DETECTION DEVICE AND METHOD FOR CORONAVIRUS AND INFLUENZA VIRUS

Abstract

The present application is related to a detection device and method for coronavirus and influenza virus. The device comprises a detection module (20), a signal processing circuit (30), a controller (40), a displayer (50), a digital-to-analog conversion circuit (60), and a clock (70). Noticeably, the detection module (20) comprises a sample cell, and a transistor sensor combination integrated in the sample cell and used for measuring different targets. The detection module (20) may comprise a sample cell array to realize simultaneous detection of a plurality of samples to be detected. The detection method comprises the following steps: adding a sample to be detected into a sample cell, reading an electrical signal response of each transistor sensor in the sample cell to judge whether the sample to be detected contains a virus to be detected or not. The present application belongs to the technical field of biological detection.

Claims

1. A detection method for a coronavirus and an influenza virus, wherein a detection device for a coronavirus and an influenza virus is used for detection, the detection device for a coronavirus and an influenza virus comprises a detection module, a signal processing circuit, a controller, a displayer, a digital-to-analog conversion circuit, and a clock, wherein input ends of the detection module are separately connected with an output end of the digital-to-analog conversion circuit and the clock; an output end of the detection module is connected with an input end of the signal processing circuit; an input end of the controller is connected with an output end of the signal processing circuit; and output ends of the controller are separately connected with an input end of the displayer, the digital-to-analog conversion circuit, and the clock; the detection module comprises a sample cell, and transistor sensor combination integrated in the sample cell and used for measuring different targets, the transistor sensor combination comprises a plurality of transistor sensor units, and each of the transistor sensor units modify different biorecognition molecules separately and is integrated in the same sample cell; the detection method specifically comprises the following steps: step 1, adding a sample to be detected into the sample cell of the detection module; step 2, obtaining an electrical signal response of all transistor sensor units in each of the sample cell of the detection module; step 3, obtaining the number n of the transistor sensor units whose electrical signal response is greater than detection value A and the number m of the transistor sensor units whose electrical signal response is less than detection value B in each of the sample cell of the detection module; and step 4, determining whether the sample to be detected contains a corresponding virus to be detected through the number n and m of the transistor sensor units; wherein, in step 3, the detection value A is equal to 3 times of an electrical signal response ΔI.sub.detection% of a negative control; the detection value B is equal to the electrical signal response ΔI.sub.detection% of the negative control, wherein, in step 4, specifically comprising the following steps: adding a sample to be detected and obtaining an electrical signal response of a transistor sensor combination in a sample cell of the detection module; obtaining an electrical signal response ΔI.sub.detection% of each of the transistor sensor units; comparing the ΔI.sub.detection% of the transistor sensor unit with the detection values A and B, if the ΔI.sub.detection% is greater than the detection value A, determining that the number n of the transistor sensor units greater than the detection value A increases by 1; if the ΔI.sub.detection% is less than the detection value B, determining that the number m of the transistor sensor units less than the detection value B increases by 1; and if the ΔI.sub.detection% is between the detection values A and B, determining that the number n of the transistor sensor units greater than the detection value A equal to 0 and the number m of the transistor sensor units less than the detection value B equal to 0; if the number n of the transistor sensor units is greater than or equal to 2, determining detected/positive, namely the sample to be detected contains a virus to be detected; if the number m of the transistor sensor units is greater than or equal to 1 and n is equal to 0, determining not detected/negative, namely the sample to be detected does not contain a virus to be detected; and if the numbers m and n of the transistor sensor units are other values, determining that the detection result is in a gray area and the detection needs to be repeated; wherein process of calculating the electrical signal response ΔI.sub.detection% of the transistor sensor unit specifically comprises: obtaining an initial current value I.sub.0 of the transistor sensor unit after adding the negative control; obtaining a measured current value I.sub.detection of the transistor sensor unit after adding a sample to be detected; obtaining a current change value ΔI.sub.detection (ΔI.sub.detection=I.sub.detection−I.sub.0) by subtracting the initial current value I.sub.0 of the transistor sensor unit from the measured current value I.sub.detection; and obtaining the electrical signal response ΔI.sub.detection% (ΔI.sub.detection%=ΔI.sub.detection/I.sub.0×100%) of the transistor sensor unit by dividing the current change value ΔI.sub.detection of the transistor sensor unit by the initial current value I.sub.0.

2. The detection method for a coronavirus and an influenza virus according to claim 1, wherein each of the transistor sensor unit comprises an insulating substrate, electrodes arranged on the insulating substrate, a sensitive material arranged on the insulating substrate and located between the electrodes, and the biorecognition molecules are anchored on the sensitive material.

