CN113899899A - Virus detection device based on full screen printing OECT and its preparation and detection method - Google Patents
Virus detection device based on full screen printing OECT and its preparation and detection method Download PDFInfo
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Abstract
本发明公开了一种基于全丝网印刷OECT(organic electrochemical transistor)的病毒检测装置及其制备和检测方法，制备方法包括通过丝网印刷技术在沟道层周围的源极和漏极表面涂覆光固化涂料以形成绝缘层，得到基底；通过巯基烷酸类分子栅极表面自组装原理固定抗体，得到固定有目标抗体的栅极；将管段底部封闭固定于基底上表面，并形成用于承装电解液的检测液槽；栅极下部伸入检测液槽内部并能与电解液接触，组装后得到基于全丝网印刷OECT的病毒特异性检测装置。本发明采用的OECT具有较强的信号放大功能，在流感病毒检测中可以将微小的抗原浓度变化转化成较大的沟道电流变化，在低栅极电压下即体现出非常高的灵敏度与极低的检测极限(10‑9M)，在流感病毒检测领域有很广阔的应用前景。
The invention discloses a virus detection device based on full screen printing OECT (organic electrochemical transistor) and a preparation and detection method thereof. The preparation method includes coating the source and drain surfaces around a channel layer by screen printing technology. Photo-curing the coating to form an insulating layer to obtain a substrate; immobilize the antibody through the principle of surface self-assembly of the mercaptoalkanoic acid molecular grid to obtain a grid immobilized with the target antibody; seal the bottom of the pipe section and fix it on the upper surface of the substrate, and form a surface for supporting the target antibody. The detection liquid tank containing the electrolyte; the lower part of the grid extends into the detection liquid tank and can be in contact with the electrolyte. After assembly, a virus-specific detection device based on full screen printing OECT is obtained. The OECT used in the present invention has a strong signal amplification function, and can convert a small antigen concentration change into a large channel current change in the detection of influenza virus, and shows very high sensitivity and extremely high sensitivity under low gate voltage. The low detection limit (10 ‑9 M) has broad application prospects in the field of influenza virus detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a virus detection device based on full-screen printing OECT and a preparation method and a detection method thereof.
Background
Influenza is a common acute respiratory infectious disease caused by influenza virus infection, and has the characteristics of high transmission speed, strong transmission capacity and various types. The current influenza virus detection methods mainly include molecular diagnostic techniques (PCR) based on polymerase chain reaction and immunodiagnostic techniques for detecting antigens or antibodies in patients. The molecular diagnosis and detection usually needs professional technicians and laboratories, has high detection cost and low speed, and is difficult to meet the requirements of mass detection and field detection. In the past, influenza virus infection is mainly detected by methods such as serum diagnosis, cell culture, chick embryo culture virus isolation, polymerase chain reaction-based molecular diagnosis (PCR) method and the like, but the methods usually need professional technicians and laboratories, have the problems of complex operation, higher detection cost, longer time consumption, incapability of quantitatively reacting infection and the like, and are difficult to meet the clinical requirements of mass detection and field detection. The immunodiagnosis technology has the characteristics of high sensitivity, strong specificity, quickness and convenience, and has important significance in early diagnosis of patients and immune state tracking of recovered patients.
Oect (organic Electrochemical transistor) is an organic Electrochemical transistor that combines the advantages of organic semiconductor materials with the inherent characteristics of the transistor. In the OECT, the current flowing through the source and drain can be modulated by applying a weak gate voltage. This signal amplification mechanism enables detection of higher sensitivity and lower concentration in biological assays. In addition, compared with the traditional field effect transistor, the OECT has the characteristics of low-temperature processing and flexible processing, and the special physical structure and performance of the OECT enable the OECT to achieve high current response in a low-voltage (generally less than 1V) solution, so that the OECT breaks through the limitation of the traditional field effect transistor in a solution system. The advent of OECT has injected viability for the development of highly sensitive, highly specific bioanalytical assays. At present, OECT biosensors are successfully applied to detection of human biochemical metabolites such as glucose and lactic acid, but virus-related detection is rarely reported.
