Virus detection device, preparation method, and virus detection method

Abstract

The virus detection device, preparation method, and virus detection method provided by the embodiments of the present invention can directly modify the gate metal in the top metal material layer of the metal-oxide semiconductor field-effect transistor chip layer, using nucleic acid probes, viral antibodies, or viral antigens for virus detection. Compared with existing electrochemical biosensors, this device does not require the introduction of electrochemically active identifiers as markers and is compatible with current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a virus detection device, a preparation method and a virus detection method. Background Art

[0002] Currently, the most common method for virus detection is through electrochemical biosensors. The basic principle of using electrochemical biosensors for virus detection is to use a fixed electrode as a base electrode, immobilize an electrochemically active recognition substance on the electrode surface, and then capture the target molecule on the electrode surface through specific recognition between biomolecules. The base electrode converts the concentration signal into an electrical signal as a response signal, thereby achieving quantitative or qualitative analysis of the target (such as a virus).

[0003] However, the existing electrochemical biosensors not only require the introduction of electrochemically active recognition substances, but also require a special preparation process for preparation, and the preparation process has poor compatibility. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a virus detection device, a preparation method thereof, and a virus detection method, so as to improve the compatibility of the preparation of the virus detection device.

[0005] To achieve the purpose of the embodiment of the present invention, the embodiment of the present invention provides a virus detection device, comprising: a metal-oxide semiconductor field effect transistor chip layer and a microfluidic channel layer;

[0006] The metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged sequentially from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; the top metal material layer is provided with a gate metal, a source metal, and a drain metal correspondingly connected to the gate, source, and drain; the gate metal is modified with a nucleic acid probe, a viral antibody, or a viral antigen for virus detection;

[0007] The microfluidic layer is arranged on the top metal material layer; a microfluidic reactor located on the gate metal is arranged in the microfluidic layer, and the microfluidic reactor is used to react the detected sample with the modified nucleic acid probe or viral antibody or viral antigen on the top metal material layer.

[0008] Preferably, the metal material of the top metal material layer is aluminum or gold.

[0009] Preferably, when the metal material of the top metal material layer is aluminum, the gate metal is modified with a nucleic acid probe, a viral antibody, or a viral antigen through an amide bond constructed by the reaction of a bridging molecule carried on the surface of the aluminum metal and a nucleic acid probe, a viral antibody, or a viral antigen modified with a carboxyl group at the end group;

[0010] The bridging molecules include: siloxane molecules; preferably 3-aminopropyltriethoxysilane.

[0011] Preferably, when the metal material of the top metal material layer is gold, the gate metal is modified with a nucleic acid probe through a gold-sulfur bond formed by the reaction of gold metal with a nucleic acid probe modified with a sulfur group at the end; or, the gate metal is modified with a viral antibody or viral antigen through a gold-sulfur bond formed by the reaction of gold metal with a viral antibody or antigen that itself has a sulfur group;

[0012] In order to achieve the purpose of the embodiments of the present invention, the embodiments of the present invention further provide two methods for preparing the above-mentioned virus detection device.

[0013] A first method for preparing a virus detection device includes:

[0014] Prepare a metal-oxide semiconductor field-effect transistor chip layer, wherein the metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged in sequence from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; and the top metal material layer is provided with a gate metal, a source metal, and a drain metal connected to the gate, the source, and the drain respectively;

[0015] The metal material of the top metal material layer is aluminum;

[0016] A microfluidic layer is provided on the top metal material layer; a microfluidic reactor located on the gate metal is provided in the microfluidic layer, and the microfluidic reactor is used for reacting the sample to be tested with the nucleic acid probe or viral antibody or viral antigen modified on the top metal material layer;

[0017] Connecting bridging molecules to the aluminum metal surface;

[0018] The bridging molecule reacts with a nucleic acid probe or a viral antibody or a viral antigen modified with a carboxyl group at the end to construct an amide bond, so that the gate metal is modified with the nucleic acid probe or the viral antibody or the viral antigen;

[0019] The bridging molecules include: siloxane molecules; preferably 3-aminopropyltriethoxysilane.

