Carbon nanotube FET biosensor for detecting lung cancer marker p53 and preparation method thereof

Abstract

The invention discloses a carbon nanotube FET biosensor for detecting a lung cancer marker p53 and a preparation method of the carbon nanotube FET biosensor, and belongs to the field of biosensors. The preparation method comprises the following steps: sequentially preparing an insulating layer, a carbon nanotube, a source electrode and a drain electrode on a substrate; carrying out surface modification on the carbon nano tube by using polyethylene glycol; carrying out activating treatment on the surface of the carbon nano tube by using EDC and NHS; a p53 antibody is added, the activated surface of the carbon nano tube reacts with amido on the antibody to form a covalent bond, so that the antibody is fixed on the surface of the sensor; and treating the sensor by using bovine serum albumin. According to the present invention, the good response performance is maintained in the high-salt environment, the high-specificity p53 recognition capability is provided, the sensor can produce the significant signal change under different p53 concentrations, and the good response is provided under the low concentration, such that the important effect is provided for the early diagnosis.

Description

Technical Field

[0001] The present invention mainly relates to the field of biosensors, in particular to a carbon nanotube FET biosensor for detecting lung cancer marker p53 and a preparation method thereof. Background Art

[0002] Currently, early diagnosis of lung cancer mainly relies on imaging examinations and serum marker detection. However, these methods often have problems with insufficient sensitivity and specificity. Therefore, it is of great significance to develop a highly sensitive and specific lung cancer marker detection method.

[0003] The p53 protein is one of the important biomarkers known to be associated with various cancers, especially lung cancer. However, there is currently a lack of efficient detection methods for the p53 protein on the market. Summary of the invention

[0004] The technical solution of the present invention aims at the technical problem that the existing technical solutions are too single, and provides a solution that is significantly different from the existing technology. It mainly provides a carbon nanotube FET biosensor for detecting the lung cancer marker p53 and a preparation method thereof, so as to achieve efficient detection of p53 protein.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: A method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 comprises the following steps: S1, sequentially preparing an insulating layer, a carbon nanotube, a source electrode and a drain electrode on a substrate; S2, using polyethylene glycol to modify the surface of carbon nanotubes; S3, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the surface of carbon nanotubes; S4, adding p53 antibody, the surface activated by carbon nanotubes reacts with the amine groups on the antibody to form a covalent bond, thereby fixing the antibody on the sensor surface; S5. The sensor was treated with bovine serum albumin to prepare a carbon nanotube FET biosensor for detecting lung cancer marker p53.

[0006] Further, step S1 includes: S11, select a high-quality silicon wafer as a substrate, and deposit a layer of insulating material thereon as an insulating layer; S12, growing carbon nanotubes on the insulating layer using a chemical vapor deposition method; S13. Then, electron beam evaporation technology is used to deposit a layer of highly conductive material on both ends of the carbon nanotube to form a source and a drain.

[0007] Furthermore, in step S11, the insulating material is silicon dioxide; And / or, the highly conductive material is gold.

[0008] Furthermore, in step S13, the surface of the carbon nanotubes is first cleaned to ensure that there are no impurities, and then the source and drain electrodes are deposited.

[0009] Furthermore, in step S13, the thickness of the source electrode and the drain electrode are equal, both of which are 10-100 nanometers.

[0010] Further, after the carbon nanotubes are formed, Y 2 O 3 Layer deposition: First, Y 2 O 3 The powder was dissolved in deionized water to prepare a precursor solution. The precursor solution was then uniformly deposited on the carbon nanotubes. Finally, the deposited Y 2 O 3 The samples of the layer were annealed.

[0011] Further, step S3 includes: S31, dispersing the PEG-modified carbon nanotubes in PBS buffer; S32, add EDC and NHS, and react at room temperature for 10-60 minutes; S33. Wash the carbon nanotubes with PBS buffer to remove excess EDC and NHS.

[0012] Further, step S4 includes: S41, dispersing the activated carbon nanotubes in PBS buffer; S42, add p53 antibody and react at room temperature for 1-3 hours; S43. Wash the carbon nanotubes with PBS buffer to remove unbound antibodies.

[0013] Further, step S5 includes: S51, dispersing the antibody-immobilized carbon nanotubes in PBS buffer; S52, add BSA, and react at room temperature for 0.5-3 hours; S53. Wash the carbon nanotubes with PBS buffer to remove excess BSA.

[0014] The present invention also provides a carbon nanotube FET biosensor for detecting lung cancer marker p53, which is prepared by the above-mentioned preparation method.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention improves the sensitivity and stability of the sensor by modifying a layer of polyethylene glycol (PEG) on the surface of carbon nanotubes, utilizing the shielding effect of PEG, improving water solubility, reducing nonspecific adsorption, and regulating interface interactions; the p53 antibody is fixed on the sensor surface after activation treatment with EDC / NHS to achieve specific recognition of the lung cancer marker p53; the sensor surface is sealed by BSA treatment to reduce nonspecific adsorption. Finally, the sensor maintains good response performance in a high-salt environment while also having the ability to recognize p53 with high specificity. The sensor can produce significant signal changes at different p53 concentrations and has a good response at low concentrations, which is particularly important for early diagnosis.

