the scanning electrode, and it was found that a large amount of VLDL was uniformly attached to the surface of the ITO electrode after soaking in VLDL for 24 hours. When the VLDL-adsorbed ITO electrode is immersed in the polymerization solution, it is wrapped with a monomer, forming a firm MIP film. After removal of VLDL, visible imprinted voids can be seen on the electrode surface, indicating successful removal of the template molecule.
Application example 3 evaluation of the 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 DA 2 O 2 To investigate the selectivity of the system. 1. Mu.M pure H according to ECL assay 2 O 2 And responses of the mixed sample containing the same concentration of the interfering substances respectively to 50. Mu.M pure H by colorimetry 2 O 2 And the response of the mixed sample containing the same concentrations of the interfering substances, respectively, are shown in fig. 12. As can be seen from fig. 12, in the presence or absence of the interfering substance, the difference in detection results was negligible at a significant level of more than 0.05, confirming its excellent selectivity. Notably, colorimetric methods have been selectively studied for two time periods of 15s and 2min, with color changes being exacerbated as the reaction time is extended. The results are shown in fig. 13. As can be seen from FIG. 13, the reaction time was 15s or 2min, pure H 2 O 2 And samples containing interfering substances all caused uniform conversion of Prussian white color, indicating that the reaction time did not affect the selectivity of the system. Thus, analysis of low concentration samples can be satisfied by appropriately extending the reaction time.
To verify the reproducibility of the system, the device of the invention detects 50. Mu. M H9 consecutive times 2 O 2 The collected detection signals were found not to significantly decrease with increasing number of detections, and the relative standard deviations of ECL and colorimetric detection results were 5.82% and 3.21%, respectively (fig. 14), indicating good reproducibility of the sensor. Subsequently, the same concentration of 50 mu M H was detected for 13 consecutive days by using the device of the present invention 2 O 2 No significant deviation of the same signal occurred, and the relative standard deviation of ECL and colorimetric detection results were 3.84% and 8.12%, respectively (fig. 15), demonstrating that the device of the present invention has acceptable long-term stability.
Application example 4
To evaluate the feasibility of the device in the detection of actual samples, glucose in neonatal bovine serum and NADH and H in HeLa cells were determined using standard sample addition methods 2 O 2 The content is as follows. The same samples were simultaneously assayed using a quantitative kit and the results are shown in table 1.
The glucose determination sample treatment process comprises the following steps: the serum samples were centrifuged at 12000rpm for 5min, and 1ml of the supernatant was mixed with 9ml of PBS.
At H 2 O 2 And NADH detection is performed by 5×10 6 Hela cells were treated with 0.5mL of 0.6ng mL each - 1 PMA and NADH extracts. The resulting lysate was centrifuged at 12000rpm for 5min, and the final supernatant was taken and stored at 0 ℃. As shown in Table 1, the results obtained for the device and kit of the present invention were consistent, with the t-test showing negligible differences at the 95% confidence interval. The relative standard deviations of these independent tests were all within satisfactory limits. 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 would be obvious to persons skilled in the art to which the invention pertains from the foregoing description and teachings. Therefore, the invention is not limited to the specific embodiments disclosed and described above, but some modifications and changes of the invention should be also included in the scope of the claims of the invention. In addition, although specific terms are used in the present specification, these terms are for convenience of description only and do not limit the present invention in any way.

Claims (10)

1. Use of an electrochemiluminescence-colorimetric dual-mode sensing detection device based on a closed bipolar electrode for detecting glucose, characterized in that it comprises the following steps:
(1) Drawing an ECL standard curve and a colorimetric standard curve: adding a series of glucose standard solutions with different concentrations into phosphate buffer solution containing glucose oxidase, and reacting at room temperature to generate H 2 O 2 Obtaining an enzymolysis mixed solution; adding the carbonic acid buffer solution containing luminol into a second detection tank to generate H 2 O 2 The enzymolysis mixed solution is also added into a second detection tank, and then the phosphoric acid buffer solution containing halide is added into a first detection tank, and then a driving voltage is applied to a system by adopting cyclic voltammetry according to the following conditionsDrawing an ECL standard curve according to the linear relation between the collected ECL signals and the standard solution concentration; removing the solution in the first detection cell after each ECL signal of one standard solution is detected, H is generated 2 O 2 Adding the enzymolysis mixed solution into 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 the linear relation between the RGB blue values and the concentration of a standard solution;
(2) Detecting a sample to be detected: adding glucose sample solution into phosphate buffer solution containing glucose oxidase, reacting at room temperature to generate H 2 O 2 Obtaining a sample enzymolysis mixed solution; adding the carbonic acid buffer solution containing luminol into a second detection tank to generate H 2 O 2 Adding the sample enzymolysis mixed solution into a second detection pool, adding a phosphate buffer solution containing halide into a first detection pool, applying a driving voltage to a system by adopting cyclic voltammetry, detecting to obtain an ECL signal, comparing the ECL signal with an ECL standard curve, and calculating to obtain the concentration of glucose in a sample to be detected; after ECL mode detection is completed, removing the solution in the first detection pool, adding another enzymolysis mixture of the same sample to be detected to the surface of the first electrode in the first detection pool, recognizing RGB blue values after a period of time, obtaining colorimetric signals, comparing the colorimetric signals with a colorimetric standard curve for calculation to obtain the glucose concentration in the sample to be detected, so as to realize the dual-mode method for detecting glucose,
the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode comprises a detection tank, a detection tank partition board, a first working electrode, a second working electrode and a conductor, wherein the detection tank partition board divides the detection tank into two areas which are not communicated, namely the first detection tank and the second detection tank, the first working electrode is positioned in the first detection tank, the second working electrode is positioned in the second detection tank, 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 insulating materials are coated on the non-reaction area of the second working electrode and the surface of the conductor.