[0020] The second method for preparing a virus detection device includes:

[0021] Prepare a metal-oxide semiconductor field-effect transistor chip layer, wherein the metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged in sequence from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; and the top metal material layer is provided with a gate metal, a source metal, and a drain metal connected to the gate, the source, and the drain respectively;

[0022] The metal material of the top metal material layer is aluminum;

[0023] Replace the gate metal with gold;

[0024] A microfluidic layer is provided on the top metal material layer; a microfluidic reactor located on the gate metal is provided in the microfluidic layer, and the microfluidic reactor is used for reacting the sample to be tested with the nucleic acid probe or viral antibody or viral antigen modified on the top metal material layer;

[0025] The gold metal is reacted with a nucleic acid probe modified with a sulfur group at the end to construct a gold-sulfur bond, so that the gate metal is modified with the nucleic acid probe.

[0026] or,

[0027] Gold metal is reacted with viral antibodies or antigens that have sulfur groups to construct a gold-sulfur bond, so that the gate metal is modified with viral antibodies or antigens.

[0028] In order to achieve the purpose of the embodiment of the present invention, the embodiment of the present invention further provides a virus detection method using the above-mentioned virus detection device, including:

[0029] Applying a test voltage between the source metal and the drain metal, and between the source metal and the gate metal of the virus detection device;

[0030] Adding a sample to be tested into a microfluidic reactor of a virus detection device;

[0031] Detecting changes in electrical signals in the conductive channel between the source metal and the drain metal before and after the test sample is added;

[0032] Based on the change of the electrical signal and the nucleic acid probe, viral antibody or viral antigen modified on the gate metal for virus detection, it is determined whether the sample being tested contains the target virus.

[0033] Beneficial effects of the embodiments of the present invention:

[0034] The embodiments of the present invention provide a virus detection device, a preparation method, and a virus detection method, which can directly modify the gate metal in the top metal material layer in the metal-oxide semiconductor field-effect transistor chip layer to form a nucleic acid probe, a viral antibody, or a viral antigen for virus detection, without the need to introduce an electrochemically active identifier as a marker.

[0035] At the same time, since the current commercial metal-oxide semiconductor field-effect transistor chips usually have a top metal material layer, there is no need to perform particularly complex modifications on the commercial metal-oxide semiconductor field-effect transistor chips. The metal-oxide semiconductor field-effect transistor chip layer of the virus detection device provided in the embodiment of the present application can be prepared, which is compatible with the current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 A schematic structural diagram of a virus detection device provided by an embodiment of the present invention;

[0038] Figure 2a A diagram illustrating a specific structure of a virus detection device provided by an embodiment of the present invention;

[0039] Figure 2b This is a structural example diagram of a microfluidic microreactor;

[0040] Figure 2c for Figure 2a A top view of the metal-oxide semiconductor field effect transistor chip layer 100 of the virus detection device shown;

[0041] Figure 3a Schematic diagram of modifying nucleic acid probes on gate metal when the gate metal is aluminum;

[0042] Figure 3b Schematic diagram of modifying viral antigens on gate metal when the gate metal is aluminum;

[0043] Figure 3c Schematic diagram of modifying viral antibodies on gate metal when the gate metal is aluminum;

[0044] Figure 4 Schematic diagram of modifying nucleic acid probes on gate metal when the gate metal is gold;

[0045] Figure 5 A schematic flow chart of a method for preparing a virus detection device with aluminum gate metal provided by an embodiment of the present invention;

[0046] Figure 6A schematic flow chart of a method for preparing a virus detection device with a gold gate metal provided by an embodiment of the present invention;

[0047] Figure 7 A schematic flow chart of a virus detection method provided in an embodiment of the present invention;

[0048] Figure 8 A schematic diagram of the principle of a virus detection method provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] In order to reduce the process complexity of preparing a virus detection device, an embodiment of the present invention provides a virus detection device, a preparation method, and a virus detection method, which are described in detail below.

