CN112485313A - Electrochemical sensor for detecting virus and preparation method thereof - Google Patents
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
本发明提供一种检测病毒的电化学传感器、制备电化学传感器的方法、一种检测病毒的装置及运用上述电化学传感器检测病毒的方法。一种用于检测病毒的电化学传感器，包括：基片，位于所述基片部分表面的工作电极层，位于所述工作电极层表面的电子介体层，位于所述电子介体层背向工作电极层的表面的敏感膜；参比电极层，所述参比电极层位于所述工作电极层的侧部；位于所述参比电极层和所述工作电极层上方的样品承载容器，所述样品承载容器中适于放置待测样品。上述电化学传感器能够快速、准确和灵敏地检测SARS‑CoV‑2。
The present invention provides an electrochemical sensor for detecting viruses, a method for preparing the electrochemical sensor, a device for detecting viruses, and a method for detecting viruses using the electrochemical sensor. An electrochemical sensor for detecting viruses, comprising: a substrate, a working electrode layer on the surface of a part of the substrate, an electron mediator layer on the surface of the working electrode layer, and an electron mediator layer on the back of the electron mediator layer A sensitive film on the surface of the working electrode layer; a reference electrode layer, the reference electrode layer is located on the side of the working electrode layer; a sample carrying container located above the reference electrode layer and the working electrode layer, so The sample carrying container is suitable for placing the sample to be tested. The electrochemical sensor described above enables rapid, accurate and sensitive detection of SARS‑CoV‑2.
Description
Technical Field
The invention relates to the field of microbial detection, in particular to an electrochemical sensor for detecting viruses and a preparation method thereof.
Background
The 2019 coronavirus disease (COVID-19) is a novel infectious disease caused by SARS-CoV-2. SARS-CoV-2 has stronger human transmissibility than other two famous coronaviruses SARS-CoV and MERS-CoV, often causes asymptomatic infection, increases the difficulty of finding new infection, and promotes the spread of virus. In order to detect SARS-CoV-2, various diagnostic methods such as serology, antigen, nucleic acid and the like are developed, which play an important role in resisting COVID-19 pandemics, and the detection method based on real-time PCR (qPCR) has higher sensitivity and specificity and is recognized as the gold standard for SARS-CoV-2 infection confirmation in the global range. The high dependence on precision equipment and the relative time consumption (about 1.5-2h) limit its detection capability to meet the need for rapid detection for screening suspected infections and close contact.
The electrochemical nucleic acid sensor has the advantages of no mark, high sensitivity, easy integration, less analyte amount and the like, and the important characteristic of the electric biosensor is that the electric signal generated in the reaction process of a detected sample can be directly detected without being influenced by light transmission behaviors. Therefore, the electrical method has special application value in the fields of gene sequences, tumor variation, molecular diagnosis, scientific research and the like, and provides another great potential for in vitro diagnosis, physical examination and personalized medical treatment compared with the optical method. As a label-free sensitive platform, nucleic acid sensing based on electrical methods can be conveniently integrated with an extendable channel and a signal sensor. In particular, a planar flexible biosensor having the advantages of high sensitivity, long-term measurement stability, easy integration, small volume and the like, which is particularly combined with an electric sensor and a nano system for label-free detection, can be applied to the sensing field, has attracted extensive attention in the scientific research field in recent years in consideration of the advantages of nanomaterials, such as efficient charge carrier transmission, materials with large specific surface area, small size and low dimension, such as graphene, carbon nanotubes and the like, while a silicon nanowire is generally explored and applied to the fields of energy storage, biological motion, biological sensing and the like through charge-discharge process feedback. These nano materials have the characteristics of certain or all of ultrahigh carrier mobility, high-efficiency electronic conductivity, high internal gain, good biocompatibility and the like, and are widely applied in recent years. Among them, one-dimensional carbon nanotubes (1D-CNTs) are one of star-shaped nanomaterials with excellent carrier transport properties and extremely high ion-electron capture and transition aspect ratios. The carbon nanotube-based bioelectricity sensor can perform low-noise detection on the ion change of the functionalized surface in the reaction system through a signal sensor of the bioelectricity sensor. Thus, carbon nanotube-based electrical sensing technology is very attractive for label-free nucleic acid diagnostics with high sensitivity.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to overcome the defects of long time, low accuracy and low sensitivity of virus detection (such as SARS-CoV-2) in the prior art, thereby providing an electrochemical sensor for virus detection, a method for preparing the electrochemical sensor, a device for virus detection and a method for virus detection by using the electrochemical sensor.
