Carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, preparation method, application and detection method

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to a preparation method and application of a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, the carbon nanotube transistor sensor comprises a substrate, a contact electrode and a channel; the channel is arranged on the substrate, and the contact electrodes are arranged at the two ends of the channel; the channel consists of a carbon nano tube of which the surface is modified with an enzyme probe; and the enzyme probe is an enzyme participating in an enzyme cascade reaction. Compared with the existing enzyme modified transistor sensor, the invention solves the problem that a sensitive and efficient biosensor suitable for realizing small molecular biomarkers under low concentration is lacked in the prior art. According to the scheme, complicated sample treatment and marking processes are not needed, the detection time is greatly shortened, the sensitivity is high, the specificity is good, and the application prospect is good.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, its preparation method, application, and detection method. Background Technology

[0002] Biosensors based on field-effect transistors (FETs) made of low-dimensional nanomaterials are widely considered important candidates for high-throughput, ultra-sensitive, and cost-effective sensors in the field of miniaturized medical diagnostics. Because transistors have high sensitivity to charge disturbances and efficient signal transduction capabilities, they can be used as a sensing method to detect the presence and concentration changes of substances by monitoring current and voltage.

[0003] Enzyme-modified transistor biosensors are now widely used due to their excellent reaction efficiency, reusability, and significant detection specificity. Although these transistor sensors outperform commonly used diagnostic methods such as ELISA, PCR, and mass spectrometry in detection performance, achieving high sensitivity and selectivity for biomarkers in physiological fluid environments remains challenging. In complex fluid detection environments, background molecules may interact with the sensor's conductive channels, causing significant interference. Simultaneously, the small Debye length and high ionic strength of clinical samples are detrimental to effective signal transduction. These factors combined may lead to insufficient sensitivity in disease detection, thus limiting the device's effectiveness. There is an urgent need to improve transistor sensors to optimize signal transduction in clinical testing. Currently, multi-enzyme catalytic cascade reactions are widely used in the synthesis of traditional high-value-added drugs, chemicals, and materials, synergistically improving reaction efficiency and accuracy. Traditional enzyme modification methods, requiring corresponding groups and reaction sites, make it difficult for enzymes participating in the cascade reaction to co-modify and produce signal enhancement.

[0004] In the prior art, CN114634968A discloses a field-effect transistor nucleic acid sensor based on Argonaute protein, its preparation method, and its application. However, this approach is mainly aimed at nucleic acid detection and is not suitable for small molecule biomarkers. CN114324521A discloses an electrochemical biosensor, its preparation method, and its application, but this approach is aimed at the detection of larger targets (such as tumor cells) and is not suitable for small molecule biomarkers. The small molecule biomarkers referred to here are low molecular weight compounds that can be measured in organisms, especially in body fluids such as blood, urine, or saliva, and used for disease diagnosis, prognostic assessment, or treatment monitoring, such as creatinine, bilirubin, and C-reactive protein. These biomarkers typically participate in or reflect the organism's metabolic processes, disease states, or responses to drugs.

[0005] Abnormal levels of small molecule biomarkers can indicate the presence of specific diseases, making them useful tools for early diagnosis. For example, elevated levels of glycated hemoglobin (HbA1c) in the blood are commonly associated with diabetes. Furthermore, the levels of small molecule biomarkers can also help predict the rate of disease progression or the effectiveness of treatment: by regularly measuring specific biomarkers, doctors can monitor a patient's disease status and treatment response, allowing for timely adjustments to the treatment plan.

[0006] Therefore, there is a need for a biosensor that is suitable for small molecule biomarkers and can be sensitively and efficiently detected. Summary of the Invention

[0007] The purpose of this invention is to address at least one of the aforementioned problems by providing a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, along with its preparation, application, and detection methods. This addresses the lack of a sensitive and efficient biosensor suitable for detecting small molecule biomarkers at low concentrations in the prior art. This solution eliminates the need for complex sample processing and labeling, significantly reduces detection time, and offers high sensitivity and specificity, demonstrating promising application prospects.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first aspect of this invention discloses a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, comprising a substrate, a contact electrode, and a channel;

[0010] The channel is disposed on the substrate, and the contact electrodes are disposed at both ends of the channel;

[0011] The channel is composed of carbon nanotubes with enzyme probes modified on their surface;

[0012] The enzyme probe is an enzyme that participates in the enzyme cascade reaction.