3. The detection method for a coronavirus and an influenza virus according to claim 2, wherein the sensitive material comprises graphene, molybdenum disulfide, tungsten disulfide, graphene oxide, a carbon nanotube, silicon, germanium, and an organic semiconductor thin film; and the biorecognition molecules comprise a molecule capable of specifically binding to different sites of a nucleic acid, a protein or a corresponding antibody of a virus to be detected.

4. The detection method for a coronavirus and an influenza virus according to claim 1, wherein the detection module contains a sample cell or an array of 3 to 256 sample cells; and the transistor sensor combination comprises 2 to 12 transistor sensor units.

5. The detection method for a coronavirus and an influenza virus according to claim 1, wherein the digital-to-analog conversion circuit comprises a power supply voltage regulator circuit, a gate voltage control circuit, and a source-drain voltage control circuit; and the signal processing circuit comprises a voltage reference, an operational amplifier, and an instrumentation amplifier.

6. (canceled)

7. The detection method for a coronavirus and an influenza virus according to claim 1, wherein the coronavirus comprises a coronavirus capable of infecting human beings in four genera of α, β, γ, and δ, and specifically comprises HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, and SARS-CoV-2; and the influenza virus comprises an influenza A virus, an influenza B virus and an influenza C virus.

8. The detection method for a coronavirus and an influenza virus according to claim 1, wherein the sample to be detected comprises a single sample, a mixed sample, and a hybrid sample, and a processing method of the sample to be detected comprises: (1) detection of nucleic acid contained in virus: after adding a nucleic acid extraction reagent to release virus RNA, inactivating the sample to be detected at a certain temperature; and (2) detection of protein contained in virus and corresponding antibody: inactivating the sample to be detected at a certain temperature.

9. The detection method for a coronavirus and an influenza virus according to claim 1, wherein the electrical signal response ΔI.sub.detection% of the negative control is prepared by a detection personnel according to a type of a virus to be detected and a total positive rate of the virus to be detected in a population, and specifically comprises a virus preservation solution, artificial saliva, and healthy human serum.

10. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a schematic diagram of the operation flow of the detection device and method for a coronavirus and an influenza virus of the present disclosure;

[0037] FIG. 2 is a step-by-step diagram of the detection method for a coronavirus and an influenza virus of the present disclosure;

[0038] FIG. 3 is a connection diagram of the detection device for a coronavirus and an influenza virus of the present disclosure;

[0039] FIG. 4 shows the detection module of the detection device for a coronavirus and an influenza virus of the present disclosure;

[0040] FIG. 5 shows the detection device for a coronavirus and an influenza virus of the present disclosure;

[0041] FIG. 6 is the detection result of the single sample of the detection device for a coronavirus and an influenza virus in example 1 of the present disclosure;

[0042] FIG. 7 is the detection result of the mixed sample of the detection device for a coronavirus and an influenza virus in example 2 of the present disclosure; and

[0043] FIG. 8 is the statistical detection result of the hybrid sample of the detection device for a coronavirus and an influenza virus in example 3 of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The present disclosure will be further explained in detail below with reference to the accompanying drawings.

[0045] FIG. 1 shows the operation flow of the detection device and method for a coronavirus and an influenza virus of the present disclosure: [0046] adding a sample to be detected into a sample cell of a detection module; [0047] obtaining an electrical signal response of all transistor sensor units in each sample cell of the detection module; [0048] obtaining the number n of the transistor sensor units whose electrical signal response is greater than detection value A and the number m of the transistor sensor units whose electrical signal response is less than detection value B in each sample cell of the detection module; and [0049] determining whether the sample to be detected contains a corresponding virus through the number n and m of the transistor sensor units.

[0050] Furthermore, the sample to be detected comprises a single sample, a mixed sample, and a hybrid sample. The sample relates to a serum sample, a nasal swab sample, a pharyngeal swab sample, a respiratory tract aspirate, a bronchial lavage fluid, an alveolar lavage fluid, or a nasopharyngeal aspirate. Among them, when collecting serum samples, the blood of a user to be detected are required to be stood for several hours and then the serum is put into a sterile tube and packaged according to biosafety; and after the nasal swab sample, the pharyngeal swab sample, the respiratory tract aspirate, the bronchial lavage fluid, the alveolar lavage fluid, or the nasopharyngeal aspirate are collected, the collected samples are preserved using virus preservation tubes and packaged according to biosafety.

[0051] A processing method of the sample to be detected comprises: [0052] (1) detection of nucleic acid contained in virus: after adding a nucleic acid extraction reagent to release virus RNA, inactivating the sample to be detected at 56° C. for 30 min; and [0053] (2) detection of protein contained in virus and corresponding antibody: inactivating the sample to be detected at 56° C. for 30 min.

