Anti-nonspecific adsorption reagent for FET biosensor and screening method thereof, FET biosensor and application thereof

Abstract

The present invention provides a method for screening anti-nonspecific adsorption reagents for FET biosensors. By combining reagents with different terminal functional groups, charges and hydrophilicity, and through the synergistic effects of hydrogen bonds, steric effects and electrostatic repulsion, multi-attribute and multi-ratio anti-nonspecific adsorption reagents for target detection systems are screened. The process is controllable and highly targeted, and anti-nonspecific adsorption reagents with excellent effects are screened more accurately for target detection systems, providing guarantees for subsequent detection. The anti-nonspecific adsorption reagents screened by the method of the present invention can improve the hydrophilicity of the interface, produce steric effects, and enhance electrostatic repulsion through the interaction between the different terminal functional groups of the thiol compound reagents, thereby reducing the adsorption of non-targets in the target detection system and showing strong anti-fouling performance. The modified FET biosensor has a wider linear detection range and an extremely low detection limit, strong anti-interference ability, high sensitivity, and fast response time.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensor antifouling, and in particular to an anti-nonspecific adsorption reagent for FET biosensors and a screening method thereof, and realizes the application of carbon nanotube field effect transistor biosensors with thiol antifouling layers. Background Art

[0002] Field-effect transistor (FET) biosensors based on low-dimensional semiconductor materials offer inherent advantages such as high sensitivity, rapid response, label-free operation, and scalable integration. Through electrostatic coupling between charged biomolecules and semiconductor channels, the concentration of target biomolecules can be highly sensitively detected and transmitted as readable electrical signals. However, due to the extremely high sensitivity of FET biosensors, even small amounts of nonspecific adsorption can cause significant signal responses, leading to the appearance of false signals and affecting detection results.

[0003] FET biosensors face significant challenges in detecting biomolecules in complex biological systems. Severe nonspecific adsorption in complex biological environments can lead to degradation of FET device performance and lifespan, while also severely impacting the reliability of FET biosensors. Consequently, anti-fouling technologies for FET biosensors have garnered increasing attention and research.

[0004] Existing antifouling technologies for FET biosensors mainly include: modifying the sensing interface with polymers and blocking redundant recognition sites with macromolecules. However, these two mainstream biosensor antifouling technologies sacrifice the sensitivity of FET biosensors, preventing them from realizing their advantages. On the other hand, current antifouling technologies do not take into account the complexity and specificity of the target detection system. Different systems often have different characteristics. For example, compared with other systems, milk samples contain large amounts of fat, phospholipids, proteins, and small molecules. These substances vary in molecular weight, functional groups, isoelectric points, and hydrophilicity. Using traditional single-functional group antifouling reagents, it is difficult to achieve good antifouling performance and meet the needs of biosensors through a single hydrophilic-hydrophobic interaction.

[0005] In current practical applications, the selection of antifouling agents for FET biosensors is essentially determined by experience or direct laboratory testing. This selection is random and uncontrollable, and the specific detection system is not considered. As a result, the final FET biosensor has poor antifouling effect when used in complex systems, which affects the final detection effect. Summary of the Invention

[0006] The present invention aims to address the problem that the selection of anti-fouling reagents for FET biosensors in the prior art is random and does not take into account the specific detection system, resulting in poor anti-fouling effect of FET biosensors when used in complex systems, thereby affecting the ultimate detection effect. The present invention provides a method for screening anti-nonspecific adsorption reagents for FET biosensors. The method can more accurately screen out anti-nonspecific adsorption reagents with excellent effects for the target detection system. FET biosensors modified with the screened anti-nonspecific adsorption reagents have excellent resistance to nonspecific signal interference, as well as excellent sensitivity and stability.

[0007] The first aspect of the present invention relates to a method for screening an anti-nonspecific adsorption agent for a FET biosensor, comprising the following steps:

[0008] S1. Preliminary screening

[0009] Sample preparation: Dissolve thiol compound reagents with different terminal functional groups in PBS to prepare corresponding first solutions;

[0010] Depositing a gold film on a silicon oxide substrate using an electron beam coating apparatus, and modifying the prepared gold film with each set of the first solution to obtain a corresponding self-assembled monolayer;

[0011] Hydrophilicity and hydrophobicity testing: The target detection system was added to each group of self-assembled monolayers, and the contact angle of the target detection system on the membrane layer was measured and compared with that of a first control group. The first solution with a smaller contact angle than that of the first control group was selected. The first control group consisted of the target detection system added to an unmodified gold film.

[0012] Nonspecific adsorption investigation: The adsorption of each group of self-assembled monolayers to non-targets in the target detection system was investigated and compared with a second control group. The first solution with a smaller adsorption amount than that of the second control group was selected. The second control group was the adsorption amount of the unmodified gold film to non-targets in the target detection system.

[0013] Selecting a first solution that meets both the hydrophilicity and hydrophobicity requirements and the nonspecific adsorption requirements, and preliminarily screening the corresponding first solution;

[0014] S2. Fine screening

[0015] The first solution selected in step S1 is modified on a floating gate field effect transistor with gold particles deposited on the channel surface, and the transfer curves of the target detection system before and after incubation are measured to determine the current response of the first solution to the interferent, and the corresponding nonspecific response values are calculated, and the order is based on the size of the nonspecific response values;

[0016] The thiol compound reagent corresponding to the group with the smallest non-specific response value is defined as group 1, and the corresponding thiol compound reagents are defined as group 2, group 3, etc. according to the increasing non-specific response value, until the Nth group, wherein N ≥ 2;

[0017] Dividing N groups of thiol compound reagents into M items, each item containing a mixed solution composed of solutions of the N groups of thiol compound reagents, and the groups in each item are consecutive groups starting from group 1, wherein M = N-1 and N ≥ 2;

[0018] S3. Optimized screening

[0019] The M items obtained in step S2 are tested according to the first method to screen out the desired anti-nonspecific adsorption reagent;

[0020] Among them, the first method is as follows:

[0021] The groups in each item are prepared into solutions of different concentrations and mixed according to different molar ratios to obtain multiple groups of mixed reagents. Each group of mixed reagents is modified on a floating-gate field-effect transistor with gold particles deposited on the channel surface. The transfer curves before and after incubation of the target detection system are measured to determine the current response of the mixed reagent to the interferent, and the corresponding nonspecific response value is calculated. The mixed reagent is selected when the nonspecific response value is less than 5%.

[0022] As an optional example, the specific process of optimization screening is as follows:

[0023] Starting from the first item obtained in step S2, test according to the first method and judge according to the second method; wherein the second method is as follows:

[0024] The mixed reagent used when the nonspecific response value is less than 5% is used as the required anti-nonspecific adsorption reagent, and the screening is completed;

[0025] When there is no non-specific response value less than 5% in the test result, continue to test the next item according to the first method and judge according to the second method until the required anti-non-specific adsorption reagent is screened out.