[0013] Preferably, the enzymes participating in the cascade reaction include one or more combinations of oxidoreductases, transferases, hydrolases, isomerases, and ligases. Specifically, this includes, but is not limited to, one or more combinations of enzymes such as sarcosine oxidase, catalase, horseradish catalase, lactate oxidase, and acetylcholine oxidase.

[0014] The oxidase probe selected for its ability to participate in cascade reactions specifically binds to the analyte and induces an enzymatic cascade reaction. The charged body absorbed by the redox reaction at the carbon nanotube / electrolyte interface sufficiently n-type dopes the semiconductor carbon nanotube, thereby altering its conductivity. Furthermore, the excellent reaction efficiency, reusability, and detection specificity of the oxidase itself significantly improve the sensor's performance.

[0015] Preferably, the carbon nanotubes are semiconducting carbon nanotubes, which are prepared by the following method: carbon nanotube raw materials and conjugated polymers are co-dispersed in a dispersion, and a semiconducting carbon nanotube solution is obtained after ultrasonic crushing and ultracentrifugation, followed by static deposition to obtain semiconducting carbon nanotubes.

[0016] Preferably, the conjugated polymer includes polyfluorene, polythiophene, and polycarbazole; the ultrasonic crushing power is 100-500 watts; the ultracentrifugation speed is 30,000-100,000 revolutions / minute; and the deposition time is 2-48 hours.

[0017] A second aspect of this invention discloses a method for preparing a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as described above, comprising the following steps:

[0018] S1: Carbon nanotubes and contact electrodes are sequentially fabricated on a substrate and then patterned.

[0019] S2: Modify enzyme probes on carbon nanotubes.

[0020] Preferably, in step S2, the enzyme probe is modified onto carbon nanotubes using a direct modification method, a physical adsorption method, or an embedding method.

[0021] Preferably, the direct modification method includes the following steps:

[0022] S21: Modifying the linking molecules: Immerse the semi-finished product obtained in step S1 in a medium containing linking molecules, or spray the medium containing linking molecules onto the surface of the semi-finished product obtained in step S1, so that the linking molecules modify the surface of the carbon nanotubes.

[0023] S22: Modifying the enzyme probe: The intermediate product obtained in step S21 is immersed in a solution containing the enzyme probe. The enzyme probe is modified onto the surface of the carbon nanotube by esterification, condensation or gold-sulfur covalent bond formation between the linker molecule and the enzyme probe. Then, it is washed with buffer or fetal bovine serum.

[0024] Preferably, in step S1, after the contact electrode is fabricated on the substrate, a layer of photoresist is coated on the surface of the contact electrode by spin coating to form a passivation layer.

[0025] A third aspect of this invention discloses an application of a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as a biosensor, as described above. This biosensor is used to detect substrates of enzyme probes. Specifically, it includes, but is not limited to, substrates of oxidoreductases, transferases, hydrolases, isomerases, and ligases. These substrates are often considered small molecule disease biomarkers, but sometimes their low concentrations make them difficult to measure. This method can achieve signal enhancement through enzyme cascade reactions, thereby obtaining more accurate detection data.

[0026] The fourth aspect of this invention discloses a detection method using a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as described above. A liquid tank is fabricated on the carbon nanotube transistor sensor, and a reaction buffer is added to the liquid tank. During detection, a portion of the reaction buffer is extracted and an analyte solution of the same volume as the extracted amount is added. The enzyme probe undergoes an enzyme cascade reaction with the analyte to enhance charge transfer, thereby generating a signal amplification effect.