2. The use according to claim 1, wherein the detection cell and the detection cell separator are made of a colorless transparent material.
3. The use according to claim 1, wherein the number of the first working electrode and the second working electrode is 1 or more.
4. The use according to claim 1, characterized in that the first and second working electrode are made of platinum, platinum alloy, platinized material, stainless steel, carbon, gold, ITO conductive glass.
5. The use according to claim 1, characterized in that the conditions for electrodeposition of prussian blue on the surface of the first working electrode are: the electrolyte is 0.1M hydrochloric acid solution containing 5.0mM K 3 [Fe(CN) 6 ]、5.0mM FeCl 3 And 0.1M KCl, applied potential 0.4V, deposition time 240s.
6. Use of an electrochemiluminescence-colorimetric dual-mode sensing detection device based on closed bipolar electrodes for detecting very low density lipoproteins (very low density lipoprotein, VLDL) and acetylcholinesterase (AChE), characterized in that it comprises the steps of:
(1) Modification of the second working electrode: modifying a molecularly imprinted polymer in a reaction zone of a second working electrode, firstly immersing the second working electrode in ethanol containing (3-aminopropyl) trimethoxysilane, then drying the surface of the second working electrode by airflow, immersing the second working electrode in VLDL solution with the concentration of 0.05-2g/dL for incubation, then sealing in acrylamide solution, polymerizing in phosphoric acid buffer containing acrylamide, N' -methylenebisacrylamide and ammonium persulfate, and immersing in oxalic acid solution to remove embedded VLDL in a polymer film;
(2) Drawing an ECL standard curve of VLDL: respectively soaking the second working electrode in a series of VLDL standard solutions with different concentrations, and soaking the second working electrode in a solution containing luminol and H 2 O 2 After the carbonic acid buffer solution containing the halide is added into the first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, and drawing an ECL standard curve 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 phosphate 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 each ECL signal of one standard solution is detected, adding the enzymolysis mixed solution into the first detection pool, identifying RGB blue values after a period of time, obtaining a colorimetric signal, and drawing a colorimetric standard curve by obtaining the linear relation between the RGB blue values and the concentration of the standard solution;
(4) Detection of VLDL-containing test samples: immersing the modified second working electrode in a sample solution containing VLDL, immersing the modified second working electrode in a sample solution containing luminol and H 2 O 2 Adding a carbonic acid buffer solution containing halide into a first detection tank, applying a driving voltage to a system by adopting cyclic voltammetry, detecting to obtain an ECL signal, comparing the ECL signal with an ECL standard curve, and calculating to obtain the concentration of VLDL in a sample to be detected;
(5) Detection of test samples containing AChE: adding a sample to be tested containing AChE into a phosphate buffer solution containing choline oxidase and acetylcholine, and reacting at room temperature to obtain an enzymolysis mixed solution; then adding the enzymolysis mixed solution to the surface of a first working electrode in the first detection cell after removing the solution in the first detection cell after completing the detection of the solution containing VLDL, identifying RGB blue values after the same time as the standard curve is drawn, obtaining a colorimetric signal, comparing the colorimetric signal with the colorimetric standard curve to obtain the concentration of AChE in a sample to be detected,
the electrochemical luminescence-colorimetric dual-mode sensing detection device based on the closed bipolar electrode comprises a detection tank, a detection tank partition board, a first working electrode, a second working electrode and a conductor, wherein the detection tank partition board divides the detection tank into two areas which are not communicated, namely the first detection tank and the second detection tank, the first working electrode is positioned in the first detection tank, the second working electrode is positioned in the second detection tank, 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 insulating materials are coated on the non-reaction area of the second working electrode and the surface of the conductor.
7. The use according to claim 6, wherein the detection cell and the detection cell separator are made of a colorless transparent material.
8. The use according to claim 6, wherein the number of the first working electrode and the second working electrode is 1 or more.
9. The use according to claim 6, wherein the first and second working electrodes are made of platinum, platinum alloy, platinized material, stainless steel, carbon, gold, ITO conductive glass.
10. The use according to claim 6, wherein the conditions for electrodeposition of prussian blue on the surface of the first working electrode are: the electrolyte is 0.1M hydrochloric acid solution containing 5.0mM K 3 [Fe(CN) 6 ]、5.0mM FeCl 3 And 0.1M KCl, applied potential 0.4V, deposition time 240s.
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