Hyperbranched DNA rolling circle amplification-based virus detection sensor and test method

Abstract

The invention provides a hyper-branched DNA rolling circle amplification-based virus detection sensor and a test method, the hyper-branched DNA rolling circle amplification-based virus detection sensor comprises a carbon-based field effect transistor biosensor, the carbon-based field effect transistor biosensor is a P-type device and sequentially comprises a substrate S I layer, an S < 2 > layer, a CNT channel layer and an HfO2 dielectric layer from bottom to top, and a source electrode and a drain electrode are symmetrically arranged on the S < 2 > layer; wherein the surface of the CNT channel layer is modified and fixed with a primer, and the primer is used for carrying out HRCA reaction on the interface of the channel layer to cause the current rise of the device; the biosensor based on the carbon-based field effect transistor is used for detecting viruses, the device reaches the micron level, batch production is easy, cost is low, carrying is convenient, the ultrasensitivity of the carbon-based field effect transistor to surface charges is utilized, a large number of negatively charged DNA molecules can be generated through the HRCA reaction, the detection process is short in time consumption, and the detection efficiency is high. And the defect of long detection time caused by low concentration of DNA of a virus sample in the existing detection technology is overcome.

Description

Technical Field

[0001] The invention relates to a virus detection sensor, in particular to a virus field effect transistor sensor based on hyperbranched DNA rolling circle amplification, belonging to the technical field of biosensors. Background Art

[0002] A biosensor is a device that combines a biological recognition element with a physical sensor to detect and analyze biochemical substances. These sensors are widely used in many fields such as medical diagnosis, virus detection, environmental monitoring, food safety, and military reconnaissance. The core of a biosensor is its biological recognition element, which is usually a biological molecule such as an enzyme, antibody, receptor, nucleic acid, or whole cell that can specifically bind to a specific target molecule (analyte). When this binding occurs, a detectable signal is generated, usually a physical or chemical signal, which is converted into an electrical signal by the sensor.

[0003] Common virus testing methods include enzyme-linked immunosorbent assay, RT-PCR, nucleic acid sequence-based detection, gene chip technology, etc. However, these testing methods are relatively complex to operate, costly, and time-consuming. Therefore, a testing method that is simple to operate, less time-consuming, and less costly is needed.

[0004] In recent years, field effect transistor-based biosensors have become very attractive for the detection of target biomolecules due to their fast response speed, high detection sensitivity and label-free. However, due to the low concentration of viral sample DNA, a signal amplification strategy is needed to meet the needs of real sample detection. Therefore, a virus detection sensor and test method based on hyperbranched DNA rolling circle amplification are proposed. Summary of the invention

[0005] In view of this, the present invention provides a virus detection sensor and a testing method based on hyperbranched DNA rolling circle amplification to solve or alleviate the technical problems existing in the prior art and at least provide a beneficial choice.

[0006] The technical solution of the embodiment of the present invention is implemented as follows: a virus detection sensor based on hyperbranched DNA rolling circle amplification, comprising a carbon-based field effect transistor biosensor, wherein the carbon-based field effect transistor biosensor is a P-type device, which comprises, from bottom to top, a substrate SI layer, a SiO2 layer, a CNT channel layer and a HfO2 dielectric layer, wherein a source electrode and a drain electrode are symmetrically arranged on the SiO2 layer;

[0007] The surface of the CNT channel layer is modified with primers fixed thereon, which are used to generate HRCA reaction at the channel layer interface, causing the device current to rise, so as to detect viral DNA molecules;

[0008] The HfO2 dielectric layer is used to isolate the CNT channel layer from the charged material and perform electrostatic regulation, and the primer is modified on the HfO2 dielectric layer.

[0009] Further preferably, the charged substances include a primer solution with a complementary target strand, an amplification solution for performing HRCA reaction, and a target solution to be detected.

[0010] Further preferably, the viral DNA molecule is an RNA or DNA viral molecule.