An electrochemical sensor for detecting a virus, comprising: the device comprises a substrate, a working electrode layer positioned on part of the surface of the substrate, an electronic medium layer positioned on the surface of the working electrode layer, and a sensitive film positioned on the surface of the electronic medium layer, which is opposite to the working electrode layer; a reference electrode layer on a side of the working electrode layer; and the sample bearing container is positioned above the reference electrode layer and the working electrode layer, and is suitable for placing a sample to be tested.
Optionally, the sensitive membrane is a polymer membrane containing an ion carrier and an ion exchanger; preferably, the ions in the ionophore and the ion exchanger refer to the same ion; the proportion of the ion carrier and the ion exchanger is (100- & lt 300-): 0.3-1.5, the proportion relation is mg/ml; the material used by the electronic medium layer is carbon nano tube, graphene, nano gold or nano platinum black.
Optionally, the polymer film is a polyvinyl chloride (PVC) film, a Polyurethane (PU) film, a polyvinyl acetate (PVA) film, or a polymethyl methacrylate (PMMA) film; the ionophore is a sodium ionophore, a hydrogen ionophore, a calcium ionophore or a potassium ionophore; the ion exchanger is polystyrene, cellulose, agarose or potassium tetraborate; preferably, the carbon nanotube is a multi-wall carbon nanotube or a single-wall carbon nanotube, and the diameter of the carbon nanotube is 1nm-20 nm.
Optionally, the virus is an RNA virus or a DNA virus; preferably, the virus is an AIDS virus, a hepatitis B virus, a coronavirus or an Ebola virus; more preferably, the coronavirus is SARS-CoV-2.
Optionally, the method further includes: a conductive line layer on the substrate, the conductive line layer being connected to the reference electrode layer; a protective layer on the conductive line layer and exposing the working electrode layer and the reference electrode layer; the sample support container also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
Optionally, the reference electrode layer is in a semi-annular structure, and the working electrode layer is in a circular or elliptical shape; the reference electrode layer surrounds the working electrode layer.
A method for preparing the electrochemical sensor for detecting the virus comprises the following steps: providing a substrate; forming a working electrode layer on a part of the surface of a substrate; forming a reference electrode layer on the surface of the substrate part, wherein the reference electrode layer is positioned at the side part of the working electrode layer; forming an electronic medium layer on the surface of the working electrode layer; forming a sensitive film on the surface of the electronic mediator layer opposite to the carbon working electrode layer; after the sensitive membrane is formed, a sample bearing container is arranged above the reference electrode and the working electrode, and the sample bearing container is suitable for placing a sample to be tested.
Optionally, the method further includes: forming a conductive line layer on the substrate, the conductive line layer being connected to the reference electrode layer; forming a protective layer on the conductive line layer before forming the sensitive film and the electronic dielectric layer, wherein the protective layer exposes the working electrode layer and the reference electrode layer; after the sample support vessel is provided, it also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
An apparatus for detecting a virus, comprising: the above electrochemical sensor; a detection module comprising at least one of a first detection module and a second detection module; the first detection module is suitable for detecting the voltage difference between the reference electrode layer and the working electrode layer; an electrical potential; the second detection module is suitable for detecting the pH value of the sample to be detected.
A method for detecting viruses adopts the device for detecting viruses, and comprises the following steps: adding a sample to be detected into a sample bearing container; and (3) acquiring the change of the voltage difference between the reference electrode layer and the working electrode layer along with time by adopting a first detection module, and/or detecting the change of the pH value of the sample to be detected along with time by adopting a second detection module.