[0026] As an optional example, in the process of continuing to test the next item according to the first method, the items are tested in order from the 2nd item to the Mth item.

[0027] As an optional example, in the first method, the groups in each item are prepared into solutions with different concentrations, and the concentration of the substance in each group is controlled at (1-4) mM.

[0028] As an optional example, in step S1, the concentration of each group of first solutions is 10 mM.

[0029] As an optional example, the preparation process of the self-assembled monolayer is as follows:

[0030] The prepared gold film was incubated with a candidate antifouling solution at 25°C until the thiol compound was fully bound to the gold film through the gold-thiol bond. After the incubation, the gold film was rinsed with PBS and ultrapure water by lateral flow and dried with nitrogen to complete the preparation of the self-assembled monolayer film.

[0031] As an alternative example, nonspecific adsorption was investigated using inverted fluorescence microscopy.

[0032] As optional examples, the thiol compound reagents having different terminal functional groups are sodium 2-mercaptoethanesulfonate, mercaptoethanol, methyl thioglycolate, thioglycolic acid, cysteine, adamantanethiol and perfluorodeoxythiol.

[0033] As an optional example, when the target detection system is milk, the anti-nonspecific adsorption reagent screened out is sodium 2-mercaptoethanesulfonate and mercaptoethanol in a molar ratio of 1:(1-4).

[0034] The second aspect of the present invention relates to an anti-nonspecific adsorption reagent for FET biosensor screened by the above method, wherein the anti-nonspecific adsorption reagent comprises at least two thiol compounds, and the terminal functional groups of the thiol compounds are different from each other.

[0035] The third aspect of the present invention relates to a FET biosensor modified with the aforementioned anti-nonspecific adsorption reagent for FET biosensor.

[0036] A fourth aspect of the present invention relates to the use of the aforementioned FET biosensor in detecting glucose, protein, antibody, antigen, enzyme, virus, DNA or RNA in a complex physiological environment.

[0037] The present invention proposes a method for screening anti-nonspecific adsorption reagents for FET biosensors. By combining reagents with different terminal functional groups, charges, and hydrophilicity, and through the synergistic effects of hydrogen bonding, steric effects, and electrostatic repulsion, the method can screen multi-attribute, multi-ratio anti-nonspecific adsorption reagents for the target detection system. The process is controllable and highly targeted, and can more accurately screen out anti-nonspecific adsorption reagents with excellent effects for the target detection system, providing a guarantee for subsequent detection.

[0038] The anti-nonspecific adsorption reagent screened out by the method of the present invention can improve the hydrophilicity of the interface through the interaction between the different terminal functional groups of the thiol compound reagent, making the modified surface more hydrophilic, and mutually repelling the hydrophobic areas of the non-target in the target system, thereby exhibiting better anti-fouling performance; on the other hand, the amount of charge carried by different terminal functional groups is different, and local areas of different charges will be formed, showing a certain dual charge (positive charge and negative charge), and it is difficult to produce electrostatic interactions with the non-target of the target system; in addition, the molecular weight and size of different terminal functional groups will be different, which will form a certain height difference, produce a certain steric effect, reduce the adsorption of non-targets in the target detection system, and show good anti-fouling performance.

[0039] The FET biosensor modified with the anti-nonspecific adsorption reagent screened by the method of the present invention has a high-density and strong stability anti-nonspecific adsorption layer of the sensor, the signal is further suppressed, the sensor interface has more sensing sites, a wider linear detection range and an extremely low detection limit are achieved, the sensor performance is optimized, the sensor has a stronger anti-interference ability, higher sensitivity, and faster response time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic structural diagram of an exemplary FET biosensor of the present invention.

[0041] Figure 2 This invention Figure 1 Schematic diagram of the various components of the biosensor as viewed from the top of the device.

[0042] Figure 3 The present invention is a flow chart of a method for screening an anti-nonspecific adsorption agent for a FET biosensor.

[0043] Figure 4 Schematic diagram of the structures of seven exemplary thiol compounds of the present invention.

[0044] FIG5 is a schematic diagram showing a comparison of contact angles of seven thiol compounds in PBS and milk in Example 1 of the present invention; wherein 5a is a contact angle actual test diagram; and 5b is a statistical diagram of 5a.

[0045] FIG6 is a schematic diagram showing the comparison of the nonspecific adsorption strength of seven thiol compounds on fluorescently labeled casein in Example 1 of the present invention; wherein 6a is a fluorescence intensity display diagram; and 6b is a statistical diagram of the fluorescence intensity results.

[0046] Figure 7 is a schematic diagram of the transfer curves and statistical results of the response sizes of seven thiol reagents used as anti-fouling layers for field-effect transistors before and after incubation in milk in Example 1 of the present invention; wherein, 7a is the transfer curve before and after incubation, and 7b is a schematic diagram of the statistical results of the response size.

[0047] Figure 8 This is a schematic diagram of the statistical results of the response changes of a field effect transistor before and after incubation with milk when two thiol reagents, sodium 2-mercaptoethanesulfonate and mercaptoethanol, in different molar ratios selected in Example 1 of the present invention are applied.

[0048] Figure 9 This is a schematic diagram of resistance to nonspecific modification in Example 1 of the present invention.

[0049] Figure 10 Schematic diagram of the detection principle of the modified biosensor in Example 1 of the present invention.

[0050] FIG11 is a schematic diagram of the specificity and anti-interference test results of the modified biosensor in Example 1 of the present invention; wherein 11a is a schematic diagram of the specificity test results, and 11b is a schematic diagram of the anti-interference test results.

[0051] Figure 12 12a and 12b are schematic diagrams of the test states before and during the test when the anti-nonspecific modification field effect transistor is used as the anti-fouling layer, and 12c and 12d are schematic diagrams of the test states before and during the test when the anti-nonspecific modification field effect transistor is used as the anti-fouling layer, respectively.

[0052] Figure 13 is a schematic diagram of the results of kanamycin sensitivity detection and linear analysis of the modified biosensor in Example 1 of the present invention tested in a milk environment; wherein 13a is the transfer curve of the FET biosensor as the kanamycin concentration changes, and 13b is the linear fitting result of the source-drain current change rate of the device as the kanamycin concentration changes in the logarithmic coordinate system under the condition of a gate voltage of -1.5V.

[0053] Figure 14 is a schematic diagram of the results of kanamycin sensitivity detection and linear analysis of the biosensor without anti-fouling modification in a milk environment in Example 1 of the present invention; wherein 14a is the transfer curve of the FET biosensor as the kanamycin concentration changes, and 14b is the linear fitting result of the source-drain current change rate of the device as the kanamycin concentration changes under the condition of a gate voltage of -1.5V in a logarithmic coordinate system. DETAILED DESCRIPTION

[0054] In order to better understand the technical content of the present invention, specific embodiments are given and described below with reference to the accompanying drawings.

[0055] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to be comprehensive. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of a number of ways.