[0051] See also Figure 1 , Figure 1 A schematic structural diagram of a virus detection device provided by an embodiment of the present invention includes a metal-oxide semiconductor field effect transistor chip layer 120 and a microfluidic channel layer 110 .

[0052] like Figure 1 As shown, the metal-oxide semiconductor field effect transistor chip layer 120 includes: a field effect transistor function layer 121 and a top metal material layer 122 arranged in sequence from bottom to top; the field effect transistor function layer 121 includes a gate, a source and a drain ( Figure 1 The top metal material layer 122 is provided with a gate metal, a source metal and a drain metal correspondingly connected to the gate, the source and the drain, and the gate metal is modified with a nucleic acid probe or a viral antibody or a viral antigen for virus detection;

[0053] The microfluidic layer 110 is arranged on the top metal material layer 122; a microfluidic reactor located on the gate metal is provided in the microfluidic layer 110, and the microfluidic reactor is used to react the detected sample with the modified nucleic acid probe or viral antibody or viral antigen on the top metal material layer.

[0054] Depend on Figure 1As can be seen from the virus detection device shown, the virus detection device provided by the embodiment of the present invention can directly modify the nucleic acid probe or virus antibody or virus antigen used for virus detection on the gate metal in the top metal material layer in the metal-oxide semiconductor field-effect transistor chip layer, and can achieve virus detection without introducing electrochemically active identifiers as markers.

[0055] At the same time, since the currently commercially available metal-oxide semiconductor field-effect transistor chips, abbreviated as MOSFET (Metal-Oxide-Semiconductor Field-Effect-Transistor), usually have a top metal material layer, it is possible to prepare the metal-oxide semiconductor field-effect transistor chip layer of the virus detection device provided in the embodiment of the present application without the need for particularly complex modifications to the commercial metal-oxide semiconductor field-effect transistor chips. This is compatible with the current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production.

[0056] In practical applications, a metal-oxide semiconductor field-effect transistor chip with a top metal layer can be purchased from a chip manufacturer, and then a microfluidic layer can be prepared on the chip to obtain the virus detection device provided in the embodiment of the present invention.

[0057] See also Figure 2a , Figure 2a This is a diagram illustrating a specific structure of a virus detection device provided in an embodiment of the present invention.

[0058] Figure 2a The virus detection device shown is a simplified schematic diagram of a device based on an NMOS structure in a six-layer silicon-based MOSFET process. The virus detection device includes a metal-oxide semiconductor field-effect transistor chip layer and a microfluidic layer 110. ①, ⑤, and ⑥ represent metal layers 1, 5, and 6, respectively. The layer containing ⑥ is called the top metal material layer, primarily composed of aluminum. One ⑥ is an extended gate metal, and the other ⑥ are the source and drain terminals, respectively. ② is the polysilicon gate, or gate. ③ is the connection hole between the layers. ④ is the insulating silicon dioxide isolation layer, or oxide layer. ⑦ is the P-type doped silicon substrate. ⑧ and ⑨ are the N-doped source and drain. ⑩ is the P-type doped substrate terminal. It is a microfluidic reactor in the microfluidic layer 110 manufactured in the subsequent process.

[0059] In the virus detection device of this embodiment, the metal-oxide-semiconductor field effect transistor is an insulated gate type, whose main feature is that there is an insulating layer between the gate and the channel, such as a silicon dioxide insulating layer.

[0060] See also Figure 2b , Figure 2bThis is a diagram illustrating the structure of a microfluidic microreactor. The microfluidic microreactor is provided with a sample reaction chamber that runs through the upper and lower sides of the microfluidic microreactor. The reaction chamber is used for the sample to react with the nucleic acid probe, viral antibody, or viral antigen modified on the top metal layer.