The sample to be detected comprises virus DNA, LAMP primers (4), DNA polymerase, dNTP, reaction buffer solution and indicator;
the sample to be detected comprises RNA of the virus, LAMP primers (4), strand displacement active DNA polymerase, dNTP, reaction buffer solution, reverse transcriptase and indicator;
the indicator is methyl orange, methyl red, litmus or phenolphthalein.
The method is a non-disease diagnostic treatment method.
The technical scheme of the invention has the following advantages:
1. the present invention provides an electrochemical sensor for detecting viruses, comprising: the device comprises a substrate, a working electrode layer positioned on part of the surface of the substrate, an electronic medium layer positioned on the surface of the working electrode layer, and a sensitive film positioned on the surface of the electronic medium layer, which is opposite to the working electrode layer; a reference electrode layer on a side of the working electrode layer; and the sample bearing container is positioned above the reference electrode layer and the working electrode layer, and is suitable for placing a sample to be tested. The sensor contains an ion carrier and an ion exchanger; a continuous conductive layer is formed between the electronic medium layer and the working electrode, and the continuous conductive layer has certain surface roughness so as to facilitate the capture and transmission of charges. Placing a sample to be detected containing virus nucleic acid in a sample bearing container to carry out loop-mediated isothermal amplification reaction, wherein the concentration of hydrogen ions is changed in the reaction process, the hydrogen ions contact a hydrogen ion selective sensitive membrane and are captured by an ion exchanger in the hydrogen ion selective sensitive membrane, then the captured hydrogen ions are transported and transmitted to the surface of an electron mediator layer through an ion carrier and are converted into electrons to form electric potential, and electric potential difference is formed at two ends of a working electrode and a reference electrode to reflect the ion concentration, so that the voltage rise and fall between the two electrodes can be measured by adopting an open circuit voltage method to judge whether the amplification reaction occurs.
The electrochemical biosensor can be used for detecting virus nucleic acid, and has the characteristics of rapidness (about 20 minutes) and sensitivity (the lowest detection limit can reach several copies) and accuracy in detection.
The electrochemical sensor for detecting viruses of the present invention is a small device that has been developed to provide a promising avenue for rapid, accurate and sensitive detection of SARS-CoV-2 and other emerging and emerging pathogens (e.g., ebola), particularly in resource-limited environments.
2. The invention provides a device for detecting viruses, which comprises an electrochemical sensor and a detection module, wherein the detection module comprises at least one of a first detection module and a second detection module; the first detection module is suitable for detecting the voltage difference between the reference electrode layer and the working electrode layer; an electrical potential; the second detection module is suitable for detecting the pH value of the sample to be detected. The first detection module can display a detection curve on line in real time. In addition, after the indicator is added into the sample to be detected, the electrochemical sensor is transparently packaged. The color change of the sample to be measured can be observed by naked eyes.
4. The method for detecting a virus of the present invention has a short time (about 20 minutes) for detecting a virus. Existing methods for detecting pathogen-associated RNA are based on PCR, and use real-time RT-PCR for molecular diagnosis, usually requiring at least 3 hours.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram showing a construction process of an electrochemical nucleic acid sensor in example 1 of the present invention;
FIG. 2 is the test results of example 3 of the present invention;
FIG. 3 is a schematic diagram of an electrochemical nucleic acid sensor in example 1;
1 is a substrate, 2 is a working electrode layer, 3 is a reference electrode layer, 4 is an electrode protection layer, and 5 is an electrode lead layer.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
An electrochemical sensor for detecting a virus, comprising: the device comprises a substrate, a working electrode layer positioned on part of the surface of the substrate, an electronic medium layer positioned on the surface of the working electrode layer, and a sensitive film positioned on the surface of the electronic medium layer, which is opposite to the working electrode layer; a reference electrode layer on a side of the working electrode layer; and the sample bearing container is positioned above the reference electrode layer and the working electrode layer, and is suitable for placing a sample to be tested.