[0054] The specific steps for collecting and processing a sample to be tested should refer to the “National Influenza Surveillance Technical Guidelines (2017 Edition)” of the Chinese Center for Disease Control and Prevention, and the “Technical Specifications for Detection of 10-in-1 Mixed Collection of Nucleic Acids of Novel Coronavirus” and “Technical Guidelines for Detection of Diluted Hybrid for Novel Coronavirus Nucleic Acid Testing” of the National Health Commission of the People's Republic of China.

[0055] Furthermore, whether the sample to be detected contains a corresponding virus is determined through the number n and m of the transistor sensor units. Specifically, the result is obtained by the steps shown in FIG. 2: [0056] S10, adding a sample to be detected and obtaining an electrical signal response of a transistor sensor combination in a sample cell of a detection module; [0057] S20, calculating an electrical signal response ΔI.sub.detection% of each transistor sensor unit; [0058] S30, determining whether the ΔI.sub.detection% of the transistor sensor unit is greater than detection value A and determining whether the ΔI.sub.detection% of the transistor sensor unit is less than detection value B; [0059] S40, obtaining the number n of the transistor sensor units whose electrical signal response is greater than detection value A and the number m of the transistor sensor units whose electrical signal response is less than detection value B in each sample cell of the detection module; [0060] S50, determining whether n is greater than or equal to 2 and determining whether n is equal to 0 and whether m is greater than or equal to 1; and [0061] S60, determining whether the sample to be detected is detected/positive, not detected/negative, or is in a gray area.

[0062] Furthermore, calculating the electrical signal response ΔI.sub.detection% of the transistor sensor unit specifically comprises: [0063] obtaining an initial current value I.sub.0 of the transistor sensor unit after adding the negative control; [0064] obtaining a measured current value I.sub.detection of the transistor sensor unit after adding a sample to be detected; [0065] obtaining a current change value ΔI.sub.detection (ΔI.sub.detection=I.sub.detection−I.sub.0) by subtracting the initial current value I.sub.0 of the transistor sensor unit from the measured current value I.sub.detection; and [0066] obtaining the electrical signal response ΔI.sub.detection% (ΔI.sub.detection%=ΔI.sub.detection/I.sub.0×100%) of the transistor sensor unit by dividing the current change value ΔI.sub.detection of the transistor sensor unit by the initial current value I.sub.0.

[0067] Furthermore, the detection value A is equal to 3 times of an electrical signal response ΔI.sub.detection% of a negative control; and the detection value B is equal to the electrical signal response ΔI.sub.detection% of the negative control. The negative control is prepared by a detection personnel according to a type of a virus to be detected and a total positive rate of the virus to be detected in the population, and specifically comprises a virus preservation solution, artificial saliva and healthy human serum.

[0068] FIG. 3 is a connection diagram of the detection device for a coronavirus and an influenza virus of the present disclosure; and the detection device comprises a detection module 20, a signal processing circuit 30, a controller 40, a displayer 50, a digital-to-analog conversion circuit 60, and a clock 70. Input ends of the detection module 20 are connected with an output end of the digital-to-analog conversion circuit 60 and the clock 70; an output end of the detection module 20 is connected with an input end of the signal processing circuit 30; an output end of the signal processing circuit 30 is connected with an input end of the controller 40; and output ends of the controller 40 are connected with an input end of the displayer 50, the digital-to-analog conversion circuit 60, and the clock 70. Since specific recognition molecules are attached to a conductive channel of each transistor sensor unit in the detection module 20, different types and concentrations of virus detection samples can change the current carrier mobility of the sensitive material, thereby generating electrical signals. The signal processing circuit 30 collects current output by the detection module 20 and outputs the current to the controller to calculate to obtain a detection signal of a virus detection sample 10.

[0069] Furthermore, the detection module comprises a sample cell 201 and a transistor sensor combination 202 integrated in the sample cell and used for measuring different targets. The detection module is as shown in FIG. 4. The detection module contains a sample cell or an array of 3 to 256 sample cells to realize detection of one sample to be detected or a combination of 3 to 256 samples to be detected.

[0070] Furthermore, the transistor sensor combination comprises 2 to 12 transistor sensor units. Each transistor sensor unit modifies different biorecognition molecules separately and is integrated in the same sample cell. Each transistor sensor unit comprises an insulating substrate, electrodes arranged on the insulating substrate, a sensitive material arranged on the insulating substrate and located between the electrodes, and the biorecognition molecule anchored on the sensitive material. The sensitive material comprises graphene, molybdenum disulfide, tungsten disulfide, graphene oxide, a carbon nanotube, silicon, germanium, and an organic semiconductor thin film; and the biorecognition molecule comprises a molecule capable of specifically binding to different sites of a nucleic acid, a protein or a corresponding antibody of a virus to be detected.