[0061] The virus detection device provided by the embodiment of the present invention can eventually be made into a chip, and its gate, source and drain can be led out of the chip through the top metal layer to form the gate terminal, source terminal and drain terminal on the chip.

[0062] See also Figure 2c , Figure 2c for Figure 2a FIG. 1 is a top view of the metal-oxide semiconductor field effect transistor chip layer 120 of the virus detection device. Figure 2c As shown, Figure 2a Correspondingly, the top metal layer of the MOSFET chip layer includes source metal, drain metal, and an extended gate. The rest of the top metal layer is insulating silicon dioxide. The source metal and drain metal lead to the source and drain terminals, respectively. The metal of the extended gate is decorated with nucleic acid probes, viral antibodies, or viral antigens. The microfluidic reactor in the microfluidic layer 110 will be located on the extended gate.

[0063] The microfluidic microreactor in this embodiment is mounted on the top metal layer via insulating silicon dioxide within the top metal layer. The sample reaction chamber of the microfluidic microreactor is located within the extended gate metal portion of the top metal layer, allowing the sample to react with the nucleic acid probe, viral antibody, or viral antigen modified on the top metal layer.

[0064] The virus detection device provided by the embodiments of the present invention can detect a variety of viruses, including influenza virus and the novel coronavirus. For example, modifying the gate metal with a nucleic acid probe, influenza virus antibody, or influenza virus antigen for influenza virus detection can detect influenza virus; modifying the gate metal with a nucleic acid probe, novel coronavirus antibody, or novel coronavirus antigen for novel coronavirus detection can detect novel coronavirus.

[0065] For example: When detecting the new coronavirus, the gate metal can be modified with the new coronavirus (SARS-CoV-2) antigen, or SARS-CoV-2 antibody, or a nucleic acid probe for detecting SARS-CoV-2.

[0066] Specifically, the SARS-CoV-2 antigens are: IgM and IgG antigen fragments;

[0067] The IgM and IgG antigen fragments are a 1:1 mixture of S protein and N protein;

[0068] Among them, the S protein is: S1-RBD, and its amino acid sequence is:

[0069] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT GCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF;

[0070] The amino acid sequence of the N protein is:

[0071] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPAR MAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAF FGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAADLDDFSKQLQQSMSSADSTQA ;

[0072] The SARS-CoV-2 antibodies are: IgM and IgG antibody fragments;

[0073] The nucleic acid probe for detecting the new coronavirus can be one of the following nucleic acid probes: (wherein each probe is 5'-3' from left to right, and the 5' is modified with SH-C6)

[0074] ORF1ab probe:

[0075] Probe 1: TTAAGTGTAAAACCCACAGGG

[0076] Probe 2: ACGATTGTGCATCAGCTGA

[0077] N protein probe:

[0078] Probe 1: ATTCTAGCAGGAGAAGTTCCCC

[0079] Probe 2: CAGACATTTTGCTCTCAAGCTG.

[0080] Figure 2c The main component of the material of the top metal material layer of the virus detection device shown is aluminum, that is, the extended gate metal is aluminum. In other embodiments of the virus detection device, the extended gate metal can be gold.

[0081] In practical applications, when the metal material of the top metal material layer is aluminum, the gate metal can be modified with a nucleic acid probe, viral antibody or viral antigen through an amide bond constructed by the reaction of a bridging molecule loaded on the surface of the aluminum metal with a nucleic acid probe, viral antibody or viral antigen modified with a carboxyl group at the end.

[0082] The bridging molecules may include: siloxane molecules; preferably 3-aminopropyltriethoxysilane.

[0083] Specifically, such as Figure 3a to Figure 3c As shown, 3-aminopropyl silicon is first loaded on the metal aluminum surface, and then an amide bond is constructed by a condensation reaction between a carboxyl group and an amino group of a nucleic acid probe or viral antibody or viral antigen modified with a carboxyl group at the end, and the gate metal aluminum is modified with a nucleic acid probe or viral antibody or viral antigen.