Specifically, the sensitive membrane is a polymer membrane containing an ion carrier and an ion exchanger; specifically, the ions in the ion carrier and the ion exchanger are the same ions; the ratio of ionophore to ion exchanger was 200:1 with the relationship mg/ml ═ L, as an alternative embodiment of the invention, the ratio was (100-: 0.3-1.5, wherein the proportion relation is that mg/ml is L; specifically, the material used for the electronic medium layer is carbon nanotubes, and as an alternative embodiment of the present invention, the material used for the electronic medium layer is graphene, nanogold or nano platinum black;
specifically, the polymer film is a polyvinyl chloride (PVC) film, and as an alternative embodiment of the present invention, the polymer film may also be a Polyurethane (PU) film, a polyvinyl acetate (PVA) film, or a polymethyl methacrylate (PMMA) film.
Specifically, the ionophore is a hydrogen ionophore, and as an alternative embodiment of the present invention, the ionophore may be selected according to the type of the detection sample, and may also be a sodium ionophore, a calcium ionophore, or a potassium ionophore; specifically, the hydrogen ion carrier can be used to obtain a hydrogen ion selective sensitive membrane, and the sodium ion carrier, the calcium ion carrier or the potassium ion carrier can be used to obtain a sodium ion selective sensitive membrane, a calcium ion selective sensitive membrane and a potassium ion selective sensitive membrane.
Specifically, the hydrogen ionophore is tri-n-dodecylamine, specifically, hydrogen ionophore I, and as an alternative embodiment of the present invention, hydrogen ionophore II or hydrogen ionophore V may also be used.
Specifically, the ion exchanger is potassium tetraborate, and as an alternative embodiment of the present invention, the ion exchanger may also be polystyrene, cellulose or agarose.
Specifically, the carbon nanotube is a single-walled carbon nanotube, and as an alternative embodiment of the present invention, the carbon nanotube may also be a multi-walled carbon nanotube; specifically, the diameter of the carbon nanotube is 15 nm; as an alternative embodiment of the present invention, the carbon nanotubes have a diameter of 1nm to 20 nm.
The virus is RNA virus or DNA virus; preferably, the virus is an AIDS virus, a hepatitis B virus, a coronavirus or an Ebola virus; this example is the coronavirus SARS-CoV-2.
Specifically, the electrochemical sensor for detecting viruses further comprises: a conductive line layer on the substrate, the conductive line layer being connected to the reference electrode layer; a protective layer on the conductive line layer and exposing the working electrode layer and the reference electrode layer; the sample support container also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
Specifically, the shape of the reference electrode layer is a semi-annular structure, the shape of the working electrode layer is a circle, and as an alternative embodiment of the present invention, the shape of the working electrode layer is an ellipse.
In particular, the reference electrode layer surrounds the working electrode layer.
Specifically, the substrate material is a PET polyester material, and as an alternative embodiment of the present invention, the substrate material may also be a PVC sheet, a PP sheet, an ABS sheet, a ceramic material, and the like; specifically, the substrate is used for printing other electrode layers;
in particular, the PET polyester material has a thickness of 0.5mm, as an alternative embodiment of the invention the PET polyester material has a thickness of 0.2mm to 0.5mm, in particular 0.3mm or 0.4 mm.
Specifically, the material used for the working electrode layer is conductive carbon ink, and as an alternative embodiment of the present invention, the material used for the working electrode layer may also be gold, silver, conductive graphene ink, or the like.
Specifically, the material used for the reference electrode layer is Ag/AgCl ink, and as an alternative embodiment of the present invention, the material used for the working electrode layer may also be Ag ink.
Specifically, the material used for the conductive line layer is Ag/AgCl ink, and as an alternative embodiment of the present invention, the material used for the conductive line layer may also be Ag ink.
Specifically, the material used for the protective layer is insulating ink.
The manufacturing process of the electrochemical sensor comprises the following steps: providing a substrate; forming a working electrode layer on a part of the surface of a substrate; forming a reference electrode layer on a surface of the substrate portion, the reference electrode layer being on a side portion of the working electrode layer; forming an electronic medium layer on the surface of the working electrode layer; forming a sensitive film on the surface of the electronic mediator layer opposite to the carbon working electrode layer; after the sensitive membrane is formed, a sample bearing container is arranged above the reference electrode and the working electrode, and the sample bearing container is suitable for placing a sample to be tested.