[0011] A testing method for a virus detection sensor based on hyperbranched DNA rolling circle amplification, characterized in that it comprises the following steps:

[0012] S1. Primer fixation: The target primer is modified on the surface of the HfO2 dielectric layer on the CNT channel layer of the sensor by using CNT-FET. The target primer is divided into primer 1 and primer 2. After the virus mediates the RCA template molecule into a ring, primer 1 amplifies around the RCA template molecule, primer 2 continues to amplify with the amplification product of primer 1 as a template, and primer 1 continues to amplify with the amplification product of primer 2 as a template;

[0013] S2, HRCA reaction - drip the test liquid on the surface of the HfO2 dielectric layer of the sensor to carry out the connection reaction and amplification reaction; wherein the connection and amplification are carried out separately, firstly, the RCA template molecule and the linking enzyme are connected for 30 minutes in the presence of the virus; after inactivation at 90° for 10 minutes (this part is completed in a test tube), the liquid and the amplification liquid are mixed and dripped on the sensor surface of the channel modified primer 1 for amplification;

[0014] S3. Fluorescence test: using the property of syber green to bind to double-stranded DNA, syber green fluorescent dye is added to the surface of the sensor HfO2 dielectric layer and irradiated with laser to observe whether fluorescence is generated. If so, it indicates that the required DNA primer is successfully fixed on the HfO2 dielectric layer and HRCA reaction occurs, otherwise, it indicates that HRCA reaction does not occur;

[0015] S41, detection test - repeat S1 operation and modify the primer;

[0016] S42, adding a buffer solution to the surface of the HfO2 dielectric layer of the sensor, and performing measurement, and after obtaining an initial curve, performing amplification on the surface of the HfO2 dielectric layer of the sensor;

[0017] S43, washing away the buffer solution added in S42, and adding the target solution to be tested to the HfO2 dielectric layer area, and starting the measurement after the DNA hybridization is completed to obtain a comparison curve, wherein the gate is fixed with a sequence complementary to the virus fragment;

[0018] S44, repeat the operation of S41 to obtain a new initial curve;

[0019] S45, washing away the buffer solution added in S44, and dropping the amplification solution on the HfO2 dielectric layer area. After the amplification reaction is completed, the surface of the HfO2 dielectric layer is cleaned with pure water and blown dry with nitrogen. Then, the buffer solution is added again, and measurement is performed to obtain a test curve, wherein the gate is fixed with primer 1 in HRCA amplification;

[0020] S46. Compare the curves measured in S44 and S45. If the curve current increases, it means that the target DNA is detected to be amplified on the surface of the sensor HfO2 dielectric layer. Compare the curves measured in S43 and S45. If the S45 curve has a large response, it indicates that amplification detection on FET is better than direct detection.

[0021] Further preferably, the method of modifying the target primer on the surface of the sensor CNT channel layer by using CNT-FET comprises the following steps:

[0022] S11, connecting APTES to the interface of the sensor CNT channel layer with -OH;

[0023] S12, modifying MBS on the interface of the CNT channel layer with -NH;

[0024] S13. Finally, the primer with -SH is modified on the interface of the CNT channel layer with -NH to form a complementary target chain.

[0025] Further preferably, the ligation reaction is to connect an unblocked circular padlock probe (PLP) and the target DNA into a circle under the action of a ligase, and the ligation reaction time is 30-90 minutes;

[0026] The amplification reaction is that the forward primer is paired with the circular template under the action of polymerase to synthesize a linear single-stranded DNA sequence, and then a reverse primer is added to combine with the linear single-stranded DNA sequence to form a multi-branch amplification reaction, causing it to grow exponentially. The amplification reaction time is 30-60 minutes.

[0027] Further preferably, in S43, the DNA hybridization time is 5-10 minutes and the waiting time is 20-30 seconds.

[0028] Further preferably, the buffer solution is 0.01×PBS phosphate buffer solution.

[0029] Further preferably, the response calculation formula of the sensor is:

[0030]

[0031] Where ΔI is the change in drain-source current, I O is the initial drain-source current value.

[0032] Since the embodiment of the present invention adopts the above technical solution, it has the following advantages: the present invention detects viruses through a biosensor based on a carbon-based field-effect transistor. The device reaches the micron level, is easy to mass-produce, is low-cost and easy to carry, and utilizes the ultra-sensitivity of the carbon-based field-effect transistor to surface charge. The HRCA reaction will produce a large number of negatively charged DNA molecules, which makes the detection process time-saving, making up for the defect of the existing detection technology that the detection time is long due to the low concentration of the virus sample DNA.