[0056] In order to better inhibit the nonspecific adsorption of FET biosensors and improve the detection effect of FET biosensors in actual complex systems, the present invention designs a screening method for anti-nonspecific adsorption reagents for FET biosensors. The screening process is controllable and highly targeted, and can more accurately screen out anti-nonspecific adsorption reagents with excellent effects for the target detection system, providing a guarantee for subsequent detection and also providing a theoretical basis for the selection of anti-nonspecific adsorption reagents.

[0057] FET biosensors

[0058] Combine Figure 1 The FET biosensor of the exemplary embodiment shown includes a silicon-based substrate 10 , a silicon dioxide dielectric layer 20 , a CNT channel layer 30 , a drain electrode 41 , a source electrode 42 , a passivation layer 43 , a dielectric layer 50 , and a connection layer 60 .

[0059] The silicon-based substrate 10 defines a first surface and an opposite second surface. Figure 1 As shown, for the sake of convenience, the first surface is used as the growth surface to be prepared subsequently. The silicon-based substrate 10 can be made of highly doped P-type silicon.

[0060] Combine Figure 1 The silicon dioxide dielectric layer 20 is located on the first surface of the silicon-based substrate 10. As an optional example, the silicon dioxide dielectric layer 20 is used as a deposition base for carbon nanotubes (CNTs), and electrical insulation between the CNTs and the silicon-based substrate 10 is achieved.

[0061] like Figure 1 In the example shown in FIG. 1 , the CNT channel layer 30 is located on the side of the silicon dioxide dielectric layer 20 away from the silicon substrate (i.e., the upper surface of the silicon dioxide dielectric layer 20). The CNT channel layer 30 has a certain size and is randomly laid flat on the upper surface of the silicon dioxide dielectric layer 20. The diameter of each carbon nanotube is controlled to be 1-3 nm.

[0062] The drain electrode 41 and the source electrode 42 are located on a surface of the CNT channel layer 30 away from the substrate 10. The drain electrode 41 and the source electrode 42 are disposed opposite to each other and spaced apart, with a dielectric layer 50 deposited therebetween.

[0063] The dielectric layer 50 is located above the CNT channel layer 40 on a side away from the silicon-based substrate 10 and between the drain electrode 41 and the source electrode 42. The dielectric layer must completely cover the entire CNT channel area, and the entire area between the drain electrode 41 and the drain electrode 42 is covered by the dielectric layer.

[0064] Combine Figure 2 As shown, the drain electrode 41 and the source electrode 42 are both connected to the corresponding two output electrodes 100 via their respective corresponding metal leads 90 to achieve signal output.

[0065] The solution gate and the silver wire that applies the gate voltage are located on the upper surface of the dielectric layer 50 and the connection layer 60, away from the substrate. Gate signal input and output are transmitted via the silver wire inserted into the solution. The diameter of the silver wire can be 0.4 mm, and the solution gate can be made of 0.01×PBS with a volume of 10 μL.

[0066] In an optional embodiment, the output electrode 100 can be electrically connected to an external device, such as a semiconductor analyzer, and the voltage signal input can be applied to the output electrode through a wire, and the output signal can be transmitted to the external device through the lead and the output electrode for signal analysis.

[0067] It can be understood that the metal leads 90 are all subjected to insulation packaging treatment, such as photoresist S1813, and are insulated from the conductive parts during detection.

[0068] Combine Figure 3 As shown, for the preparation process of the FET biosensor, the drain electrode 41 and the source electrode 42 on the Si / SiO2 interface are designed to be spaced apart, for example, the drain electrode 41 and the source electrode 42 are designed to be strip electrodes with a certain thickness.

[0069] The sensing interface is formed by stacking Y2O3 with HfO2, which is then modified with gold nanoparticles and incubated with the probe layer after passivation.

[0070] Gold nanoparticles constitute the connecting layer 60 , which has a theoretical thickness of 0.3 nm (corresponding to a diameter of a single particle of approximately 0.3 nm), and adjacent gold nanoparticles do not contact each other.

[0071] like Figure 2 As shown, a passivation layer 43 is also prepared on the surface of the drain electrode 41 and the source electrode 42 and the outer surface of the metal lead 90, for example, a passivation layer formed by S1813 photoresist packaging, so that the conductive area of the biosensor is not affected by the solution environment when detecting biological molecules in the solution, and only the CNT channel layer and the silver wire to which the voltage is applied are exposed to the liquid environment.

[0072] As an optional example, the thickness of the source electrode and the drain electrode is the same and is controlled to be 60-80 nm.

[0073] As a preferred example, the metal oxide dielectric layer is made of yttrium oxide stacked with hafnium oxide having a high dielectric constant, and the average thickness thereof is greater than or equal to 10 nm.

[0074] As an optional example, the length of the source electrode 41 and the drain electrode 42 is designed to be greater than 50 μm, and the width is greater than 5 μm. The distance between the source and drain electrodes is 20 μm. In designs where the drain electrodes 41 and the source electrodes 42 have the same or different lengths, the length of the source electrode 41 and the drain electrode 42 can reach 50 μm, 100 μm, or even greater than 500 μm.

[0075] In an optional embodiment, the widths of the drain electrode 41 and the source electrode 42 may be designed to have the same size or different sizes.

[0076] In a preferred embodiment, the drain electrode 41 and the source electrode 42 are made of three metals: Ti / Pd / Au, with a total thickness controlled to be 60-80nm. Ti has good wettability, which allows the contact metal to better adhere to the substrate. Pd with a high work function can form a P-type channel after contacting the CNT film, and Au is used as a conductive metal.

[0077] As an optional example, the metal lead is made of the same metal material as the source and drain electrodes, and has a length of more than 1000 μm.

[0078] As an optional example, the output electrode 100 (pad) is made of the same metal material as the source and drain electrodes, and its side length is 300 μm.

[0079] Combine Figure 2 As shown, the size of the output electrode 100 (pad) can be designed to be 300*300 μm, or the size can be designed according to needs, with a thickness of more than 60 nm.

[0080] Preparation process of FET biosensor

[0081] 1. Deposit a silicon dioxide dielectric layer on the substrate to form a Si / SiO2 structure;

[0082] 2. Using the silicon dioxide dielectric layer as the CNT growth surface, deposit the CNT film;

[0083] 3. Use a spin coater to coat the surface of the Si / SiO2 structure with the CNT film deposited thereon, use a photolithography process to expose the corresponding pattern, and etch the CNT film to obtain a CNT channel layer;

[0084] 4. Use a photolithography machine to expose the source electrode, drain electrode, metal lead and output electrode to which voltage is applied, and use an electron beam evaporation coating device to deposit metal to form the source electrode, drain electrode, metal lead and output electrode;

[0085] 5. Perform coating and exposure again, and use electron beam evaporation to evaporate metal yttrium on the upper surface of the CNT channel layer. The device is oxidized in air on a 270-degree hot plate to form yttrium oxide. Then, a layer of hafnium oxide is deposited on the yttrium oxide interface using an atomic layer deposition system to form a dielectric layer.