The manufacturing process of the electrochemical sensor further comprises the following steps: forming a conductive line layer on the substrate, wherein the conductive line layer is connected with the reference electrode layer; forming a protective layer on the conductive line layer before forming the sensitive film and the electronic dielectric layer, the protective layer exposing the working electrode layer and the reference electrode layer; after the sample support vessel is provided, it also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
The process of forming the working electrode layer, the reference electrode layer, the protective layer and the electrode lead layer is as follows:
(1) the surface of the substrate material is thoroughly cleaned with alcohol to remove contaminants from the surface of the material, followed by cleaning with ultrapure water (and ultrasonic cleaning in ultrapure water to remove residues, if necessary).
(2) Printing carbon paste ink by adopting a screen printing mode, drying for 40min at 130 ℃, and forming a working electrode layer with the film thickness of 20 mu m on the substrate, wherein the diameter of the working electrode layer can be changed according to the amount of a reaction system, and the working electrode layer is two circular working electrodes with the diameter of 4mm in the example. The working electrode layer is a reaction area for reagent modification (carbon nano tube and sensitive film modification) and sample amplification. In other embodiments, the working electrode layer and the thickness and diameter may be selected from other values without limitation.
(3) Printing Ag/AgCl ink on the basis of the step (2), drying for 20min (optional 10min-20min) at 120 ℃ (optional 90-120 ℃), and then forming a reference electrode layer and an electrode lead layer with the film thickness of 15 microns, wherein the reference electrode layer and the working electrode layer form an electrochemical signal testing loop, and the electrode lead layer is used as an electronic conducting lead in the testing loop.
(4) Printing insulating ink on the basis of the step (3), and drying for 1.5-2h at the temperature of 100-120 ℃ to form a protective layer; in each printing process, deionized water is adopted to wash for multiple times in advance to remove surface impurities, and after printing of each layer of printing ink is finished, ultraviolet disinfection is carried out, and the printing ink is stored under a sealed and dry condition.
An electrochemical sensor comprising a working electrode layer, a reference electrode layer, an electrode lead layer and a protective layer is manufactured through (1) (2) (3) (4), and a schematic diagram of the sensor is shown in fig. 3, wherein 1 is a substrate, 2 is the working electrode layer, 3 is the reference electrode layer, 4 is the electrode protective layer, and 5 is the electrode lead layer.
The process of forming the electron mediator layer and the sensitive film is as follows:
1. in this embodiment, the material of the electronic dielectric layer is SWCNTs, which is used to realize fast transmission of electronic signals.
The carbon nano tube modification method can adopt a chemical adsorption method, a composite reagent method, a direct coating method, an electrochemical polymerization method and the like, the example adopts the direct coating method, 5 mu L of single-wall carbon nano tube dispersion liquid (wherein the concentration of the single-wall carbon nano tube is 10 wt%) is uniformly coated on the surface of the working electrode, the working electrode is completely covered, and then a carbon nano tube modification layer is formed on the surface of the working electrode as an electronic medium layer after natural drying;
2. and forming a sensitive film on the surface of the electron mediator layer, which is opposite to the carbon working electrode layer. Specifically, the carbon nanotube modification layer serves as an electronic medium layer, and accordingly, a sensitive film is formed on the surface of the carbon nanotube modification layer. The method for modifying the sensitive film on the carbon nano tube modification layer comprises the following steps:
A. preparing a polyvinyl chloride (PVC) membrane solution, specifically, preparing a PVC solution (optionally 10 wt% -30 wt%) with polyvinyl chloride concentration of 20 wt% by using cyclohexanone as a solvent and polyvinyl chloride as a solute.