Therefore, it is highly desirable to provide an influenza virus detection sensor based on OECT and immunodiagnosis technology, so as to realize large-scale detection and rapid detection of various influenza viruses.
Disclosure of Invention
The invention provides a virus detection device based on full-screen printing OECT (organic electronic tomography), and a preparation method and a detection method thereof, and aims to solve the problems of complex operation, long detection time consumption, difficult low-concentration detection and the like of the conventional virus antigen detection technology. The invention relates to a virus detection device based on OECT, which adopts materials such as PET and the like as a substrate and combines a silk-screen printing technology to prepare a source electrode, a drain electrode, a channel layer and an insulating layer, wherein the conductive channel layer adopts doped PEDOT (PEDOT: PSS) printable slurry, and preferably utilizes a three-dimensional plane gold electrode attached with an antibody as a grid electrode. This is because the cubic planar gold electrode has good conductivity, a large surface area, excellent electrochemical properties and good biocompatibility, and can efficiently immobilize an antiviral antibody on the surface of the electrode through the strong interaction of Au — S bonds. The specific combination of the antigen and the antibody finally causes the current change of the source electrode and the drain electrode, and in the process, the virus antigen concentration and the current change of the source electrode and the drain electrode are in positive correlation, so that the true concentration of the influenza virus to be detected can be obtained according to the magnitude of the current change of the source electrode and the drain electrode. The virus detection device has strong universality, and can realize the detection of various subtype influenza viruses only by replacing the antibody adsorbed by the grid aiming at the influenza viruses of different subtypes.
The invention adopts the following specific technical scheme:
in a first aspect, the invention provides a preparation method of a virus detection device based on full-screen printing OECT, which comprises the following specific steps:
s1: depositing source and drain layers on the cleaned substrate by a screen printing technology, coating a channel layer between the source and drain layers by the screen printing technology, connecting a source electrode and a drain electrode by the channel layer, and then placing the substrate in inert atmosphere such as nitrogen for annealing heat treatment; coating photocuring paint on the surfaces of the source electrode and the drain electrode around the channel layer by a screen printing technology, and curing under an ultraviolet lamp to form an insulating layer to obtain a substrate;
s2: soaking the cleaned and dried original electrode in a self-assembly layer solution to form a self-assembly layer on the surface of the original electrode; activating the terminal carboxyl group of the self-assembled layer by using an activating agent, quickly transferring the terminal carboxyl group into a target antibody solution after the activation is finished under a light-shielding condition, and connecting the target antibody with the activated carboxyl group under a refrigeration condition to obtain a grid electrode fixed with the target antibody;
s3: sealing and fixing the bottom of the cleaned pipe section on the upper surface of the substrate, and forming a detection liquid tank for containing electrolyte; the detection liquid tank covers all the channel layers and part of the source drain layers covered with the insulating layers, so that the electrolyte can only be in direct contact with the channel layers;
s4: and fixing the grid above the detection liquid tank, wherein the lower part of the grid extends into the detection liquid tank and can be contacted with the electrolyte, and obtaining the virus detection device based on the full-screen printing OECT after assembly.
Preferably, the substrate is made of glass or PET; the material of the source electrode and the drain electrode is one of printable gold, silver or graphite slurry; the primary electrode is a gold electrode or a glassy carbon electrode, and is preferably a gold electrode; the material of the channel layer is PEDOT PSS slurry which can be printed and is doped.
Preferably, the annealing heat treatment temperature is 100-120 ℃, and the refrigeration treatment temperature is 2-8 ℃.
Preferably, the photocureable coating comprises the following components in concentration ratio of 1: 5-10 of a photosensitizer and a diluent.
Further, the photosensitizer is one of benzophenone or benzoin alkyl ether; the diluent is styrene or acrylate.