[0084] In practical applications, when the metal material of the top metal material layer is gold, gold can directly react with a nucleic acid probe modified with a sulfur group at the end group or a viral antibody or viral antigen that itself carries a sulfur group to construct a gold-sulfur bond, thereby modifying the nucleic acid probe.

[0085] Specifically, such as Figure 4 As shown, gold directly reacts with a nucleic acid probe or viral antibody or viral antigen modified with a sulfur group at the end to construct a gold-sulfur bond, thereby modifying the nucleic acid probe.

[0086] As can be seen from the above examples, the virus detection device provided by the embodiments of the present invention, compared to existing electrochemical biosensors, does not require the introduction of electrochemically active identifiers as markers and is compatible with current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production. In other words, the embodiments of the present invention can combine mature MOSFET processing technology, chemical / biological modification, and microfluidics technology, while simultaneously possessing many advantages such as miniaturization, low cost, easy integration, high-throughput detection, and rapid on-site detection.

[0087] The present invention provides two methods for preparing virus detection devices, one for preparing a virus detection device with aluminum gate metal and the other for preparing a virus detection device with gold gate metal, which are described in detail below.

[0088] See also Figure 5 , Figure 5 A schematic flow chart of a method for preparing a virus detection device with aluminum gate metal provided in an embodiment of the present invention includes the following steps:

[0089] Step 501, preparing a metal-oxide semiconductor field-effect transistor chip layer, wherein the metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged in sequence from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; the top metal material layer is provided with a gate metal, a source metal, and a drain metal correspondingly connected to the gate, the source, and the drain; the metal material of the top metal material layer is aluminum.

[0090] In practical applications, chip manufacturers can use mature MOSFET processing technology to prepare the metal-oxide semiconductor field-effect transistor chip layer.

[0091] Step 502, setting a microfluidic layer on the top metal material layer; a microfluidic reactor located on the gate metal is set in the microfluidic layer, and the microfluidic reactor is used to react the detected sample with the modified nucleic acid probe or viral antibody or viral antigen on the top metal material layer.

[0092] like Figure 2c As shown, the top metal layer of the metal-oxide semiconductor field effect transistor chip layer includes: source metal, drain metal and extended gate metal; the rest of the top metal layer is insulating silicon dioxide.

[0093] Thus, the microfluidic microreactor in this embodiment can be mounted on the top metal layer via the insulating silicon dioxide in the top metal layer. The sample reaction chamber of the microfluidic microreactor is located at the position of the extended gate metal in the top metal layer, and is used for the sample to react with the nucleic acid probe, viral antibody, or viral antigen modified on the top metal material layer.

[0094] Step 503: Connecting bridging molecules to the aluminum metal surface.

[0095] In step 504, a bridging molecule is reacted with a nucleic acid probe or viral antibody or viral antigen modified with a carboxyl group at the end to construct an amide bond, so that the gate metal is modified with the nucleic acid probe or viral antibody or viral antigen; the bridging molecule includes: a siloxane molecule; preferably 3-aminopropyltriethoxysilane.

[0096] Figure 5 In the process of step 503 to step 504, nucleic acid probes, viral antibodies or viral antigens are modified on the extended gate metal. Figure 3a to Figure 3c .

[0097] Below is Figure 3a As a specific embodiment, the process of modifying the gate metal aluminum with a nucleic acid probe is described in detail.

[0098] Step 1: Place a metal-oxide MOSFET chip whose gate metal is aluminum in a solution of 0.2 mol / L 3-aminopropyltriethoxysilane.

[0099] Step 2: heating to above 100°C and reacting for 12 hours.

[0100] Step three: washing with an organic solvent (such as ethanol) to obtain a chip with 3-aminopropyl silicon loaded on the surface of metal aluminum.

[0101] In step 4, the chip is placed in a solution of a single-stranded DNA probe modified with a terminal carboxyl group (in other embodiments, it can be an antibody probe or an antigen probe), and a condensation agent (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide) is added to construct an amide bond by a condensation reaction between the carboxyl group and the amino group.