[0027] The working principle of this invention is as follows: more charge transfer is generated through cascade reactions, which dopes the carbon nanotubes, produces changes in electrical signals, thereby amplifying the sensing signal and improving the sensor's detection activity.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The field-effect transistor (FET) sensor proposed in this invention has the advantages of high sensitivity and high specificity. By employing universal enzyme binding sites and utilizing the high specific surface area of ​​carbon nanotube networks, the modification efficiency and density of enzymes in the cascade reaction are improved, ultimately achieving signal enhancement. During detection, a liquid tank is fabricated on the FET, and the sample solution to be tested is added to it. This allows the enzyme probe modified on the FET to bind to the target substance through the enzyme cascade reaction, producing a doping effect, thereby amplifying changes in the electrical signal.

[0030] Compared with traditional testing methods, the advantages of this invention are: it constructs a carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement. This sensor array utilizes a carbon nanotube film as an ultrathin active channel and uses the two enzymes participating in the enzyme cascade reaction as selective acceptors; through the enzyme cascade reaction, more charge transfer is generated, doping the carbon nanotubes and producing changes in electrical signals, thereby amplifying the sensed signal and improving the sensor's detection activity. The enzyme cascade reaction signal enhancement carbon nanotube transistor sensor has the advantages of simple processing, label-free operation, high sensitivity, high specificity, portability, and low cost, broadening the detection range of traditional aptamer field-effect transistor sensors. Attached Figure Description

[0031] Figure 1This is a schematic diagram of the carbon nanotube transistor sensor structure of this scheme;

[0032] Figure 2 This is the current-voltage response curve for detecting creatine in Example 1;

[0033] Figure 3 This is the time-current response curve for detecting creatine in Example 1;

[0034] Figure 4 This is a schematic diagram of the specificity test for detecting creatine in Example 1;

[0035] Figure 5 This is the current-voltage response curve for detecting lactic acid in Example 4;

[0036] Figure 6 This is a schematic diagram of the specific test for detecting lactic acid in Example 4;

[0037] Figure 7 This is the current-voltage response curve for detecting acetylcholine in Example 5;

[0038] Figure 8 This is a schematic diagram of the specific test for detecting acetylcholine in Example 5;

[0039] Figure 9 This is the current-voltage response curve for glucose detection in Example 6;

[0040] Figure 10 This is a schematic diagram of the specific test for detecting glucose in Example 6. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] Unless otherwise specified, the reagents used in the following description are commercially available products, and the methods used are conventional techniques in the field.

[0043] A carbon nanotube transistor sensor that enhances the signal of an enzyme cascade reaction, such as... Figure 1 As shown, this is a field-effect transistor (FET) sensor. The FET sensor includes: an insulating substrate; carbon nanotubes (serving as channels) disposed on the insulating substrate; source and drain electrodes at both ends of the carbon nanotubes; and oxidases modified on the surface of the carbon nanotubes to participate in enzyme cascade reactions. During detection, the FET sensor is placed in a detection solution and connected to a semiconductor testing instrument. The analyte is then added to the solution, enabling specific and ultrasensitive detection of the analyte.

[0044] Preferably, the insulating substrate is made of silicon dioxide / silicon substrate, or it can be selected from a variety of insulating materials such as polyimide, polydimethylsiloxane, polyethylene terephthalate, and polyethylene naphthalate.

[0045] Preferably, the working channel material is carbon nanotubes with a thickness of 1–5 nanometers.

[0046] Preferably, the electrode is a patterned electrode, and the electrode material is selected from conductive metals such as gold, silver, copper, nickel, titanium, iron, and aluminum, or a conductive polymer may also be selected, with a thickness of 10 to 500 nanometers.