Preferably, the self-assembly layer solution is a mercaptoalkanoic acid solution with a concentration of 10mM, preferably mercaptoundecanoic acid or mercaptododecanoic acid.
Preferably, the activating agent is one of a mixed solution of 10mM NHS/5mM EDC, dicyclohexylcarbodiimide or 4-dimethylaminopyridine.
Preferably, the pipe section is a plastic hose, preferably a PE pipe or a PVC pipe.
In a second aspect, the invention provides a virus detection device based on the full screen printing OECT, which is obtained by the preparation method of any one of the first aspect.
In a third aspect, the present invention provides a method for detecting a virus antigen concentration by using the virus detection device of the second aspect, which comprises the following steps:
adding electrolyte to be detected into a detection liquid tank, wherein the electrolyte contains a virus antigen matched with a target antibody; applying a voltage between the source electrode and the drain electrode, forming a channel current through the channel layer, applying a voltage between the gate electrode and the substrate, and regulating and controlling the channel current through the formed gate voltage; due to the specific combination of the virus antigen and the target antibody on the grid, the interface potential of the grid is changed, so that the channel current is changed; the detection of the virus antigen concentration is realized by detecting different change values of the channel current and according to the change relation between the channel current and the virus antigen concentration.
Compared with the prior art, the invention has the following beneficial effects:
1) the virus detection device of the invention has high detection accuracy and strong specificity: the HA antibody capable of specifically recognizing the virus antigen is fixed on the grid electrode, only when the virus to be detected is specifically combined with the antibody on the grid electrode, the impedance and the interface potential of the grid electrode can be changed, further, ions in the solution are influenced to enter a channel layer of the organic electrochemical transistor, and finally, the existence and the concentration of the virus are detected by measuring the change of the channel current.
2) The virus detection device of the invention has strong signal amplification function and high detection sensitivity: the OECT has a good signal amplification function, channel current and antigen concentration change are in positive correlation in influenza virus detection, and tiny antigen concentration can be presented in a larger channel current mode through the detection device after weak grid voltage is applied to a grid interface. The method shows very high sensitivity and extremely low detection limit (10) under low grid voltage-9M)。
3) The virus detection device of the invention has high packaging quality and small signal pollution: the exposed parts of the source electrode and the drain electrode, which are positioned at the periphery of the PEDOT, PSS channel, are sealed by the photocuring coating, and the small detection liquid tank is used for packaging, so that the direct contact of electrolyte and a source and drain electrode layer is avoided, the influence of the electrolyte on the source electrode and the drain electrode is reduced, and the final output signal (channel current) can more accurately reflect the antigen concentration change.
4) The virus detection device has the advantages of simple preparation structure, low manufacturing cost, repeated use, safety, simplicity and convenience, and convenient carrying. After a certain antigen sample is detected, the grid electrode is only needed to be washed by strong acid/alkali or high-concentration salt solution, and the new antigen sample can be measured again after being washed.
Drawings
FIG. 1 is a top view of a virus detection device;
FIG. 2 is a front view of the virus detection apparatus;
FIG. 3 is a schematic structural diagram of a gate;
FIG. 4 is a graph showing the output characteristics of the OECT device of the virus detection apparatus in the embodiment;
FIG. 5 is an electrochemical impedance spectrum of gate-immobilized mercaptoundecanoic acid and an antibody according to the example;
FIG. 6 shows the detection of viral antigen by the grid electrode having immobilized viral antibody in example IDS-a T-plot;
FIG. 7 is a diagram showing an embodiment of a virus detection apparatus;
the reference numbers in the figures are: the detection device comprises a substrate 10, a substrate 11, a source electrode 12, a drain electrode 13, a channel layer 14, an insulating layer 15, a source-drain layer 16, a grid electrode 20, a self-assembly layer 21, a target antibody 22, a detection liquid tank 30 and an electrolyte 40.
Detailed Description
The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.