[0071] Furthermore, according to Table 1, site 1 of a main control board and site 2 of a collection board are connected to obtain the virus detection device (FIG. 5) with a test function; and then the virus detection device is connected to a notebook computer through a USB connecting line.

TABLE-US-00001 TABLE 1 Wiring table of detection device for coronavirus and influenza virus of the present disclosure Name Site 1 Name Site 2 Main control board AP4 Collection board VG Main control board AN4 Collection board GND Main control board AP3 Collection board VD3 Main control board AN3 Collection board GND Main control board AP2 Collection board VD2 Main control board AN2 Collection board GND Main control board AP1 Collection board VD1 Main control board AN1 Collection board GND Main control board AGND Collection board GND Main control board DAC4 Collection board VGPK Main control board DAC3 Collection board VGDC Main control board DAC2 Collection board MODE Main control board DAC1 Collection board VGIT Main control board DAC8 Collection board EN Main control board DAC7 Collection board VD3 Main control board DAC6 Collection board VD2 Main control board DAC5 Collection board VD1 Main control board DGND USB interface GND Main control board Tx USB interface Rxd Main control board Rx USB interface Txd

Example 1

[0072] The example presented a detection result of a single sample of a novel coronavirus and specifically comprised the following steps:

[0073] Step 1, a detection module 20 for a novel coronavirus was prepared: processing of a transistor sensor array comprised the following seven steps: [0074] (a) a photoresist was spin-coated on a single-polished silicon oxide wafer (including 300 nm SiO.sub.2 in an upper layer and 500 μm P-type doped Si in a lower layer) and an electrode pattern was exposed and developed; [0075] (b) the electrode pattern was subjected to evaporation to obtain metal electrodes (5 nm chromium and 50 nm gold), wherein the electrodes comprised a source electrode and a drain electrode, the source electrode was a current input end and the drain electrode was a current output end; [0076] (c) 8 wt. % of polymethyl methacrylate (PMMA) was spin-coated on graphene grown on a metal substrate, the graphene/PMMA film was electrochemically transferred to a Si/SiO.sub.2 substrate with the metal electrodes, the film-coated substrate was connected between the source electrode and the drain electrode, soaked in acetone for 2 h and then washed with isopropanol/deionized water, and graphene was etched into a specific shape by photolithography to obtain a device to be modified; [0077] (d) the device to be modified was annealed at a current of 500 μA for 10 min; [0078] (e) a connection molecule was modified in a graphene channel region (non-metallic electrode component) on the device to be modified, and an example was: after graphene was soaked in 5×10.sup.−3 mol/L of 1-pyrenebutyric acid N-hydroxysuccinimide ester solution for 2 h, the graphene was washed with absolute ethanol and deionized water; [0079] (f) after the graphene was soaked in a novel coronavirus specific antibody solution for 6 h, the graphene was washed with a 1×phosphate buffered saline solution; and [0080] (g) after the graphene was soaked in 200×10.sup.−3 mol/L of an ethanolamine solution for 2 h, the graphene was washed with absolute ethanol and a phosphate buffered saline solution to obtain the detection module.

[0081] Step 2, a virus detection device with a test function was obtained by the connection according to the steps described in the specific embodiments.

[0082] Step 3, a novel coronavirus sample to be detected was prepared: [0083] (a) bilateral pharyngeal tonsils and a posterior pharyngeal wall were wiped with 2 plastic-rod swabs with polypropylene fiber tips at the same time, the swab tips were soaked in a tube containing 3 mL of a virus preservation solution (or an isotonic saline solution, a tissue culture solution and a phosphate buffer solution), tails of the swabs were discarded, the tube cap was tightened, and the sample was packaged according to biosafety requirements; [0084] (b) 0.5 mL of the sample to be detected in the sampling tube was taken, placed in a centrifuge tube, and thoroughly mixed (shaken on a shaker for 30 s) to obtain a hybrid sample, and the sample was packaged according to biosafety requirements; and [0085] (c) the sample to be detected was inactivated at 56° C. for 30 min.