[0033] The above summary is for illustrative purposes only and is not limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The structure diagram of the biosensor based on carbon-based field effect transistor of the present invention;

[0036] Figure 2 This is a schematic diagram of the primer modification principle of the present invention;

[0037] Figure 3 It is the principle diagram of HRCA amplification and on-channel amplification of the present invention;

[0038] Figure 4 It is the fluorescence image of the channel before and after amplification of the present invention;

[0039] Figure 5 A comparison chart of the sensor detection data of the present invention;

[0040] Figure 6 It is a linear fitting diagram of the current change of the DNA direct test and HRCA test of the present invention;

[0041] Figure 7 It is a comparison diagram of random strand and target strand amplification reaction data of the present invention;

[0042] Figure 8It is a comparison chart of the polymerase and dNTP concentration optimization data of the present invention. DETAILED DESCRIPTION

[0043] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0044] It should be noted that the terms "first", "second", "symmetrical", "array", etc. are only used to distinguish between description and position description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the definition of "first", "symmetrical", etc. can explicitly or implicitly include one or more of these features; similarly, when the number of certain features is not limited in the form of words such as "two" or "three", it should be noted that this feature also explicitly or implicitly includes one or more feature quantities.

[0045] Related terms:

[0046] CNT-FET, or Carbon Nanotube Field-Effect Transistor, is a field effect transistor that uses carbon nanotubes (CNTs) as a conductive channel. Carbon nanotubes are one-dimensional carbon materials with unique structures and excellent electronic properties. They are made of single or multi-layer graphene curls and have extremely high carrier mobility and good mechanical properties; Field Effect Transistor (FET) is an important semiconductor device that uses the electric field effect to control the flow of current.

[0047] HRCA, or Hyperbranched Rolling Circle Amplification, is a DNA-based biotechnology that is an improved rolling circle amplification (RCA) method. Rolling circle amplification is a technique used to amplify circular DNA molecules, while hyperbranched rolling circle amplification further optimizes this process to increase amplification efficiency and yield.

[0048] Syber green is a fluorescent dye used to detect double-stranded DNA (dsDNA). It emits weak fluorescence in the free state, but the fluorescence intensity is significantly enhanced after binding to double-stranded DNA. The maximum absorption wavelength of this dye is about 497nm, and the maximum emission wavelength is about 520nm.

[0049] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0050] Embodiment 1

[0051] like Figure 1 As shown, an embodiment of the present invention provides a virus detection sensor based on hyperbranched DNA rolling circle amplification, including a carbon-based field effect transistor biosensor, characterized in that the carbon-based field effect transistor biosensor is a P-type device;

[0052] Since DNA molecules are negatively charged, when DNA molecules are adsorbed on the dielectric layer of the device, the absolute value of the device threshold voltage will decrease, thereby causing the current of the device to increase. Therefore, this characteristic is used to detect viral DNA.

[0053] From bottom to top, there are substrate SI layer, SiO2 layer, CNT channel layer and HfO2 dielectric layer, and the source and drain are symmetrically arranged on the SiO2 layer;

[0054] The surface of the CNT channel layer is modified with primers fixed thereon, which are used to generate HRCA reaction at the interface of the channel layer, causing the device current to rise, thereby detecting viral DNA molecules.

[0055] The virus detection sensor detects viruses by fixing the primers required for HRCA on the field effect transistor channel. Before detection, the HRCA amplification solution is added to the channel to cause an HRCA reaction. Since the HRCA reaction will produce a large number of long DNA chains with branched structures, a large number of DNA molecules will be captured on the device surface, causing the device current to increase, thereby realizing the detection of viral DNA molecules.

[0056] Among them, the HfO2 dielectric layer is used to isolate the CNT channel layer from the charged material and perform electrostatic regulation;

[0057] Charged substances can shift the threshold voltage (VTH) of carbon-based devices in two main ways: one is that the charged substances directly transfer charges to carbon nanotubes (CNTs), and the other is that the charged substances are adsorbed on CNTs for electrostatic regulation. The carbon-based field effect transistor biosensor used in the present invention is a hafnium oxide (HfO2) dielectric layer plated in the CNT channel. Because the charged substances cannot directly contact the CNTs, only electrostatic regulation can occur.

[0058] In one embodiment, the charged substances include a primer solution with a complementary target strand, an amplification solution for performing an HRCA reaction, and a target solution to be detected.