The invention provides a virus detection device based on full-screen printing OECT and a preparation method thereof, which mainly adopts the screen printing technology to manufacture a source drain layer, a channel layer and an insulating layer of the substrate part of an organic electrochemical transistor, fixes an antibody by the self-assembly principle of the surface of a mercaptoalkanoic acid molecular grid, and realizes functional application by the whole device through the encapsulation of a small detection liquid tank. The preparation method mainly comprises four steps, namely preparation of a substrate device, fixation of an antibody by a grid electrode, fixation of a detection liquid tank and assembly of a virus monitoring device, which are specifically described below.
S1: a source-drain layer 16 is deposited on the cleaned substrate 11 by a screen printing technique, a channel layer 14 is coated between a source electrode 12 and a drain electrode 13 of the source-drain layer 16 by a screen printing technique, the source electrode 12 and the drain electrode 13 are connected by the channel layer 14, and then annealing heat treatment is performed under an inert atmosphere such as nitrogen. The light-curing paint is coated on the surfaces of the source electrode 12 and the drain electrode 13 around the channel layer 14 by a screen printing technique, and cured under an ultraviolet lamp to form the insulating layer 15, resulting in the substrate 10.
In practical application, the substrate material is one of glass or PET; the source electrode and the drain electrode are both made of printable conductive materials such as gold, silver, graphite and the like; the gate electrode is one of electrodes such as a gold electrode and a glassy carbon electrode, and is preferably a gold electrode. The temperature of the annealing heat treatment is 100-120 ℃. The material of the channel layer 14 may be PEDOT: PSS, which is available directly from Hereaus under the model PEDOT: PSS SV 3.
In this example, a substrate was prepared using the following method, using the following specific steps:
and cleaning the PET substrate by using alcohol and ultrapure water in sequence, and blow-drying the residual moisture on the surface of the substrate by using nitrogen. And (3) attaching the PET substrate to the first template with the designed pattern, printing the template by using carbon paste, and placing the PET with the carbon paste in a drying oven at 100-120 ℃ for heating and annealing to prepare the PET plate with the printed source electrode and the printed drain electrode. The source and drain electrodes obtained at this time were both made of conductive graphite. And tightly attaching the PET plate to a second template with a designed pattern, printing the template by using doped PEDOT (PSS), and then placing the PET plate in a nitrogen environment for heating and annealing to form a channel layer made of the PEDOT (PSS). And (3) closely attaching the PET plate to a third template with a designed pattern, printing the template by using a photocuring coating, and finally, placing the template under an ultraviolet lamp to irradiate until the coating is cured, so as to form an insulating layer on the surfaces of the source electrode 12 and the drain electrode 13 around the channel layer 14, thereby finally obtaining the substrate 10.
S2: and soaking the cleaned and dried original electrode in a self-assembly layer solution to form a self-assembly layer 21 on the surface of the original electrode. Activating the terminal carboxyl group of the self-assembled layer 21 with an activator, after activation under a dark condition, rapidly transferring the activated terminal carboxyl group to a target antibody 22 solution, and connecting the target antibody 22 with the activated carboxyl group under a refrigeration condition to obtain the grid 20 on which the target antibody 22 is immobilized. The resulting gate is shown in fig. 3.
In practical application, the temperature of the refrigeration treatment can be 2-8 ℃. The photocureable coating can be prepared from the following components in a concentration ratio of 1: 5-10 of a photosensitizer and a diluent, wherein the photosensitizer is preferably one of benzophenone or benzoin alkyl ether, and the diluent is preferably styrene or acrylate. The self-assembly layer solution may be a mercaptoalkanoic acid solution with a concentration of 10mM, preferably mercaptoundecanoic acid or mercaptododecanoic acid. The activator may be one of a mixed solution of 10mM NHS (N-hydroxysuccinimide)/5 mM EDC ((1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), 10mM DCC (dicyclohexylcarbodiimide), or 10mM DMAP (4-dimethylaminopyridine).