[0086] 6. Perform the coating, photolithography, and development steps again, and use an electron beam evaporation coating device to deposit a connection layer on the surface of the prepared dielectric layer;

[0087] 7. Perform the photolithography and development step again to encapsulate the source electrode, the drain electrode, and the metal lead, so that when the biosensor measures the target analyte, only the biosensor interface and the silver wire for applying the gate voltage are exposed to the liquid environment; thus, the desired FET biosensor is obtained.

[0088] Screening method for anti-nonspecific adsorption reagents for FET biosensors

[0089] Combine Figure 3 As shown, in a preferred embodiment of the present invention, based on the above-mentioned FET biosensor, a method for screening an anti-nonspecific adsorption agent for a FET biosensor is provided, comprising the following steps:

[0090] S1. Preliminary screening

[0091] Sample preparation: Dissolve thiol compound reagents with different terminal functional groups in PBS to prepare corresponding first solutions;

[0092] Depositing a gold film on a silicon oxide substrate using an electron beam coating apparatus, and modifying the prepared gold film with each set of the first solution to obtain a corresponding self-assembled monolayer;

[0093] Hydrophilicity and hydrophobicity testing: The target detection system was added to each group of self-assembled monolayers, and the contact angle of the target detection system on the membrane layer was measured and compared with that of a first control group. The first solution with a smaller contact angle than that of the first control group was selected. The first control group consisted of the target detection system added to an unmodified gold film.

[0094] Nonspecific adsorption investigation: The adsorption of each group of self-assembled monolayers to non-targets in the target detection system was investigated and compared with a second control group. The first solution with a smaller adsorption amount than that of the second control group was selected. The second control group was the adsorption amount of the unmodified gold film to non-targets in the target detection system.

[0095] A first solution that meets both the hydrophilicity and the nonspecific adsorption requirements is selected, and the corresponding first solution is preliminarily screened.

[0096] S2. Fine screening

[0097] Modifying the first solution screened in step S1 on the aforementioned FET biosensor, measuring the transfer curve before and after incubation of the target detection system, determining the current response of the first solution to the interferent, and calculating the corresponding nonspecific response value, and ranking according to the size of the nonspecific response value;

[0098] The thiol compound reagent corresponding to the group with the smallest non-specific response value is defined as group 1, and the corresponding thiol compound reagents are defined as group 2, group 3, etc. according to the increasing non-specific response value, until the Nth group, wherein N ≥ 2;

[0099] N groups of thiol compound reagents are divided into M items, each item is a mixed solution composed of solutions of N groups of thiol compound reagents, and the groups in each item are consecutive groups starting from the first group, wherein M=N-1, N≥2.

[0100] S3. Optimized screening

[0101] The M items obtained in step S2 are tested according to the first method to screen out the desired anti-nonspecific adsorption reagent;

[0102] Among them, the first method is as follows:

[0103] The groups in each item are prepared into solutions of different concentrations and mixed according to different molar ratios to obtain multiple groups of mixed reagents. Each group of mixed reagents is modified on the aforementioned FET biosensor, and the transfer curves before and after incubation of the target detection system are measured to determine the current response of the mixed reagent to the interfering substance, and the corresponding nonspecific response value is calculated. The mixed reagent is selected when the nonspecific response value is less than 5%.

[0104] As an optional example, the specific process of optimization screening is as follows:

[0105] Starting from the first item obtained in step S2, test according to the first method and judge according to the second method; wherein the second method is as follows:

[0106] The mixed reagent used when the nonspecific response value is less than 5% is used as the required anti-nonspecific adsorption reagent, and the screening is completed;

[0107] When there is no non-specific response value less than 5% in the test result, continue to test the next item according to the first method and judge according to the second method until the required anti-non-specific adsorption reagent is screened out.

[0108] As an optional example, in the process of continuing to test the next item according to the first method, the items are tested in order from the 2nd item to the Mth item.

[0109] As an optional example, in the first method, the groups in each item are prepared into solutions of different concentrations, and the concentration of the substances in each group is controlled at (1-4) mM; the concentration value needs to be controlled within a reasonable range to ensure that the substances between different groups can work together.

[0110] In an exemplary embodiment, after preliminary screening, it is assumed that four groups of first solutions are preliminarily screened out and are labeled as Group 1, Group 2, Group 3, and Group 4;

[0111] In the fine screening process, each group of first solutions is modified on the aforementioned FET biosensor to obtain corresponding nonspecific response values. It is assumed that the order of the nonspecific response values is group 1 < group 2 < group 3 < group 4.

[0112] The four groups of first solutions are divided into three items, wherein the first item is a mixed solution composed of solutions of the thiol compound reagents of the first and second groups, the second item is a mixed solution composed of solutions of the thiol compound reagents of the first, second and third groups, and the third item is a mixed solution composed of solutions of the thiol compound reagents of the first, second, third and fourth groups.

[0113] Then the optimization screening process is carried out:

[0114] In item 1: the solutions of the thiol compound reagents of group 1 and group 2 are mixed in a molar ratio of (1 to 4):(1 to 4) to obtain a first mixed solution having a different mixing ratio;

[0115] In item 2: the solutions of the thiol compound reagents of group 1, group 2, and group 3 are mixed in a molar ratio of (1-4):(1-4):(1-4) to obtain a second mixed solution having a different mixing ratio;

[0116] In item 3: the solutions of the thiol compound reagents of group 1, group 2, group 3 and group 4 are mixed in a molar ratio of (1-4):(1-4):(1-4):(1-4) to obtain a third mixed solution of different mixing ratios;

[0117] Each mixed solution in each item was modified on the aforementioned FET biosensor, and the transfer curves before and after incubation of the target detection system were measured to determine the current response of the mixed solution to the interferent, and the corresponding nonspecific response value was calculated. The mixed solution was selected when the nonspecific response value was less than 5%.

[0118] In a preferred embodiment, the optimization screening process is carried out as follows to improve the screening efficiency:

[0119] Each mixed solution in item 1 is modified on the aforementioned FET biosensor, and the transfer curves before and after incubation of the target detection system are measured to determine the current response of the mixed solution to the interferent, and the corresponding nonspecific response value is calculated. The mixed solution with a nonspecific response value of less than 5% is selected for use;

[0120] At this point, if a mixed solution can be selected in item 1 when the nonspecific response value is less than 5%, the mixed solution is the screened anti-nonspecific adsorption reagent, and the screening process is completed;

[0121] When each group of mixed solutions in item 1 cannot meet the requirement of non-specific response value less than 5%, each group of mixed solutions in item 2 is tested. If a mixed solution can be selected in item 2 for use when the non-specific response value is less than 5%, the mixed solution is the required anti-non-specific adsorption reagent screened out, and the screening process ends; if the requirement of non-specific response value less than 5% cannot be met, each group of mixed solutions in item 3 is tested.