B. Uniformly mixing a PVC solution, an ion carrier and an ion exchanger to prepare a mixed solution, specifically, using the PVC solution as a solvent, using the hydrogen ion carrier and the ion exchanger as solutes, wherein the concentration of the ion exchanger in the mixed solution is 1.5mg/mL (optionally 1-3 mg/mL); a hydrogen ionophore concentration of 1% (v/v) (optionally 0.3% to 1.5% (v/v)); the hydrogen ion carrier in this embodiment is tri-n-dodecylamine, and specifically, a hydrogen ion carrier i, a hydrogen ion carrier ii, or a hydrogen ion carrier v may be used, and this embodiment is the hydrogen ion carrier i.
C. In the embodiment, the PVC solution, the ionophore and the ion exchanger are uniformly mixed to prepare a mixed solution of 2 μ L (optionally 0.5-2 μ L), a drop coating method (optionally a spin coating method or a screen printing method) is adopted to cover the mixed solution on the surface of the carbon nanotube modified layer, and the mixed solution is dried at 37 ℃ to obtain the sensitive membrane, wherein the thickness of the finally formed sensitive membrane is 10 μm-30 μm.
3. The sample loader (also called sample bearing container) can be used as a micro-upgrading sample bearing container and a sample reaction observation window, the sample loader is made of transparent materials such as PMMA materials, quartz glass, PE materials or PDMS materials, the quartz glass materials are adopted in the embodiment, the sample loading pool can be fixed above the reference electrode and the working electrode by means of adhesives, thermocompression bonding and the like, the embodiment is fixed above the reference electrode and the working electrode by means of adhesives, the bottom of the quartz glass is uniformly coated with the adhesives to form an adhesive layer, then the adhesive layer is placed around the working electrode of the sensor, so that the sample loading area covers the working electrode and the reference electrode, the sample loader can be fixedly packaged above the reference electrode and the working electrode after the adhesive layer is dried at 50-70 ℃, and the formed electrochemical nucleic acid sensor is as shown in step 4 in figure 1, the sample loader is integrally a cube, 20mm long and 10mm wide, and is provided with two sample loading pools, the aperture of each sample loading pool is 4-6mm (6 mm in the embodiment), the depth of each sample loading pool is 2-4mm (4 mm in the embodiment), and each sample loading pool can accommodate 10-40 μ L (25 μ L in the embodiment) of samples to be tested. The electrochemical nucleic acid sensor is stored under a closed drying condition, the electrochemical nucleic acid sensor is washed by deionized water for multiple times before use, the electrochemical nucleic acid sensor is dried and then is subjected to ultraviolet disinfection, a sample to be detected needs to be added into a sample loading pool by using a liquid transfer device under an aseptic condition, then the sample loading pool is subjected to aseptic sealing, the sample loading pool can be sealed by adopting a biological-grade self-adhesive sealing film, the upper surface of a sample loader is completely adhered and covered, a reagent system in the sample loading pool is ensured to avoid biological pollution, and evaporation leakage pollution caused by temperature rise of the sample is avoided during an amplification experiment.
In summary, the preparation and use process of the sensor mainly comprises 6 key preparation processes: the method comprises the steps of screen printing of a working electrode layer, a reference electrode layer, a protective layer and an electrode lead layer, forming (or modifying) of an electronic medium layer (a carbon nano tube layer), forming (or modifying) of a sensitive film, packaging of a sample loader, loading of a sample and sealing of a sensor. The designed electric biosensor can be produced in batch by methods such as screen printing, modification, packaging and the like, and has low cost and small difference. The prepared electrochemical nucleic acid sensor can be used for electrically monitoring and recording the progress of nucleic acid reaction by an electrochemical method, and can also be used for judging and processing the chromaticity change of a sample before and after the reaction by a visual observation method.
The electrochemical nucleic acid detecting sensor must form a continuous conductive layer between the electronic medium layer (such as the carbon nanotube modified layer) and the working electrode, and have a certain surface roughness for capturing and transmitting charges. In order to achieve efficient reception and transmission of signals, a dense structure with a certain surface roughness must be established to reduce the debye screening effect. Meanwhile, in order to avoid the introduction of potential ions or contaminants to affect the signal detection, in addition to removing the residual solvent, a pretreatment such as a deionized water rinse or an ultraviolet sterilization, etc. is required for the sensor after each step of the manufacturing and application process of the biosensor. FIG. 1 shows a schematic diagram of the construction and functional modification process of an electrochemical sensor for SARS-CoV-2 detection, and demonstrates the sample encapsulation reaction process. During the nucleic acid reaction, the electrochemical technology can record the change process of the amplification reaction in the nucleic acid sensor in real time.