In this embodiment, a gate is prepared by the following method, which specifically includes the following steps:
and soaking the gold electrode in a piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide: 3: 1), performing ultrasonic treatment for 5min, performing electrochemical cleaning in a 1M sulfuric acid solution, taking out, cleaning with distilled water, and drying to obtain a cleaned and dried gold electrode. 2mL of 10mM mercaptoundecanoic acid is prepared, and the gold electrode is placed in the solution to react for 4h at room temperature, so that a self-assembly layer is formed on the surface of the gold electrode. After the electrode was taken out and washed with ethanol, 2mL of a 10mM NHS/5mM EDC solution (pH 6.0) was prepared, and the gold electrode having mercaptoundecanoic acid immobilized thereon was placed in the above solution and reacted for 1 hour with exclusion of light to activate carboxyl groups on the electrode surface. And taking out the activated electrode, washing the electrode by using distilled water, immediately putting the electrode into a PBS (phosphate buffer solution) solution of the antibody, reacting for 12 hours at 4 ℃, and dehydrating and condensing an amino group at the tail end of the antibody and a carboxyl group on the surface of the grid to complete the fixation of the antibody on the grid. And finally, taking out the electrode and washing the electrode by using distilled water to obtain the gold electrode modified by the antibody as a grid electrode.
S3: the bottom of the cleaned pipe section is sealed and fixed on the upper surface of the substrate 10, and a detection liquid tank 30 for containing the electrolyte 40 is formed. The detection liquid tank 30 houses the entire channel layer 14 and a portion of the source-drain layer 16 covered with the insulating layer 15 so that the electrolyte 40 can only come into direct contact with the channel layer 14. The arrangement can isolate the electrolyte 40 from the source-drain layer 16, ensure the direct contact of the electrolyte 40 and the channel layer 14, reduce the input of a mixed signal and improve the detection precision.
In practical applications, the pipe section may be a plastic hose, preferably a PE pipe or a PVC pipe.
In this example, a fixed detection fluid bath was prepared using the following method, using the following specific steps:
a plastic hose with the length of 2cm is taken, washed by ethanol and distilled water in sequence, and is quickly fixed on an OECT photocuring coating layer after AB glue is coated at the bottom of the hose, so that the inner diameter of the hose is ensured to cover all the channel layer 14 and part of the source drain layer 16 covered with the insulating layer 15. And after the AB glue is solidified, obtaining the detection liquid tank 30 with the closed bottom connection part.
S4: and fixing the grid 20 above the detection liquid tank 30, wherein the lower part of the grid 20 extends into the detection liquid tank 30 and can be contacted with the electrolyte 40, and obtaining the virus detection device based on the full-screen printing OECT after assembly.
The prepared virus detection device is shown in figures 1-3 and mainly comprises an organic electrochemical transistor substrate 10, a grid 20 and a detection liquid tank for containing electrolyte 40. The base 10 includes a substrate 11, and a source electrode 12 and a drain electrode 13 disposed on the substrate 11. The source 12 and drain 13 are collectively referred to as a source drain layer 16 and are connected by a channel layer 14. The remaining portions of the source electrode 12 and the drain electrode 13 in the vicinity of the channel layer 14 are enclosed by the insulating layer 15.
The method for detecting the virus antigen concentration by using the virus detection device comprises the following specific steps:
before testing, the relationship between the virus antigen concentration and the channel current magnitude, namely the standard curve of the concentration-current response of the device, can be obtained by changing the concentrations of different target virus antigens contained in the electrolyte.
During testing, adding electrolyte 40 to be detected into the detection liquid tank 30, wherein the electrolyte 40 contains a virus antigen matched with the target antibody 22; subsequently, a voltage is applied between the source electrode 12 and the drain electrode 13 to form a channel current through the channel layer 14, a voltage is applied between the gate electrode 20 and the substrate 10, and the channel current is regulated by the formed gate voltage; since the virus antigen is specifically bound to the target antibody 22 on the gate 20, the interface potential of the gate 20 is changed, thereby causing a change in channel current. And detecting the virus antigen concentration by detecting different change values of the channel current according to the change relationship between the channel current and the virus antigen concentration.