[0122] It is understandable that more than one group of mixed solutions can be screened, as long as the nonspecific response value is less than 5%. On the basis of satisfying the nonspecific response value of less than 5%, the mixed solution with the smallest nonspecific response value is particularly preferred as the anti-nonspecific adsorption reagent.

[0123] As an optional example, in step S1, the concentration of each group of first solutions is 10 mM.

[0124] As an optional example, the preparation process of the self-assembled monolayer is as follows:

[0125] The prepared gold film was incubated with a candidate antifouling solution at 25°C until the thiol compound was fully bound to the gold film through the gold-thiol bond. After the incubation, the gold film was rinsed with PBS and ultrapure water by lateral flow and dried with nitrogen to complete the preparation of the self-assembled monolayer film.

[0126] As an alternative example, nonspecific adsorption was investigated using inverted fluorescence microscopy.

[0127] As optional examples, the thiol compound reagents having different terminal functional groups are sodium 2-mercaptoethanesulfonate, mercaptoethanol, methyl thioglycolate, thioglycolic acid, cysteine, adamantanethiol and perfluorodeoxythiol.

[0128] As an optional example, when the target detection system is milk, the anti-nonspecific adsorption reagent screened out is sodium 2-mercaptoethanesulfonate and mercaptoethanol in a molar ratio of 1:(1-4), and the molar ratio of sodium 2-mercaptoethanesulfonate and mercaptoethanol is particularly preferably 1:1.

[0129] In a typical embodiment, milk is used as the target detection system, and the screening of anti-nonspecific adsorption reagents is carried out based on the aforementioned FET biosensor. The specific process is as follows:

[0130] Step 1: Prepare seven thiol blocking reagents with different terminal functional groups, including sodium 2-mercaptoethanesulfonate (Mesna), mercaptoethanol (MCH), methyl thioglycolate (MTG), thioglycolic acid (TGA), cysteine (Cys), adamantanethiol (ADT), and perfluorodeoxythiol (PFDT). Dilute these 7 thiol reagents to a concentration of 10 mM with PBS. The molecular structures of the seven thiol reagents are as follows: Figure 4 shown.

[0131] Step 2: Use an electron beam coating device to deposit Ti / Au = 20 / 40 nm on the silicon oxide substrate to prepare a gold film with a size of 0.5 cm × 0.5 cm.

[0132] Step 3: Incubate the prepared gold film with seven thiol compounds at a concentration of 10 mM at 25°C for 8 hours to complete the preparation of the thiol antifouling layer. The volume of the added reagents is uniformly controlled at 10 μL. Finally, rinse the gold film with PBS and ultrapure water and blow dry with nitrogen gas.

[0133] Step 4: Add 5 μL of PBS and milk respectively on the prepared gold film with anti-fouling layer, observe with a fully automatic integral tilt contact angle analyzer, measure the contact angle in the fitted ellipse mode, and record the contact angle test results of multiple groups of PBS and milk respectively. The smaller the contact angle, the better the wettability, which means the degree of contamination of the membrane is lighter; compare the results with the control group, in which 5 μL of PBS and milk are added respectively on the gold film without modified anti-fouling layer, and select the group with smaller contact angle than the control group.

[0134] A gold film with an antifouling layer was modified with fluorescently labeled casein at a concentration of 5 mg / mL. The volume of the added reagent was uniformly controlled at 5 μL, the incubation temperature was 25°C, and the incubation time was 4 h. The gold film was then washed with PBS and ultrapure water by lateral flow and blown dry with nitrogen. The fluorescence intensity of the gold film modified with different thiol antifouling agents was observed under an inverted fluorescence microscope. The weaker the fluorescence intensity of the gold film, the smaller the amount of adsorbed protein, indicating a stronger anti-nonspecific adsorption ability. The results were compared with those of the control group, in which a gold film without an antifouling layer was modified with fluorescently labeled casein at a concentration of 5 mg / mL and incubated. The group with weaker fluorescence intensity than the control group was selected.

[0135] Step 5: Incubate the selected thiol antifouling reagent on the channel surface of the carbon nanotube field effect transistor at 25°C for 30 minutes, then incubate milk on the channel surface of the carbon nanotube field effect transistor at 25°C for 1 hour, and use a semiconductor analyzer to test the transfer characteristic curve of the transistor before and after milk incubation. Compare the current change of the device with the current change caused by nonspecific adsorption when the thiol antifouling layer is directly applied to the field effect transistor in the face of a complex milk environment, calculate the corresponding nonspecific response values, and sort them by size to select two thiol antifouling reagents with relatively small response values.

[0136] Step 6: preparing solutions of different concentrations of the two selected thiol compounds and mixing them in a molar ratio of (1-4):(1-4) to obtain multiple mixed solutions. Testing the corresponding nonspecific response values according to the method of Step 5, and selecting the mixed solution with a nonspecific response value less than 5%;

[0137] If no response value is less than 5%, three thiol antifouling reagents with relatively small response values are selected from the ranking in step 5, and mixed in a molar ratio of (1-4):(1-4):(1-4) to obtain multiple mixed solutions. The corresponding nonspecific response values are tested according to the method in step 5, and the mixed solution with a nonspecific response value less than 5% is selected;

[0138] If no response value is less than 5%, four thiol antifouling reagents with relatively small response values are selected from the ranking in step 5, and mixed in a molar ratio of (1-4):(1-4):(1-4):(1-4) to obtain multiple mixed solutions, and the mixed solutions are tested and selected when the nonspecific response value is less than 5%;

[0139] According to this rule, the screening can be terminated until a mixed solution is selected for use when the non-specific response value is less than 5%.

[0140] In another preferred embodiment of the present invention, an anti-nonspecific adsorption reagent for FET biosensor screened by the above method is provided, wherein the anti-nonspecific adsorption reagent comprises at least two thiol compounds, and the terminal functional groups of the thiol compounds are different from each other.

[0141] In another preferred embodiment of the present invention, a FET biosensor modified with the aforementioned anti-nonspecific adsorption reagent for FET biosensor is provided.

[0142] It can be understood that the anti-nonspecific adsorption reagent can be used in all types of FET biosensors. As an anti-nonspecific adsorption layer of the FET biosensor, it can reduce the nonspecific adsorption caused by hydrophobicity, electrostatic or other interactions to a certain extent through the synergistic effect of hydrogen bonding, steric effect and electrostatic repulsion, thereby improving the anti-fouling performance of the FET biosensor surface and enhancing the sensitivity and stability of the FET biosensor.

[0143] In another preferred embodiment of the present invention, a FET biosensor modified with the aforementioned anti-nonspecific adsorption reagent is provided for use in detecting glucose, protein, antibody, antigen, enzyme, virus, DNA or RNA in a complex physiological environment, and is particularly applicable to the detection of kanamycin in milk.