[0059] In one embodiment, the viral DNA molecule is an RNA or DNA viral molecule.

[0060] Embodiment 2

[0061] like Figure 1-Figure 8 As shown, the embodiment of the present invention also provides a method for testing a virus detection sensor based on hyperbranched DNA rolling circle amplification, comprising the following steps:

[0062] S1. Primer fixation - CNT-FET is used to modify the target primer on the surface of the HfO2 dielectric layer on the CNT channel layer of the sensor. The target primer is divided into primer 1 and primer 2. After the virus mediates the RCA template molecule into a ring, primer 1 amplifies around the RCA template molecule, and primer 2 continues to amplify with the amplification product of primer 1 as a template. Primer 1 continues to amplify with the amplification product of primer 2 as a template. Primer 1 is fixed in the channel, and primer 2 is in the solution.

[0063] S2, HRCA reaction - drip the test liquid on the surface of the HfO2 dielectric layer of the sensor to carry out the connection reaction and amplification reaction; wherein the connection and amplification are carried out separately, firstly, the RCA template molecule and the linking enzyme are connected for 30 minutes in the presence of the virus; after inactivation at 90° for 10 minutes (this part is completed in a test tube), the liquid and the amplification liquid are mixed and dripped on the sensor surface of the channel modified primer 1 for amplification;

[0064] The ligation reaction is to connect an unblocked circular padlock probe (PLP) and the target DNA into a circle under the action of ligase. The ligation reaction time is 30-90 minutes. The two ends of the circular padlock probe (PLP) are completely complementary to the target DNA.

[0065] The amplification reaction is that the forward primer is paired with the circular template under the action of the polymerase to synthesize a linear single-stranded DNA sequence, and then the reverse primer is added to combine with the linear single-stranded DNA sequence to form a multi-branch amplification reaction, causing it to grow exponentially. The amplification reaction time is 30-60 minutes;

[0066] Specific as Figure 3 a: HRCA amplification principle diagram, from Figure 3 b: In the schematic diagram of on-channel amplification, it can be seen that after the primers are fixed on the channel, amplification on the device surface can be achieved by adding the required amplification solution.

[0067] S3. Fluorescence test: using the property of syber green to bind to double-stranded DNA, syber green fluorescent dye is added to the surface of the sensor HfO2 dielectric layer and irradiated with laser to observe whether fluorescence is generated. If so, it indicates that the required DNA primer is successfully fixed on the HfO2 dielectric layer and HRCA reaction occurs, otherwise, it indicates that HRCA reaction does not occur;

[0068] The fluorescence effect before and after amplification is as follows Figure 4 shown.

[0069] S41, detection test - repeating the S1 operation to modify the complementary target chain on the surface of the sensor CNT channel layer;

[0070] S42, dropping a buffer solution (0.01×PBS) on the surface of the HfO2 dielectric layer of the sensor, and performing measurement, after obtaining an initial curve, performing amplification on the surface of the HfO2 dielectric layer of the sensor;

[0071] S43, washing away the buffer solution added in S42, and adding the target solution to be tested to the HfO2 dielectric layer area, after DNA hybridization is completed, the DNA hybridization time is 5-10min, the waiting time is 20-30s, and then starting the measurement to obtain a comparison curve, wherein the gate is fixed with a sequence complementary to the virus fragment;

[0072] S44, repeat the operation of S41 to obtain a new initial curve;

[0073] S45, washing off the buffer solution added in S44, and dropping the amplification solution on the HfO2 dielectric layer area. After the amplification reaction is completed, the surface of the HfO2 dielectric layer is cleaned with pure water and blown dry with nitrogen, and then the buffer solution (0.01×PBS) is added, and measurement is performed to obtain a test curve, wherein the gate is fixed with primer 1 in HRCA amplification;

[0074] S46. Compare the curves measured in S44 and S45. If the curve current increases, it means that the target DNA is detected to be amplified on the surface of the sensor HfO2 dielectric layer. Compare the curves measured in S43 and S45. If the S45 curve has a large response, it indicates that amplification detection on FET is better than direct detection.