[0102] Step 5: Rinse with an organic solvent (such as ethanol) and water to prepare an aluminum metal-oxide MOSFET chip with nucleic acid probes modified on the gate metal aluminum.

[0103] It can be seen from the above embodiments that the method for preparing a virus detection device with aluminum gate metal provided by the embodiments of the present invention can directly modify the nucleic acid probe or viral antibody or viral antigen used for virus detection on the gate metal aluminum in the top metal material layer in the metal-oxide semiconductor field-effect transistor chip layer. Compared with existing electrochemical biosensors, there is no need to introduce electrochemically active identifiers as markers, and it is compatible with the current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production.

[0104] See also Figure 6 , Figure 6 A schematic flow chart of a method for preparing a virus detection device with a gold gate metal provided in an embodiment of the present invention includes the following steps:

[0105] Step 601: Prepare a metal-oxide semiconductor field-effect transistor chip layer, wherein the metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged in sequence from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; the top metal material layer is provided with a gate metal, a source metal, and a drain metal connected to the gate, the source, and the drain; the metal material of the top metal material layer is aluminum;

[0106] Step 602, replacing the gate metal with gold metal;

[0107] In practical applications, chip manufacturers can use mature MOSFET processing technology to prepare a metal-oxide semiconductor field-effect transistor chip layer with an aluminum top metal layer, and then replace the aluminum metal of the gate metal with gold metal.

[0108] For example: You can Figure 2c The extended gate in the device is replaced with gold metal.

[0109] Due to the limitation of MOSFET processing technology, the main component of the top metal material of the device gate is aluminum. In order to achieve diversified modification methods, the aluminum metal material can be replaced by gold in this embodiment.

[0110] The specific steps are as follows:

[0111] First, the metal-oxide MOSFET chip is evenly spin-coated with photoresist, and then patterned exposure is performed and the exposed photoresist is removed by development. Figure 2c The extended gate metal portion is exposed.

[0112] Then, the exposed extended gate metal aluminum is completely etched away using an aluminum etchant.

[0113] Subsequently, gold is plated on the extended gate metal using an electron beam evaporator or a thermal evaporator.

[0114] Finally, the remaining photoresist and the film above are peeled off together, and the aluminum metal of the extended gate metal part is replaced with gold metal.

[0115] Step 603: Disposing a microfluidic layer on the top metal material layer; the microfluidic layer is provided with a microfluidic reactor located on the extended gate metal, the microfluidic reactor being used for reacting the sample to be tested with the nucleic acid probe or viral antibody or viral antigen modified on the top metal material layer;

[0116] Step 604a, reacting gold metal with a nucleic acid probe modified with a sulfur group at the end to construct a gold-sulfur bond, so that the gate metal is modified with the nucleic acid probe;

[0117] or,

[0118] Step 604b: reacting gold metal with the viral antibody or antigen having a sulfur group to construct a gold-sulfur bond, so that the gate metal is modified with the viral antibody or antigen.

[0119] Figure 6 In the process, step 603 is the process of directly modifying the gate (eg, extended gate) metal gold with a nucleic acid probe or a viral antibody or a viral antigen.

[0120] Below is Figure 4 As a specific embodiment, the process of modifying the gate metal gold with a nucleic acid probe is described in detail.

[0121] Step 1: Soak the metal-oxide MOSFET chip with gold gate metal in a single-stranded DNA probe solution modified with terminal thiol for 24 hours. The thiol and gold will directly form a gold-sulfur bond, so the DNA probe will self-assemble on the gold surface, thereby connecting the DNA probe molecule to the gold gate.

[0122] Step 2: Soak the chip in a 1 mmol / L 6-mercaptohexanol aqueous solution for 1 hour, rinse with deionized water, and blow dry with nitrogen. Alternatively, soak the chip in a 200 mmol / L sodium bromide buffer solution for half an hour, rinse with the buffer solution, and blow dry with nitrogen.