[0144] Combine Figure 1 As shown, in the process of detecting kanamycin in milk, it is preferred that the channel surface of the sensor be functionalized on the basis of the aforementioned FET biosensor that has not been modified against nonspecific adsorption reagents, that is, the nucleic acid aptamer is modified as the biological probe 70, and the nonspecific adsorption reagent is modified as the anti-fouling layer 80. The nonspecific adsorption reagent is particularly preferably sodium 2-mercaptoethanesulfonate and mercaptoethanol in a molar ratio of 1:1, thereby obtaining an aptamer biosensor, and then using the obtained aptamer biosensor for detection. After kanamycin binds to the biological probe nucleic acid aptamer, different conductance values are formed, thereby realizing the detection of the target biological molecule.

[0145] When this aptamer biosensor detects kanamycin in milk, the anti-fouling layer set up can improve the hydrophilicity of the interface through the interaction between different functional groups. For example, intermolecular hydrogen bonds may be formed between sulfonic acid groups and hydroxyl groups, and between amino groups and hydroxyl groups, making the modified surface more hydrophilic, and mutually repelling the hydrophobic regions of proteins, showing better anti-fouling performance; the amount of charge carried by different functional groups varies, forming areas of local different charges, showing a certain degree of dual charge (positive and negative charge), and it is difficult to produce electrostatic interactions with proteins; in addition, the molecular weight and size of different functional groups will vary, forming a certain height difference, resulting in a certain steric effect, reducing the adsorption of surface proteins, and showing good anti-fouling performance.

[0146] Aptamer biosensors can be integrated and made by micro-nano processing. The anti-fouling layer design makes the probe modification interval more appropriate, avoids the tipping over and entanglement of the probes, and makes the sensor interface have more sensing sites. The sensor has stronger anti-interference ability, higher sensitivity and faster response time. At the same time, compared with ordinary aptamer field-effect transistors, the anti-fouling layer makes the sensor's anti-nonspecific adsorption layer have high density and strong stability, the signal is further suppressed, and a wider linear detection range and extremely low detection limit are achieved, and the sensor performance is optimized.

[0147] The following will be combined with specific examples and experiments to conduct exemplary experiments and comparisons on the effects of the aforementioned method and the screened anti-nonspecific adsorption reagents. Of course, the embodiments of the present invention are not limited thereto.

[0148] Example 1

[0149] Taking the detection of kanamycin in pure milk system as an example, during the screening process, the anti-nonspecific adsorption mainly examines the adsorption to casein.

[0150] An ultrasensitive carbon nanotube field-effect transistor (CNT-FET) biosensor with an on-chip pseudo-reference gate was fabricated. The design dimensions are as follows:

[0151] The output electrode (pad) size is 300*300μm or more, and the thickness is 60nm;

[0152] The source and drain electrodes are Ti / Pd / Au with thicknesses of 0.6 / 20 / 60nm;

[0153] The thickness of the passivation layer is about 1 micron;

[0154] The network carbon nanotube film uses a single layer of network carbon tubes with a thickness of less than 2nm;

[0155] The thickness of the insulating layer yttrium oxide stack is about 10-11nm;

[0156] The particle size of gold nanoparticles is approximately equal to 0.6 nm;

[0157] The device adopts a liquid gate structure, with Ag / AgCl as the reference electrode to apply gate voltage, and the liquid gate environment is 0.1×PBS.

[0158] [Screening of anti-nonspecific adsorption reagents]

[0159] Step 1: Prepare seven thiol blocking reagents with different terminal functional groups, including sodium 2-mercaptoethanesulfonate (Mesna), mercaptoethanol (MCH), methyl thioglycolate (MTG), thioglycolic acid (TGA), cysteine (Cys), adamantanethiol (ADT), and perfluorodeoxythiol (PFDT), and dilute these 7 thiol reagents to a concentration of 10 mM with PBS.

[0160] Step 2: Use an electron beam coating device to deposit Ti / Au = 20 / 40 nm on the silicon oxide substrate to prepare a gold film with a size of 0.5 cm × 0.5 cm.

[0161] Step 3: Incubate the prepared gold film with seven thiol compounds at a concentration of 10 mM at 25°C for 8 hours to complete the preparation of the thiol antifouling layer. The volume of the added reagents is uniformly controlled at 10 μL. Finally, rinse the gold film with PBS and ultrapure water and blow dry with nitrogen gas.

[0162] Step 4: Add 5 μL of PBS and milk respectively on the prepared gold film with antifouling layer, observe with the fully automatic integral tilt contact angle analyzer, measure the contact angle in the fitting ellipse mode, and record multiple groups of contact angle test results in PBS and milk. One group of contact angle test results is as follows: Figure 5a The statistical results are shown in Figure 5b shown.

[0163] Gold films modified with sodium 2-mercaptoethanesulfonate and mercaptoethanol exhibited the best wettability in PBS and milk, indicating that the terminal functional groups of sodium 2-mercaptoethanesulfonate and mercaptoethanol possess a stronger hydrophilicity. These hydrophilic groups can form a water-bearing layer on the membrane surface by hydrogen bonding with water molecules, thereby weakening the hydrophobic interaction between the contaminant and the membrane surface. Therefore, membranes with strong hydrophilicity are generally less susceptible to fouling, suggesting that sodium 2-mercaptoethanesulfonate and mercaptoethanol have greater potential as antifouling agents.

[0164] Step 5: Modify the gold film with the antifouling layer with a concentration of 5 mg / mL fluorescently labeled casein. The volume of the added reagent is uniformly controlled at 5 μL. The incubation temperature is 25°C and the incubation time is 4 hours. Then, the gold film is washed with PBS and ultrapure water by lateral flow and blown dry with nitrogen. Then, the fluorescence intensity of the gold film modified with different thiol antifouling is observed by inverted fluorescence microscope. The fluorescence intensity is recorded as follows: Figure 6a The relative fluorescence intensity results are shown in Figure 6b shown.

[0165] The fluorescence intensity of the gold film modified with sodium 2-mercaptoethanesulfonate and mercaptoethanol was weak, and the amount of adsorbed protein was small, indicating that sodium 2-mercaptoethanesulfonate and mercaptoethanol had stronger anti-nonspecific adsorption ability.

[0166] Step 6: Incubate the seven thiol antifouling agents on the channel surface of the carbon nanotube field-effect transistor at 25°C for 30 minutes. Then, incubate milk on the channel surface of the carbon nanotube field-effect transistor at 25°C for 1 hour.

[0167] The transfer characteristic curve of the transistor before and after milk incubation was tested with a semiconductor analyzer. g = -1.2V at I ds The response changes of the device are compared by comparing the current changes caused by nonspecific adsorption when seven thiol antifouling layers with different terminal functional groups are directly applied to field effect transistors in the face of complex milk environments. The results are as follows Figure 7a , the statistical results are as follows Figure 7b shown.