The working principle of the sensor is as follows: after a reaction system (for example, including a SARS-CoV-2RNA sample, 4 LAMP primers, a strand displacement active DNA polymerase, dNTPs, a reaction buffer solution, a reverse transcriptase and methyl red) including a virus sample is added into a sample loading pool, an amplification reaction (such as a loop-mediated isothermal amplification reaction) is carried out at 65 ℃, the concentration of hydrogen ions is changed in the reaction process, the hydrogen ions contact with a hydrogen ion selective sensitive membrane and are captured by an ion exchanger in the hydrogen ions, then the captured hydrogen ions are transported and transmitted to the surface of an electronic mediator layer through an ion carrier and are converted into electrons to form a potential, a potential difference is formed between two ends of a working electrode and a reference electrode to reflect the ion concentration, and thus an open-circuit voltage method can be adopted to measure the voltage rise and fall between the two electrodes to judge whether the amplification reaction occurs or not.
Example 2
An apparatus for detecting a virus, comprising: the above electrochemical sensor; a detection module comprising at least one of a first detection module and a second detection module; the first detection module is suitable for detecting the voltage difference between the reference electrode layer and the working electrode layer; an electrical potential; the second detection module is suitable for detecting the pH value of the sample to be detected.
Example 3 detection Using the electrochemical sensor obtained in example 1
A method for detecting viruses adopts the device for detecting viruses, and comprises the following steps: adding a sample to be detected into a sample bearing container; and (3) acquiring the change of the voltage difference between the reference electrode layer and the working electrode layer along with time by adopting a first detection module, and/or detecting the change of the pH value of the sample to be detected along with time by adopting a second detection module.
Preparation of positive and negative sample reaction systems: the positive sample reaction system comprises SARS-CoV-2RNA, 4 LAMP primers, strand displacement active DNA polymerase, dNTP, reaction buffer solution, reverse transcriptase and indicator, wherein the indicator is methyl red in the embodiment; as an alternative embodiment of the present invention, the indicator may also be methyl orange, methyl red, litmus or phenolphthalein;
the negative sample reaction system is to replace SARS-CoV-2RNA in the positive sample reaction system with the de-RNA ddH2O。
After a 20 mu L negative sample reaction system and a 20 mu L positive sample reaction system are respectively sealed in two sample loading pools of the nucleic acid sensor, amplification reaction is carried out at 65 ℃, and voltage changes along with the reaction in the amplification process, so that the real-time voltage change of the amplification reaction system is recorded by using an electrochemical open-circuit voltage method, and the amplification reaction process is recorded. As shown in FIG. 2, the negative curve begins to show a significant rising peak after 3000s, that is, the negative amplification reaction begins to proceed after 3000s, while the positive curve begins to show a significant rising peak after about 1200s, which means that the sample amplification reaction has proceeded about 1200s, and a significant curve difference can be observed by comparing the negative results, which indicates that the specific RNA amplification product shows reactivity in the presence of a real-time curve, indicating that the designed biosensor can be used for real-time detection of SARS-CoV-2 RNA. Meanwhile, the chromaticity of the reagent system of the positive sample and the chromaticity of the negative sample are consistent in the initial amplification stage, the obvious chromaticity change appears after the positive amplification reaction, the comparison with the chromaticity of the negative sample in the same time period is obvious, the negative sample is changed into purple red transparent from yellowish transparent initially after 3000s, the color change can be clearly seen by naked eyes, and therefore, the colorimetric method can also be used for judging the negative and positive of the reaction system. Therefore, a novel method for detecting SARS-CoV-2rna is constructed based on a label-free nucleic acid biosensor by using a real-time electrochemical monitoring and colorimetry double-standard method.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. An electrochemical sensor for detecting a virus, comprising: the device comprises a substrate, a working electrode layer positioned on part of the surface of the substrate, an electronic medium layer positioned on the surface of the working electrode layer, and a sensitive film positioned on the surface of the electronic medium layer, which is opposite to the working electrode layer; a reference electrode layer on a side of the working electrode layer; and the sample bearing container is positioned above the reference electrode layer and the working electrode layer, and is suitable for placing a sample to be tested.