[0123] It can be seen from the above embodiments that the method for preparing a virus detection device with a gold gate metal provided by the embodiments of the present invention can replace the gate metal aluminum in the top metal material layer in the metal-oxide semiconductor field-effect transistor chip layer with gold, and directly modify the gate metal gold for virus detection using nucleic acid probes or viral antibodies or viral antigens. Compared with existing electrochemical biosensors, there is no need to introduce electrochemically active identifiers as markers, and it is compatible with the current mainstream MOSFET processing technology, laying a solid foundation for large-scale commercial production.

[0124] Finally, see Figure 7 , Figure 7 This is a flow chart of a virus detection method provided by an embodiment of the present invention. This method, which can be performed using any of the above-mentioned virus detection devices, includes the following steps:

[0125] Step 701: applying a test voltage between the source metal and the drain metal, and between the source metal and the gate metal of the virus detection device;

[0126] Step 702: adding a sample to be tested into the microfluidic reactor of the virus detection device;

[0127] Step 703, detecting changes in electrical signals in the conductive channel between the source metal and the drain metal before and after the test sample is added;

[0128] Step 704 : Determine whether the sample being tested contains the target virus based on the change in the electrical signal and the nucleic acid probe, virus antibody, or virus antigen modified on the gate metal for virus detection.

[0129] For example: if the voltage reaches a threshold voltage, it is determined whether the sample being tested contains the target virus.

[0130] Specifically, such as Figure 2a The basic principle of virus detection provided by the virus detection device of the embodiment of the present invention is shown in FIG. Figure 8 :

[0131] When performing virus detection, in the microfluidic reactor A reference electrode is set in the sample liquid to be tested.

[0132] When a voltage is applied between the N-doped source and drain terminals, and between the source terminal and the reference electrode (that is, between the source and gate), a current flows through the conductive channel beneath gate ②. This current is determined by the impedance of the conductive channel, and the voltage at gate ② affects the resistance of the channel. If base pairing with a nucleic acid probe or antibody-antigen binding occurs at the extended gate metal terminal ⑥, electron gain or loss or a potential difference occurs. This is equivalent to a change in the gate voltage or the transistor's threshold voltage at gate ②, thereby changing the resistance of the channel and, in turn, the current flowing through the channel. By detecting whether this current changes, it is possible to determine whether the corresponding reaction has occurred.

[0133] Therefore, in actual use, Figure 2b A test voltage is applied between the source lead and the gate lead, and between the source lead and the reference electrode, and then the patient's test sample (nucleic acid, blood specimen, throat swab, etc.) is added to the microfluidic reactor. The change in the current signal before and after the addition is used to determine whether the patient is infected with the virus.

[0134] The following experimental data illustrates the effectiveness of the virus detection method:

[0135] First, the device for virus detection is: a virus detection device with a gold gate metal, which is directly modified with the aforementioned N protein probe 1 on the gold through a gold-sulfur bond (from left to right is 5'-3', 5' is modified with SH-C6):

[0136] ATTCTAGCAGGAGAAGTTCCCC

[0137] Add different concentrations of probe complementary chain

[0138] Then, different concentrations of probe complementary chains (5'-3' from left to right) are dropped onto the chip surface (that is, the microfluidic layer of the virus detection device) for hybridization reaction:

[0139] GGGGAACTTCTCCTGCTAGAAT

[0140] The results showed that the lowest concentration that the chip could detect was 0.1 fg / mL, the detection time was less than ten minutes, and the accuracy rate reached over 99.9%.