[0075] In one embodiment, the target primer is modified on the surface of the sensor CNT channel layer using CNT-FET, comprising the following steps:

[0076] S11, connecting APTES to the interface of the sensor CNT channel layer with -OH;

[0077] S12, modifying MBS on the interface of the CNT channel layer with -NH;

[0078] S13, finally modifying the primer with -SH on the interface of the CNT channel layer with -NH to form a complementary target chain;

[0079] The specific principle is as follows Figure 2 shown.

[0080] In one embodiment, the sensor response calculation formula is:

[0081]

[0082] Where ΔI is the change in drain-source current, I O is the initial drain-source current value.

[0083] In one embodiment, the specific test data is as follows: Figure 5-Figure 6 As shown:

[0084] Figure 5 In the figure, a: direct complementary chain response; b: HRCA amplification response; c: comparison of direct complementary detection and HRCA detection response; d: comparison of direct complementary detection and HRCA signal-to-noise ratio (SNR).

[0085] Figure 6 In the figure, a: direct complementary detection linear fitting; b: HRCA detection linear fitting.

[0086] like Figure 5 As shown in a, when a target DNA solution ranging from 200aM to 2pm was introduced, the current increased from 2.7% to 10.1%. It is worth noting that after the HRCA reaction process, the current increased significantly compared to when the target DNA was directly introduced, and the current change after amplification of target DNA concentrations from 200aM to 2pm increased from 10.8% to 33%. As the target DNA concentration increased, the drift current increased, and HRCA amplification was effective at all concentrations. In addition, compared with direct detection, the signal-to-noise ratio of amplification detection on FET was improved by more than 100%, from Figure 5 It can be seen that the device is sensitive to aM concentration level (10 -18 M) DNA responded, demonstrating the high sensitivity of the device.

[0087] Figure 6 It is a linear fitting diagram of the current changes of DNA direct test and HRCA test, which can more intuitively show the response trends of the two test methods. It can be seen that the current change of HRCA detection is more obvious than that of direct complementary detection, and it is easier to detect the target.

[0088] In one embodiment, to demonstrate the high specificity of the designed biosensor, the response of random strands and target strands amplified on FET is compared. The detection results are as follows: Figure 7 As shown:

[0089] Figure 7 In the figure, a: random chain different amplification time response; b: target chain different amplification time response; c: random chain and target chain different amplification time response bar graph; d: random chain and target chain different amplification time response linear fitting;

[0090] Figure 7 a is the amplification response of the random chain on the FET. The results show that the leakage current detected does not change significantly with the extension of the amplification time. Figure 7 b It can be seen that as the amplification time of the target chain increases, the leakage current obtained on the FET becomes larger and larger, indicating that a large amount of charge is introduced during the amplification process. This shows that the amplification can only perform HRCA reaction on a specific target chain, reflecting that the designed biosensor has a high specificity.

[0091] In one embodiment, the selected parameters are optimized to obtain the best detection performance. Since HRCA is an exponential amplification reaction, the amount of Bst DNA polymerase and the concentration of dNTPs will directly affect the amount of ssDNA and dsDNA amplified by HRCA, which is optimized first. Figure 8 As shown;

[0092] Figure 8 In, a: polymerase concentration optimization; b: dNTP concentration optimization;

[0093] from Figure 8 As can be seen from a, with the increase in the amount of phi29 DNA polymerase, the response gradually increased and reached saturation at 6U polymerase concentration, indicating that 6U of phi29 DNA polymerase is sufficient for HRCA reaction. Figure 8 b As can be seen, the dNTP concentration has a great influence on the HRCA reaction. The response increases significantly with the increase of dNTP concentration and tends to be stable after 500uM, so the optimized concentration of 500uM dNTP is used.

[0094] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A virus detection sensor based on hyperbranched DNA rolling circle amplification, comprising a carbon-based field effect transistor biosensor, characterized in that: The carbon-based field effect transistor biosensor is a P-type device, which includes a substrate SI layer, a SiO2 layer, a CNT channel layer and a HfO2 dielectric layer from bottom to top, and a source electrode and a drain electrode are symmetrically arranged on the SiO2 layer; The surface of the CNT channel layer is modified with primers fixed thereon, which are used to generate HRCA reaction at the channel layer interface, causing the device current to rise, so as to detect viral DNA molecules; The HfO2 dielectric layer is used to isolate the CNT channel layer from the charged material and perform electrostatic regulation, and the primer is modified on the HfO2 dielectric layer.