[0047] Preferably, the carbon nanotubes and the enzymes modified thereon via linker molecules constitute an analyte recognition layer capable of reacting with the analyte. The enzymes include, but are not limited to, the following: oxidoreductases, transferases, hydrolases, isomerases, ligases, etc., specifically including: sarcosine oxidase; catalase; horseradish catalase; lactate oxidase; acetylcholine oxidase, etc. The above-mentioned enzymes can be used for modification as a single type or in combination.

[0048] Preferably, the carbon nanotube material is a semiconducting carbon nanotube, which can be prepared by the following method: A carbon nanotube sample (raw material) and a conjugated polymer are dispersed in a dispersion solution, followed by ultrasonic disruption to obtain a preliminary dispersion solution. This dispersion solution is then subjected to ultracentrifugation to obtain a semiconducting carbon nanotube solution, thereby separating the metallic and semiconducting carbon nanotubes. A semiconducting carbon nanotube film is then obtained through static deposition of the solution. The conjugated polymer can be selected from polyfluorene, polythiophene, polycarbazole, etc. The ultrasonic disruption power is 100–500 W, the ultracentrifugation speed is 30,000–100,000 revolutions / minute, the deposition time is 2–48 hours, and the thickness of the semiconducting carbon nanotube film is 1–5 nm. The semiconducting carbon nanotubes used in subsequent embodiments are prepared in advance according to this method; as long as the parameters of the preparation process are controlled within the specified range, the resulting semiconducting carbon nanotubes will have essentially the same performance with no significant differences, and will not affect the subsequent preparation and performance of the sensor.

[0049] A method for fabricating a carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement includes the following steps:

[0050] (1) Carbon nanotubes are grown on an insulating substrate, and source and drain electrodes are fabricated on the insulating substrate;

[0051] (2) The electrodes are patterned using photolithography to form a specific shape;

[0052] (3) Carbon nanotubes are patterned into specific shapes using photolithography and oxygen plasma etching techniques for patterning:

[0053] (4) Photoresist is coated on the surface of carbon nanotube transistors to form a passivation protective layer, which protects the electrodes.

[0054] (5) Modify the carbon nanotube channels of the prepared device with connecting molecules;

[0055] (6) Modify and fix the cascade reaction enzyme onto the surface of the carbon nanotube channel;

[0056] (7) Fabricating a liquid tank on a field-effect transistor (FET) Figure 1 The sample loading device is stored for later use. During use, the analyte is added to the liquid tank, allowing an enzyme cascade reaction to dope the carbon nanotube channels. High-sensitivity detection of the analyte is achieved through changes in the electrical signal.

[0057] Preferably, the specific method for modifying the connecting molecules in step (5) is to immerse the field-effect transistor device with the carbon nanotube material exposed in an organic small molecule solution or spray it with gold nanoparticles, and then connect the cascade reaction enzyme through esterification reaction, condensation reaction or gold-sulfur covalent bond.

[0058] Preferably, in step (6), the cascade reaction enzyme is modified and immobilized onto the surface of the carbon nanotube channel. The specific modification method is as follows:

[0059] The direct modification method involves preparing an enzyme probe solution with a concentration of 2–200 U per milliliter, immersing the transistor sensor device with carbon nanotubes in the enzyme probe solution with a concentration of 2–200 U per milliliter for 3–13 hours at room temperature, and then rinsing it clean with buffer or fetal bovine serum.

[0060] In addition, oxidases can be loaded onto the surface of carbon nanotubes using physical adsorption and encapsulation methods, in which case it is not necessary to prepare the linker molecules first.

[0061] Preferably, in step (7), the field effect transistor sensor is stored by adding 80 to 100 microliters of reaction buffer to a liquid tank when not in use, and storing it at a low temperature with 40 to 80% humidity. The preferred storage temperature is 4 to 20 degrees Celsius, and the optimal storage temperature is 4 degrees Celsius.

[0062] Preferably, the photoresist in step (4) includes, but is not limited to, S1813, LOR 3A, SU-8, etc.

[0063] Preferably, when the field-effect transistor sensor is used in step (7), the specific detection method is as follows:

[0064] (1) Connect the source of the prepared carbon nanotube transistor sensor to the positive electrode of the semiconductor tester and the drain to the negative electrode of the semiconductor tester.