[0168] According to the ranking of nonspecific response values, two thiol antifouling reagents, sodium 2-mercaptoethanesulfonate and mercaptoethanol, were selected.

[0169] Step 7: Sodium 2-mercaptoethanesulfonate and mercaptoethanol were used as antifouling layers in different ratios (molar ratios of 1:1, 1:2, 1:3, and 1:4, respectively). The I ds The statistical results of the response changes of CNT-FET after milk incubation are as follows: Figure 8 As shown, all four ratios meet the requirements, among which the molar ratio of sodium 2-mercaptoethanesulfonate and mercaptoethanol of 1:1 has the best effect.

[0170] Combine Figure 9 As shown, by comprehensively considering the hydrophilicity and hydrophobicity of the above seven thiol reagents, their resistance to non-specific protein adsorption, and their anti-interference performance in a complex milk environment when directly modified on field-effect transistors, two thiol compounds with good anti-fouling performance, sodium 2-mercaptoethanesulfonate (Mesna) and mercaptoethanol (MCH), were screened out. By exploring the anti-interference ability of field-effect transistors modified with different ratios of these two anti-fouling reagents in milk, the optimal mixed reagent of sodium 2-mercaptoethanesulfonate and mercaptoethanol in a molar ratio of 1:1 was used as the thiol reagent with the strongest anti-fouling performance applied to field-effect transistors.

[0171] [Mesna and MCH modified in a 1:1 molar ratio in aptamer field-effect transistors]

[0172] Step 1: A nucleic acid aptamer chain with a 5′-SH end modified with SH was centrifuged for 60 seconds, cleaved into short chains, and diluted with PBS to a concentration of 5 μM. The nucleic acid aptamer was then incubated on the channel surface of a carbon nanotube field-effect transistor at 4°C for 10 hours. The nucleic acid aptamer was covalently bound to the Au NPs surface through the Au-S bond to complete the fixation of the probe molecule.

[0173] Step 2: Dilute PBS to 10 mM Mesna and MCH and mix thoroughly in a 1:1 ratio. Then, add 10 μL of antifouling reagent to the CNT-FET sensing interface and incubate at 25°C for 30 minutes to complete the modification of the antifouling layer to prevent possible nonspecific binding events. Then rinse with deionized water.

[0174] Step 3: Determine the concentration of the milk sample by HPLC and dilute the milk sample with PBS to 1, 2, 5, 10, 20, 50, 100aM, 1f-100nM.

[0175] Combine Figure 10 As shown in the schematic diagram of applied voltage, during detection, the liquid to be tested is dropped onto the surface of the sensor channel to allow it to fully bind to the probe, and then the unbound target is washed away, and then 0.01×PBS is added as the gate for applying voltage.

[0176] When the isoelectric point of the target is lower than the pH of PBS, the probe molecules are negatively charged. As the concentration of the biomolecules in the solution increases or decreases, the conformation of the probe molecules tends to move toward or away from the sensing interface. This causes the number of charge holes in the semiconductor carbon nanotubes in the channel region to increase or decrease, thus affecting the sensor's current. Therefore, as the concentration of the target molecule increases, the sensor's current gradually increases.

[0177] Combine Figure 11a 、 11b In the example shown, when kanamycin is present in the test solution, the probe molecule conforms toward the sensing interface, increasing the number of charge holes in the semiconductor carbon nanotubes in the channel region, thereby boosting the sensor's current. However, the sensor's current response is weak when nonspecific molecules are added. Adding kanamycin to the nonspecific target solution increases the sensor's current again, demonstrating that the field-effect transistor modified with a 1:1 molar ratio of sodium 2-mercaptoethanesulfonate and mercaptoethanol exhibits excellent specific recognition and anti-interference capabilities.

[0178] The nonspecific solution includes the main components of milk: cholesterol (CHOL), lactose (Lactose), vitamin C (Vit.C), casein (CS), and common antibiotics: tetracycline (TC), penicillin (PG), oxytetracycline (OTC), erythromycin (EM), and chloramphenicol (CPL).

[0179] [Test comparison]

[0180] Combine Figure 12As shown, when sodium 2-mercaptoethanesulfonate and mercaptoethanol with a molar ratio of 1:1 are applied to a field effect transistor, a schematic diagram of the shape of the aptamer modified at the interface of the field effect transistor biosensor and a schematic diagram of its capturing the molecule to be measured are respectively shown.

[0181] like Figure 12 As shown in a, when the field effect transistor biosensor is modified with sodium 2-mercaptoethanesulfonate and mercaptoethanol at a molar ratio of 1:1, the shape of the aptamer modified on the channel surface of the field effect transistor biosensor is compared with that without the anti-nonspecific adsorption layer (combined with Figure 12 c), will be more perpendicular to the channel surface, the effective specific binding of aptamers will increase significantly, therefore, the binding Figure 12 In the example shown in b, the aptamer can capture more molecules to be detected (compared to Figure 12 d shows the capture result).

[0182] In the present invention Figure 13a 、 Figure 13b as well as Figure 14a 、 Figure 14b In the experiment, the test solution with the same solubility was used.

[0183] Based on this principle, combined with Figure 10 The schematic diagram of applied voltage is shown in the figure. When using the biosensor to test kanamycin in milk, a certain voltage V is applied between the source electrode and the drain electrode of the sensor. ds A certain range of scanning voltage is applied to the gate. According to the different concentrations of kanamycin and the number of probes combined, the current flowing between the source electrode and the drain electrode is different.

[0184] Each concentration of kanamycin can scan a transfer curve after binding to the probe. The transfer curves of different concentrations of kanamycin will be different. The higher the concentration, the threshold voltage of the transfer curve moves in the positive direction, and the on-state current of the curve gradually increases. It has a very wide test range, as low as 1aM and as high as 100nM. Figure 13a As shown. The sensitivity of the sensor is defined as: taking a certain V at each concentration g There will be different I ds Therefore, different I ds This is because the concentration of carrier holes in the sensor channel increases as the concentration of the target substance combined with the probe increases, thereby changing the voltage of the sensor transfer curve. ds The function between the / I0 value and the target concentration is that the concentration corresponding to 3 times the blank response is the sensor's lowest detection limit, which is calculated as 0.1aM here. The concentration corresponding to 10 times the blank response is the sensor's lowest quantification limit, which is calculated as 15.1aM here.

[0185] like Figure 13b In the example shown, V g =-1.5V as an example, scan to obtain I ds The sensitivity curve was obtained based on the value of . In the low concentration range of 1aM-100aM, the fitting curve equation is: y = 0.174lg x + 0.054, and the correlation coefficient R 2 =0.9984. In the concentration range of 100aM-100nM, it tends to be saturated, and the fitting equation is: y=0.057lg x+0.290, and the correlation coefficient R 2 =0.9995.