The principle of the detection method is as follows:
since the channel current equation on an organic electrochemical transistor is:
wherein, IDSRepresents channel current, q represents electron charge, μ represents hole mobility, p0Representing the initial hole density in the organic semiconductor layer (i.e., channel layer 14), W and L representing the effective width and length of the channel, respectively, t representing the thickness of the organic semiconductor layer (i.e., channel layer 14), VpRepresenting pinch-off voltage, VG offRepresenting the effective gate voltage, VGRepresents the gate voltage, VDSRepresents the source-drain voltage, ciEffective gate capacitance, V, representing OECToffsetRepresenting the compensation voltage.
When the virus antigen is added into the electrolyte, the virus antigen and the antibody are specifically identified and combined, the surface charge of the antibody is covered, the charge distribution condition of the electrode surface is changed, and the effective voltage (V) of the grid electrode isG off) The change results in a change in the ion flux entering the channel in the electrolyte, which ultimately affects the channel current. Under the condition of controlling the binding efficiency and time of the antibody, the concentration of the antigen can directly influence the change condition of the channel current, and the change of the channel current can directly reflect the concentration of the virus antigen.
In addition, due to the effective voltage (V) of the gateG off) The change is very small, and a material with higher conductivity is required to reflect and amplify the change of channel current, so that the doped PEDOT/PSS printing slurry is used as an organic channel semiconductor layer for connecting a source electrode and a drain electrode, so that the detection sensitivity of virus antigens can be effectively improved, and the lower limit of detection is reduced.
The virus detection device prepared in the example was tested in 3M KCl solution with an Ag/AgCl electrode as the gate. As shown in FIG. 4, is a fixed VDS＝(-0.3V，-0.4V，-0.5V，-0.6V)，VGI varying from 0 to 0.8VDS-VGGraph is shown. As can be seen from the figure, the gate of the device has a better modulating effect on the overall device, and the channel current variation is most significant when the gate voltage is set to 0.4V.
As shown in fig. 5, after some stages are completed, the electrochemical impedance spectrum of the grid electrode prepared in this embodiment is measured, it is found that before and after self-assembly, the electrochemical impedance spectrum is significantly changed, after adding antigen, the surface charge of the antibody is shielded by the antigen, and therefore the grid electrode shows lower electrochemical impedance, and each stage of the preparation process of the detection device can determine whether the device part is successfully assembled or not by measuring the electrochemical impedance.
As shown in FIG. 6, the virus antigen I of the grid prepared in this exampleDS-[HA]Graph, from which it can be seen that V is measured in 0.1M PBS solutionG＝0.4V，V DSThe detection limit of the viral antigen was 10 ═ 0.2V-9M is lower than the lower detection limit of a common virus antigen detection device, which shows that the OECT of the device can convert a weak biological signal into a stronger electric signal, more sensitively reflects the antigen concentration in a detection solution, and has a better application prospect.
In summary, the virus detection device of the present invention is formed by packaging a substrate portion on which a source/drain layer, a channel layer, and an insulating layer are printed on a substrate, and a gate portion on which an HA antibody that can specifically recognize influenza virus is immobilized, in a small detection liquid tank. When the virus to be detected is specifically combined with the antibody on the grid electrode, the impedance and the interface potential of the grid electrode can be changed, so that the process that ions in a solution enter a channel of the organic electrochemical transistor is influenced, and finally, the existence and the concentration of the virus can be detected by measuring the change of the channel current. The OECT adopted by the invention has good signal amplification function, and can amplify micro-virus in the detection of influenza virusThe antigen concentration change is converted into larger channel current change, and the high sensitivity and the extremely low detection limit (10) are shown under the low grid voltage-9M). And the device has simple preparation structure, low cost, safe and simple use and convenient carrying.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (10)
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