[0065] (2) Set up a liquid tank on the carbon nanotube channel and add 20 to 120 μL of reaction buffer to the liquid tank;

[0066] (3) There are two test modes:

[0067] ① Current-gate voltage test: fix the source-drain voltage, give the gate voltage scan range, and start the test solution test when the drain current is stable;

[0068] ② Current-time test: Adjust the output voltage of the test to keep the source and drain current constant. When the source and drain current are basically stable (the percentage change in current is less than 0.2%), the test of the device under test will begin.

[0069] (4) Draw a certain amount of reaction buffer into the liquid bath, and then add the same volume of the analyte solution. The cascade reaction enzyme captures the analyte, causing the target to dope the carbon nanotubes, thereby generating an electrical signal. When using the current-gate voltage test mode, read the signal 2-30 minutes after adding the analyte. The signal reading method is the change in drain current or threshold voltage. When using the current-time test mode, after adding the target, read the normalized current signal response value ΔI when the current response reaches equilibrium and stabilizes over time. 非待测 .

[0070] (5) The determination of the detection method of the analyte in the target is based on the detection mode and there are two types:

[0071] ① In the current-gate voltage test, a buffer solution is first added as a background solution. A given gate voltage scan range is established to keep the current constant. The change in drain current caused by current noise is recorded as ΔI. 非待测 This value is used as the signal response value of the non-analyte solution; the change in current after adding the analyte solution is recorded as ΔI. 待测 When the drain current changes by (ΔI) 待测 (greater than 3ΔI) 非待测 When ΔI is detected, it indicates that the marker is detected. 待测 Less than or equal to 3ΔI 非待测 This indicates that the marker was not detected.

[0072] ② In the current-time test, a buffer solution is first added as a background solution. A fixed gate voltage is applied to keep the drain current stable. After adding a solution other than the analyte, the drain current change value ΔI is read when the current response reaches equilibrium over time. 非待测 After adding the analyte solution, when the current response reaches equilibrium over time, the change in drain current is read: ΔI. 待测 This is used as the signal response value of the analyte. When ΔI 待测 Greater than 3ΔI 非待测 When ΔI is present, it indicates that the marker has been detected; when ΔI is present...待测 Less than or equal to 3ΔI 非待测 When the time is right, it indicates that the marker was not detected.

[0073] Example 1

[0074] A carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal was prepared for the detection of sarcosine in a buffer solution.

[0075] The first step involves fabricating a nanotube field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 5 nm chromium and 40 nm gold as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0076] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0077] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of sarcosine oxidase solution and 50 μL of horseradish catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal for detecting sarcosine.

[0078] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing sarcosine was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After each concentration of sarcosine solution was added to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0079] Figure 2 The current-voltage response curve of the field-effect transistor sensor in Example 1 for detecting sarcosine shows that the constructed sensor has a linear response to sarcosine. Figure 3 This is the current-time response curve of the carbon nanotube transistor sensor in Example 1 for detecting sarcosine. Figure 4This is a schematic diagram of the specificity test of the carbon nanotube transistor sensor.

[0080] Example 2

[0081] A carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal was prepared for the detection of L-alanine in buffer solution.

[0082] The first step involves fabricating a carbon nanotube field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 10 nm chromium and 20 nm gold as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0083] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 mmol of 1-pyrenebutyric acid N-hydroxysuccinimide ester at room temperature for 7 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0084] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of alanine transaminase solution and 50 μL of horseradish catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal for detecting L-alanine.

[0085] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing L-alanine was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After adding L-alanine solutions of each concentration to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0086] Example 3

[0087] A carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal was prepared for the detection of D-alanine in buffer solution.

[0088] The first step involves fabricating a carbon nanotube field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 5 nm chromium and 40 nm gold as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0089] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0090] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of D-alanine oxidase solution and 50 μL of horseradish catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor for detecting D-alanine.