[0186] Combine Figures 14a-14b The example shown shows the transfer curve and linear fit results for the same field-effect transistor biosensor measured in the same milk environment. The concentration corresponding to 3 times the blank response is the sensor's lower detection limit, calculated here to be 1.5 aM. The concentration corresponding to 10 times the blank response is the sensor's lower limit of quantification, calculated here to be 0.5 aM. Compared to a field-effect transistor sensor using sodium 2-mercaptoethanesulfonate in a 1:1 ratio with mercaptoethanol as an antifouling layer, the detection range is significantly narrowed, from 1 aM to 100 fM, and the range for accurate quantification is only from 0.5 fM to 100 fM.

[0187] During the detection process, a scanning voltage of -1.5 to 0.3 V is used, which is related to time. A voltage value is applied per second, and a total of 90 I ds point, and thus the transfer curve is obtained.

[0188] It can be seen that the seven thiol small molecules based on the exemplary embodiments of the present invention: sodium 2-mercaptoethanesulfonate (Mesna), mercaptoethanol (MCH), methyl thioglycolate (MTG), thioglycolic acid (TGA), cysteine (Cys), adamantanethiol (ADT), and perfluorodeoxythiol (PFDT) can be applied to FET biosensors to resist nonspecific adsorption.

[0189] According to an exemplary embodiment of the present invention, an antifouling layer comprising sodium 2-mercaptoethanesulfonate and mercaptoethanol in a 1:1 molar ratio is applied to a field-effect transistor biosensor. Using this antifouling layer in conjunction with a semiconductor process improves the sensor's anti-interference performance, thereby increasing its sensitivity. Furthermore, due to its small size, the FET biosensor can be easily integrated into various control panels, enabling portable real-time monitoring. Furthermore, using this antifouling layer in conjunction with a semiconductor process effectively improves the spatial configuration of the probe at the sensor interface by leveraging the competitive effect of thiols on the aptamer, increasing specific binding sites and enabling detection across a wide concentration range. This addresses the issue of transistor-based biosensors, which often respond severely to interference signals due to their high sensitivity.

[0190] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for screening an anti-nonspecific adsorption agent for FET biosensor, characterized in that: The following steps are involved: S1. Preliminary screening Sample preparation: Dissolve thiol compound reagents with different terminal functional groups in PBS to prepare corresponding first solutions; Depositing a gold film on a silicon oxide substrate using an electron beam coating apparatus, and modifying the prepared gold film with each set of the first solution to obtain a corresponding self-assembled monolayer; Hydrophilicity and hydrophobicity testing: The target detection system was added to each group of self-assembled monolayers, and the contact angle of the target detection system on the membrane layer was measured and compared with that of a first control group. The first solution with a smaller contact angle than that of the first control group was selected. The first control group consisted of the target detection system added to an unmodified gold film. Nonspecific adsorption investigation: The adsorption of each group of self-assembled monolayers to non-targets in the target detection system was investigated and compared with a second control group. The first solution with a smaller adsorption amount than that of the second control group was selected. The second control group was the adsorption amount of the unmodified gold film to non-targets in the target detection system. Selecting a first solution that meets both the hydrophilicity and hydrophobicity requirements and the nonspecific adsorption requirements, and preliminarily screening the corresponding first solution; S2. Fine screening Modifying a floating-gate field-effect transistor having gold particles deposited on the channel surface with the first solution screened in step S1, measuring the transfer curve before and after incubation of the target detection system, determining the current response of the first solution to the interferent, and calculating the corresponding nonspecific response value, and ranking according to the size of the nonspecific response value; The thiol compound reagent corresponding to the group with the smallest non-specific response value is defined as group 1, and the corresponding thiol compound reagents are defined as group 2, group 3, etc. according to the increasing non-specific response value, until the Nth group, wherein N ≥ 2; Dividing N groups of thiol compound reagents into M items, each item containing a mixed solution composed of solutions of the N groups of thiol compound reagents, and the groups in each item are consecutive groups starting from group 1, wherein M = N-1 and N ≥ 2; S3. Optimized screening The M items obtained in step S2 are tested according to the first method to screen out the desired anti-nonspecific adsorption reagent; Among them, the first method is as follows: The groups in each item are prepared into solutions of different concentrations and mixed according to different molar ratios to obtain multiple groups of mixed reagents. Each group of mixed reagents is modified on a floating-gate field-effect transistor with gold particles deposited on the channel surface. The transfer curves before and after incubation of the target detection system are measured to determine the current response of the mixed reagent to the interferent, and the corresponding nonspecific response value is calculated. The mixed reagent is selected when the nonspecific response value is less than 5%.

2. The method for screening an anti-nonspecific adsorption reagent for FET biosensor according to claim 1, characterized in that: The specific process of optimization screening is as follows: Starting from the first item obtained in step S2, test according to the first method and judge according to the second method; wherein the second method is as follows: The mixed reagent used when the nonspecific response value is less than 5% is used as the required anti-nonspecific adsorption reagent, and the screening is completed; When there is no non-specific response value less than 5% in the test result, continue to test the next item according to the first method and judge according to the second method until the required anti-non-specific adsorption reagent is screened out.

3. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 2, characterized in that: In the process of continuing to test the next item according to the first method, the items are tested in order from the 2nd item to the Mth item, where M=N-1 and N≥2.

4. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to any one of claims 1 to 3, characterized in that: In the first method, each group in each item is prepared into solutions with different concentrations, and the concentration of the substance in each group is controlled at (1-4) mM.

5. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 1, characterized in that: In step S1 , the concentration of each set of first solutions is 10 mM.

6. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 1, characterized in that: The preparation process of self-assembled monolayer is as follows: The prepared gold film was incubated with the first solution at 25°C until the thiol compound was fully combined with the gold film through the gold-thiol bond. After the incubation, the gold film was rinsed with PBS and ultrapure water by lateral flow and dried with nitrogen to complete the preparation of the self-assembled monolayer film.

7. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 1, characterized in that: Inverted fluorescence microscopy was used to investigate the nonspecific adsorption.

8. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 1, characterized in that: The thiol compound reagents with different terminal functional groups are sodium 2-mercaptoethanesulfonate, mercaptoethanol, methyl thioglycolate, thioglycolic acid, cysteine, adamantanethiol and perfluorodeoxythiol.

9. The method for screening an anti-nonspecific adsorption agent for FET biosensor according to claim 8, characterized in that: When the target detection system is milk, the screened anti-nonspecific adsorption reagent is sodium 2-mercaptoethanesulfonate and mercaptoethanol in a molar ratio of 1:(1-4).

10. An anti-nonspecific adsorption reagent for FET biosensor screened by the method according to any one of claims 1 to 9, characterized in that: The anti-nonspecific adsorption reagent comprises at least two thiol compounds, and the terminal functional groups of the thiol compounds are different from each other. 11 . A FET biosensor modified with the anti-nonspecific adsorption reagent for FET biosensor according to claim 10 .

12. Use of the FET biosensor according to claim 11 in detecting glucose, protein, antibody, antigen, enzyme, virus, DNA or RNA in a complex physiological environment.

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