2. The virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 1, characterized in that: The charged substances include a primer solution with a complementary target chain, an amplification solution for HRCA reaction, and a target solution to be detected.

3. The virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 1, characterized in that: The viral DNA molecule is an RNA or DNA viral molecule.

4. A testing method for a virus detection sensor based on hyperbranched DNA rolling circle amplification according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Primer fixation: The target primer is modified on the surface of the HfO2 dielectric layer on the CNT channel layer of the sensor by using CNT-FET. The target primer is divided into primer 1 and primer 2. After the virus mediates the RCA template molecule into a ring, primer 1 amplifies around the RCA template molecule, primer 2 continues to amplify with the amplification product of primer 1 as a template, and primer 1 continues to amplify with the amplification product of primer 2 as a template; S2, HRCA reaction - drip the test liquid on the surface of the HfO2 dielectric layer of the sensor to carry out the connection reaction and amplification reaction; wherein the connection and amplification are carried out separately, firstly, the RCA template molecule and the linking enzyme are connected for 30 minutes in the presence of the virus; after inactivation at 90° for 10 minutes (this part is completed in a test tube), the liquid and the amplification liquid are mixed and dripped on the sensor surface of the channel modified primer 1 for amplification; S3. Fluorescence test: using the property of syber green to bind to double-stranded DNA, syber green fluorescent dye is added to the surface of the sensor HfO2 dielectric layer and irradiated with laser to observe whether fluorescence is generated. If so, it indicates that the required DNA primer is successfully fixed on the HfO2 dielectric layer and HRCA reaction occurs, otherwise, it indicates that HRCA reaction does not occur; S41, detection test - repeat S1 operation and modify the primer; S42, adding a buffer solution to the surface of the HfO2 dielectric layer of the sensor, and performing measurement, and after obtaining an initial curve, performing amplification on the surface of the HfO2 dielectric layer of the sensor; S43, washing away the buffer solution added in S42, and adding the target solution to be tested to the HfO2 dielectric layer area, and starting the measurement after the DNA hybridization is completed to obtain a comparison curve, wherein the gate is fixed with a sequence complementary to the virus fragment; S44, repeat the operation of S41 to obtain a new initial curve; S45, washing away the buffer solution added in S44, and dropping the amplification solution on the HfO2 dielectric layer area. After the amplification reaction is completed, the surface of the HfO2 dielectric layer is cleaned with pure water and blown dry with nitrogen. Then, the buffer solution is added again, and measurement is performed to obtain a test curve, wherein the gate is fixed with primer 1 in HRCA amplification; S46. Compare the curves measured in S44 and S45. If the curve current increases, it means that the target DNA is detected to be amplified on the surface of the sensor HfO2 dielectric layer. Compare the curves measured in S43 and S45. If the S45 curve has a large response, it indicates that amplification detection on FET is better than direct detection.

5. The test method of the virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 4, characterized in that: The method of modifying the target primer on the surface of the sensor CNT channel layer by using CNT-FET comprises the following steps: S11, connecting APTES to the interface of the sensor CNT channel layer with -OH; S12, modifying MBS on the interface of the CNT channel layer with -NH; S13. Finally, the primer with -SH is modified on the interface of the CNT channel layer with -NH to form a complementary target chain.

6. The test method of the virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 4, characterized in that: The ligation reaction is to connect an unblocked circular padlock probe (PLP) and the target DNA into a circle under the action of a ligase, and the ligation reaction time is 30-90 minutes; The amplification reaction is that the forward primer is paired with the circular template under the action of polymerase to synthesize a linear single-stranded DNA sequence, and then a reverse primer is added to combine with the linear single-stranded DNA sequence to form a multi-branch amplification reaction, causing it to grow exponentially. The amplification reaction time is 30-60 minutes.

7. The test method of the virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 4, characterized in that: In the S43, the DNA hybridization time is 5-10 minutes and the waiting time is 20-30 seconds.

8. The test method of the virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 4, characterized in that: The buffer solution is 0.01×PBS phosphate buffer solution.

9. The test method of the virus detection sensor based on hyperbranched DNA rolling circle amplification according to claim 4, characterized in that: The sensor response calculation formula is: Where ΔI is the change in drain-source current, I O is the initial drain-source current value.

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