[0091] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing D-alanine was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After adding D-alanine solutions of each concentration to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0092] Example 4

[0093] A carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal was prepared for the detection of lactic acid in a buffer solution.

[0094] The first step is to fabricate a field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 6 nm chromium and 35 nm gold as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0095] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0096] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of lactate oxidase solution and 50 μL of catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor for detecting lactic acid.

[0097] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing lactic acid was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After each concentration of lactic acid solution was added to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0098] Figure 5 The current-voltage response curve of the field-effect transistor sensor in Example 4 for detecting lactic acid shows that the constructed sensor has a linear response to lactic acid. Figure 6 This is a schematic diagram illustrating the specificity test of the carbon nanotube transistor sensor for detecting lactic acid.

[0099] Example 5

[0100] A carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement was prepared for the detection of acetylcholine in a buffer solution.

[0101] The first step is to fabricate a field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 5 nm chromium and 40 nm silver as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0102] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0103] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of acetylcholinesterase solution and 50 μL of choline oxidase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor for detecting acetylcholine.

[0104] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing acetylcholine was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After each concentration of acetylcholine solution was added to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0105] Figure 7 The current-voltage response curve of the field-effect transistor sensor in Example 5 for detecting acetylcholine shows that the constructed sensor has a linear response to acetylcholine. Figure 8 This is a schematic diagram of the specificity test for detecting acetylcholine using the carbon nanotube transistor sensor.

[0106] Example 6

[0107] A carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement was prepared for the detection of glucose in a buffer solution.

[0108] The first step involves fabricating a carbon nanotube field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 5 nm chromium and 40 nm copper as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0109] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0110] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of glucose oxidase solution and 50 μL of horseradish catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement for glucose detection.

[0111] Finally, the transfer curve test was initiated. The source of the prepared semiconducting single-walled carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing glucose was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 Moles per liter. After each concentration of glucose solution was added to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0112] Figure 9 The current-voltage response curve of the field-effect transistor sensor in Example 6 for detecting lactic acid shows that the constructed sensor has a linear response to lactic acid. Figure 10 This is a schematic diagram illustrating the specificity test of the carbon nanotube transistor sensor for detecting lactic acid.

[0113] Example 7

[0114] A carbon nanotube transistor sensor with enzyme cascade reaction signal enhancement was prepared for the detection of uric acid in buffer solution.

[0115] The first step involves fabricating a carbon nanotube field-effect transistor sensor. Semiconductor carbon nanotubes are transferred onto a clean silica / silicon substrate. Patterned electrodes are then fabricated using laser direct-write lithography, followed by the deposition of 5 nm chromium and 40 nm gold as source and drain electrodes via thermal evaporation. The carbon nanotubes are then patterned using photolithography and oxygen plasma etching techniques to obtain the carbon nanotube transistor sensor.

[0116] The second step involves immersing the carbon nanotube transistors in an acetone solution containing 5 millimoles of 1-pyrene butyrate N-hydroxysuccinimide ester at room temperature for 6 hours, followed by rinsing with ethanol and ultrapure water, and finally drying with nitrogen.

[0117] Next, the fabricated PDMS liquid bath was placed above the carbon nanotube transistor channel, and 50 μL of uric acid oxidase solution and 50 μL of catalase solution were added respectively. After incubation for 6 hours, the mixture was washed with phosphate buffer solution to obtain a carbon nanotube transistor sensor with enhanced enzyme cascade reaction signal for detecting uric acid.