[0086] Step 4, a novel coronavirus sample to be detected was detected: [0087] (a) 100 μL of a negative control (virus preservation solution) was added to a sample cell of the detection module, and an output voltage of the virus detection device was set to enable a fluctuation range of the electrical signal response of the transistor sensor combination to be less than ±0.3%; and detection value A was set to ±0.9% and detection value B was set to ±0.3%; [0088] (b) the negative control was taken out, 100 μL of the sample to be detected was added in a sample cell of the detection module, and the electrical signal response of the transistor sensor combination in a sample cell of the detection module was obtained; [0089] (c) the number n of the transistor sensor units whose electrical signal response was greater than detection value A and the number m of the transistor sensor units whose electrical signal response was less than detection value B in each sample cell of the detection module were obtained according to the steps of the specific embodiments; and [0090] (d) whether the sample to be detected was detected/positive, not detected/negative, or is in a gray area was determined.

[0091] FIG. 6 was the electrical signal response generated by a transistor sensor unit 203 in the example: after a sample to be detected containing 2.5×10.sup.−8 mol/L of a spike protein of a novel coronavirus, the electrical signal response of the transistor sensor unit was close to 25% and greater than detection value A, that is, the number n of the transistor sensor units whose electrical signal response was greater than detection value A was equal to 1.

Example 2

[0092] Detection was performed according to the method of the specific embodiments. The differences between the example and example 1 were: [0093] (1) A sample to be detected used in the example was collected in accordance with the “Technical Specifications for Detection of 10-in-1 Mixed Collection of Nucleic Acids of Novel Coronavirus” of the National Health Commission of the People's Republic of China and was directly added to a sample cell of a virus detection device without pretreatment, wherein, M1 is a mixed sample of throat swabs of 10 healthy people; and M3 is a “mock positive mixed sample” obtained by adding 5×10.sup.−12 mol/L of a spike protein of a novel coronavirus to M1; and [0094] (2) 100 μL of a negative control (virus preservation solution) was added to a sample cell of a detection module, and an output voltage of the virus detection device was set to enable a fluctuation range of the electrical signal response of the transistor sensor combination to be less than ±0.5%; and detection value A was set to ±1.5% and detection value B was set to ±0.5%.

[0095] FIG. 7 was the detection result of the mixed sample of the example: after M1 was added, the number n of the transistor sensor units whose electrical signal response was greater than detection value A was equal to 0 and the number m of the transistor sensor units whose electrical signal response was less than detection value B was equal to 2, and thus M1 was determined to be not detected/negative, that is, did not contain a virus. After M3 was added, the number n of the transistor sensor units whose electrical signal response was greater than detection value A was equal to equal to 2, and thus M3 was determined to be detected/positive, that is, contained a virus.

Example 3

[0096] Detection was performed according to the method of the specific embodiments. The differences between the example and example 1 were: [0097] (1) A sample to be detected used in the example was a hybrid sample and directly added to a sample cell of a virus detection device without pretreatment, wherein, M1 is a mixed sample of throat swabs of 10 healthy people; and M2, M3, M4, M5, and M6 were “mock positive hybrid samples” obtained by adding a certain concentration of a spike protein of a novel coronavirus to M1; and after calibration, M2 contains 0.5×10.sup.−12 mol/L of the spike protein of a novel coronavirus; M3 contains 5×10.sup.−12 mol/L of the spike protein of a novel coronavirus; M4 contains 50×10.sup.−12 mol/L of the spike protein of a novel coronavirus; M5 contains 500×10.sup.−12 mol/L of the spike protein of a novel coronavirus; and M6 contains 50×10.sup.−9 mol/L of the spike protein of a novel coronavirus; and [0098] (2) 100 μL of a negative control (virus preservation solution) was added to a sample cell of a detection module, and an output voltage of the virus detection device was set to enable a fluctuation range of the electrical signal response of the transistor sensor combination to be less than ±0.13%; and detection value A was set to ±0.39% and detection value B was set to ±0.13%.

[0099] FIG. 8 was the statistical detection result of the hybrid samples in the example: after M1 was added, the electrical signal response ΔI.sub.detection% of the transistor sensor unit was 0.27%, between the detection value A and the detection value B, and in a gray area; and after M2 to M5 were added, the electrical signal response ΔI.sub.detection% of the transistor sensor unit was all greater than the detection value A, the number n of the transistor sensor units whose electrical signal response greater than the detection value A was calculated to be equal to 3, and thus M2 to M5 were detected/positive, that is, contained a novel coronavirus.

[0100] The above description of the examples is intended to facilitate a person of ordinary skill in the art to understand and use the present disclosure. Obviously, a person skilled in the art can easily make various modifications to these examples, and apply a general principle described herein to other examples without creative efforts. Therefore, the present disclosure is not limited to the above examples. All improvements and modifications made by a person skilled in the art according to implication of the present disclosure without departing from the spirit of the present disclosure should fall within the protection scope of the present disclosure.