2. The electrochemical sensor for detecting viruses according to claim 1, wherein the sensitive membrane is a polymer membrane containing an ion carrier and an ion exchanger; preferably, the ions in the ionophore and the ion exchanger refer to the same ion; the proportion of the ion carrier and the ion exchanger is (100- & lt 300-): 0.3-1.5, the proportion relation is mg/ml; the material used by the electronic medium layer is carbon nano tube, graphene, nano gold or nano platinum black.
3. The electrochemical sensor for detecting viruses according to claim 2, wherein the polymer film is a polyvinyl chloride (PVC) film, a Polyurethane (PU) film, a polyvinyl acetate (PVA) film, or a polymethyl methacrylate (PMMA) film; the ionophore is a sodium ionophore, a hydrogen ionophore, a calcium ionophore or a potassium ionophore; the ion exchanger is polystyrene, cellulose, agarose or potassium tetraborate; preferably, the carbon nanotube is a multi-wall carbon nanotube or a single-wall carbon nanotube, and the diameter of the carbon nanotube is 1nm-20 nm.
4. The electrochemical sensor for detecting a virus according to claim 1, wherein the virus is an RNA virus or a DNA virus; preferably, the virus is an AIDS virus, a hepatitis B virus, a coronavirus or an Ebola virus; more preferably, the coronavirus is SARS-CoV-2.
5. The electrochemical sensor for detecting viruses according to claim 1, further comprising: a conductive line layer on the substrate, the conductive line layer being connected to the reference electrode layer; a protective layer on the conductive line layer and exposing the working electrode layer and the reference electrode layer; the sample support container also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
6. The electrochemical sensor for detecting viruses according to claim 1, wherein the reference electrode layer has a semi-annular structure and the working electrode layer has a circular or elliptical shape; the reference electrode layer surrounds the working electrode layer.
7. A method of preparing an electrochemical sensor for detecting a virus according to any one of claims 1 to 6, comprising the steps of: providing a substrate; forming a working electrode layer on a part of the surface of a substrate; forming a reference electrode layer on the surface of the substrate part, wherein the reference electrode layer is positioned at the side part of the working electrode layer; forming an electronic medium layer on the surface of the working electrode layer; forming a sensitive film on the surface of the electronic mediator layer opposite to the carbon working electrode layer; after the sensitive membrane is formed, a sample bearing container is arranged above the reference electrode and the working electrode, and the sample bearing container is suitable for placing a sample to be tested.
8. The method of preparing an electrochemical sensor for detecting viruses according to claim 7, further comprising: forming a conductive line layer on the substrate, wherein the conductive line layer is connected with the reference electrode layer; forming a protective layer on the conductive line layer before forming the sensitive film and the electronic dielectric layer, wherein the protective layer exposes the working electrode layer and the reference electrode layer; after the sample support vessel is provided, it also extends over a portion of the protective layer surrounding the working electrode layer and the reference electrode layer.
9. An apparatus for detecting a virus, comprising: an electrochemical sensor according to any one of claims 1 to 6; a detection module comprising at least one of a first detection module and a second detection module; the first detection module is suitable for detecting the voltage difference between the reference electrode layer and the working electrode layer; an electrical potential; the second detection module is suitable for detecting the pH value of the sample to be detected.
10. A method for detecting a virus using the apparatus for detecting a virus according to claim 8, comprising: adding a sample to be detected into a sample bearing container; and (3) acquiring the change of the voltage difference between the reference electrode layer and the working electrode layer along with time by adopting a first detection module, and/or detecting the change of the pH value of the sample to be detected along with time by adopting a second detection module.
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