[0118] Finally, the transfer curve test was initiated. The source of the prepared carbon nanotube transistor sensor was connected to the positive terminal of the semiconductor tester, and the drain was connected to the negative terminal. 100 μL of phosphate buffer solution was added to the PDMS liquid bath. The current-gate voltage test was selected in the semiconductor tester, and the source-drain voltage and gate voltage scan ranges were given. The test began when the drain current or threshold voltage change was less than the instrument's voltage resolution. During the test, 50 μL of phosphate buffer solution was first withdrawn from the PDMS liquid bath, and then 50 μL of phosphate buffer solution containing uric acid was added. The test concentration started from 1 × 10⁻⁶. -18 moles / liter to 1×10 -10 mol / L. After adding uric acid solution of each concentration to the PDMS liquid bath, the signal was read after 3 minutes. The signal was read as the change in drain current.

[0119] Table 1 Summary of Examples 1-7

[0120]

[0121]

[0122] In summary, during detection, the transistor sensor is connected to a semiconductor testing instrument, and the sample to be tested is added to the sensor's sample loading chamber. The enzyme cascade reaction amplifies the signal, enhancing the detection signal and improving signal transmission efficiency, thereby achieving ultrasensitive detection of the analyte. The carbon nanotube sensor of this invention possesses the advantages of ultra-high sensitivity and specificity.

[0123] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement, characterized in that, Includes substrate, contact electrodes, and channels; The channel is disposed on the substrate, and the contact electrodes are disposed at both ends of the channel; The channel is composed of carbon nanotubes with enzyme probes modified on their surface; The enzyme probe is an enzyme that participates in the enzyme cascade reaction.

2. The carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 1, characterized in that, Enzymes involved in cascade reactions include one or more combinations of oxidoreductases, transferases, hydrolases, isomerases, and ligases.

3. A carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 1, characterized in that, The carbon nanotubes mentioned are semiconducting carbon nanotubes, which are prepared by the following method: carbon nanotube raw materials and conjugated polymers are co-dispersed in a dispersion, and after ultrasonic crushing and ultracentrifugation, a semiconducting carbon nanotube solution is obtained, followed by static deposition to obtain semiconducting carbon nanotubes.

4. A carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 3, characterized in that, The conjugated polymers include polyfluorene, polythiophene, and polycarbazole; the ultrasonic disruption power is 100–500 watts; the ultracentrifugation speed is 30,000–100,000 revolutions per minute; and the deposition time is 2–48 hours.

5. A method for preparing a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1: Carbon nanotubes and contact electrodes are sequentially fabricated on a substrate and then patterned. S2: Modify enzyme probes on carbon nanotubes.

6. The method for fabricating a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 5, characterized in that, In step S2, the enzyme probe is modified onto carbon nanotubes using a direct modification method, a physical adsorption method, or an embedding method.

7. The method for fabricating a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 6, characterized in that, The direct modification method includes the following steps: S21: Modifying the linking molecules: Immerse the semi-finished product obtained in step S1 in a medium containing linking molecules, or spray the medium containing linking molecules onto the surface of the semi-finished product obtained in step S1, so that the linking molecules modify the surface of the carbon nanotubes. S22: Modifying the enzyme probe: The intermediate product obtained in step S21 is immersed in a solution containing the enzyme probe. The enzyme probe is modified onto the surface of the carbon nanotube by esterification, condensation or gold-sulfur covalent bond formation between the linker molecule and the enzyme probe. Then, it is washed with buffer or fetal bovine serum.

8. The method for fabricating a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement according to claim 5, characterized in that, In step S1, after the contact electrode is fabricated on the substrate, a layer of photoresist is coated on the surface of the contact electrode by spin coating to form a passivation layer.

9. The application of a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as described in any one of claims 1 to 4 as a biosensor, characterized in that, The biosensor is used to detect the substrate of the enzyme probe.

10. A detection method using a carbon nanotube transistor sensor based on enzyme cascade reaction signal enhancement as described in any one of claims 1 to 4, characterized in that, A liquid tank is fabricated on a carbon nanotube transistor sensor, and a reaction buffer is added to the liquid tank. During detection, a portion of the reaction buffer is extracted and an analyte solution of the same volume as the extracted amount is added. The enzyme probe undergoes an enzyme cascade reaction with the analyte to enhance charge transfer and generate signal amplification.

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