[0141] A total of 18 clinical samples were tested using both the RT-qPCR method and the virus detection device according to the present invention. The results obtained using the RT-qPCR method were negative in 9 cases and positive in 9 cases, which were all consistent with the results obtained using the virus detection device according to the present invention. The specific experimental data are as follows:

[0142] serial number RT-qPCR test results Virus detection device test results consistency 1 Negative Negative yes 2 Negative Negative yes 3 Negative Negative yes 4 Negative Negative yes 5 Negative Negative yes 6 Negative Negative yes 7 Negative Negative yes 8 Negative Negative yes 9 Negative Negative yes 10 Positive Positive yes 11 Positive Positive yes 12 Positive Positive yes 13 Positive Positive yes 14 Positive Positive yes 15 Positive Positive yes 16 Positive Positive yes 17 Positive Positive yes 18 Positive Positive yes

[0143] As can be seen from the above examples, the virus detection methods provided by the embodiments of the present invention have a short detection time, high sensitivity, strong specificity, and high accuracy. Furthermore, the embodiments of the present invention combine established MOSFET processing techniques, chemical / biological modification, and microfluidics technology, offering numerous advantages such as miniaturization, low cost, ease of integration, high-throughput detection, and rapid on-site detection.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. A virus detection method, characterized in that: Detection using virus detection devices, including: Applying a test voltage between the source metal and the drain metal, and between the source metal and the gate metal of the virus detection device; Adding a sample to be tested into a microfluidic reactor of a virus detection device; Detecting changes in electrical signals in the conductive channel between the source metal and the drain metal before and after the test sample is added; Determining whether the sample being tested contains the target virus based on the change in the electrical signal and the nucleic acid probe, virus antibody, or virus antigen modified on the gate metal for virus detection; Wherein, the virus detection device comprises: a metal-oxide semiconductor field effect transistor chip layer and a microfluidic channel layer; The metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged sequentially from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; the top metal material layer is provided with a gate metal, a source metal, and a drain metal correspondingly connected to the gate, source, and drain; the gate metal is modified with a nucleic acid probe, a viral antibody, or a viral antigen for virus detection; The microfluidic layer is arranged on the top metal material layer; a microfluidic reactor located on the gate metal is arranged in the microfluidic layer, and the microfluidic reactor is used for reacting the sample to be tested with the nucleic acid probe or viral antibody or viral antigen modified on the top metal material layer; The metal material of the top metal material layer is aluminum or gold.

2. The method according to claim 1, characterized in that When the metal material of the top metal material layer is aluminum, the gate metal is modified with the nucleic acid probe, viral antibody or viral antigen through an amide bond constructed by the reaction of the bridging molecule carried on the surface of the aluminum metal and the nucleic acid probe, viral antibody or viral antigen modified with a carboxyl group at the end group; The bridging molecule is 3-aminopropyltriethoxysilane.

3. The method according to claim 1, characterized in that When the metal material of the top metal material layer is gold, the gate metal is modified with a nucleic acid probe through a gold-sulfur bond formed by the reaction of gold metal with a nucleic acid probe modified with a sulfur group at the end; or The gate metal is modified with viral antibodies or viral antigens through a gold-sulfur bond constructed by the reaction of gold metal with viral antibodies or antigens having sulfur groups.

4. A method for preparing a virus detection device, characterized in that: include: Prepare a metal-oxide semiconductor field-effect transistor chip layer, wherein the metal-oxide semiconductor field-effect transistor chip layer includes a field-effect transistor functional layer and a top metal material layer arranged in sequence from bottom to top; the field-effect transistor functional layer includes a gate, a source, and a drain; and the top metal material layer is provided with a gate metal, a source metal, and a drain metal connected to the gate, the source, and the drain respectively; The metal material of the top metal material layer is aluminum; A microfluidic layer is provided on the top metal material layer; a microfluidic reactor located on the gate metal is provided in the microfluidic layer, and the microfluidic reactor is used for reacting the sample to be tested with the nucleic acid probe or viral antibody or viral antigen modified on the top metal material layer; Connecting bridging molecules to the aluminum metal surface; The bridging molecule reacts with a nucleic acid probe or a viral antibody or a viral antigen modified with a carboxyl group at the end to construct an amide bond, so that the gate metal is modified with the nucleic acid probe or the viral antibody or the viral antigen; The bridging molecule is 3-aminopropyltriethoxysilane.

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