[0016] (2) The present invention provides a carbon nanotube FET biosensor for detecting the lung cancer marker p53. Experimental results show that under different p53 concentrations, the sensor of the present invention can produce significant signal changes. The lowest concentration response reaches 40%, while the highest concentration response can reach 200%, that is, it has high sensitivity; the sensor is highly specific to p53 and has high repeatability.

[0017] (3) The present invention is based on a basically formed sensor. First, PEG modification can not only introduce more functional groups such as hydroxyl groups and amino groups into the sensor (especially the surface of carbon nanotubes), but also these functional groups can form covalent bonds with the amino groups on the antibody, significantly enhancing the fixation effect of the p53 antibody. At the same time, PEG modification also greatly improves the water solubility and biocompatibility of carbon nanotubes, effectively reduces nonspecific adsorption, and thus improves the sensitivity and stability of the sensor. In this process, PEG modification makes obvious binding sites appear on the surface of the sensor, and intentionally blocks several non-target binding sites, so as to improve the accuracy of subsequent p53 antibody fixation sites. Then, EDC / NHS is used for activation treatment. EDC / NHS activation treatment can activate the carboxyl groups on the surface of carbon nanotubes, prompting them to form stable amide bonds with the amino groups on the p53 antibody, thereby ensuring that the p53 antibody can be accurately and firmly fixed on the sensor surface. This process not only avoids nonspecific adsorption, but also significantly improves the specificity of the sensor. Finally, BSA treatment is used to block the remaining binding sites on the sensor surface that are not bound to the antibody. BSA treatment effectively prevents nonspecific adsorption and ensures that only p53 antigen can specifically bind to the fixed antibody. This step is crucial to improving the signal-to-noise ratio and specificity of the sensor, further ensuring the precise binding of p53 antigen and antibody, thereby improving the overall performance and reliability of the sensor.

[0018] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the structure of the carbon nanotube FET biosensor provided by the present invention; Figure 2 A schematic diagram of the preparation process of the carbon nanotube FET biosensor provided by the present invention and the process of detecting p53 markers by the sensor; Figure 3 is the X-ray photoelectron spectroscopy (XPS) test result of the sensor surface of the present invention; Figure 4 The energy dispersive spectroscopy (EDS) characterization results of the sensor of the present invention after detecting antigens prove the uniform distribution of C, O, and N elements, indicating a good combination of antigen and antibody; Figure 5 is the transfer characteristic curve of the sensor at different p53 antigen concentrations in the present invention; Figure 6 The sensor specificity detection test results of the present invention; Figure 7 It is the repeatability test result of the sensor detection response value in the present invention. DETAILED DESCRIPTION

[0020] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings, but the present invention can be implemented in different forms and is not limited to the embodiments described in the text. On the contrary, these embodiments are provided to make the content disclosed in the present invention more thorough and comprehensive.

[0021] Example 1: Please refer to the attached Figure 2 A method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 comprises the following steps: 1. Choose the base material: A high-quality silicon wafer is selected as the substrate, on which a silicon dioxide insulating layer is deposited.

[0022] 2. Preparation and modification of carbon nanotubes: (1) Preparation of carbon nanotubes: Single-walled carbon nanotubes are grown on a silicon dioxide insulating layer using chemical vapor deposition (CVD).

[0023] (2) Y 2 O 3 Layer deposition: First, Y 2 O 3The powder was dissolved in deionized water to prepare a precursor solution with a concentration of 0.1 M. The precursor solution was then evenly deposited on the carbon nanotubes using a spin coating method. 2 O 3 The samples of the layer were annealed at 550 °C for 1 h to ensure that the Y 2 O 3 Layer uniformity and stability.

[0024] Y 2 O 3 It can act as a catalyst carrier during the growth process of carbon nanotubes, helping to improve the dispersion and stability of the catalyst, thereby improving the growth quality and density of carbon nanotubes.

[0025] (3) Forming the source and drain electrodes: First, clean the surface of the carbon nanotube to ensure that there are no impurities. Then use electron beam evaporation technology to deposit a layer of gold on both ends of the carbon nanotube to form the source and drain electrodes. During the evaporation process, the thickness of the gold layer is precisely controlled to 10 nanometers to ensure good electrical contact and mechanical stability. Ensure that the gold layer evenly covers both ends of the carbon nanotube to form a stable electrical connection.

[0026] Gold was selected as the material for the source and drain electrodes because gold has excellent conductivity, chemical stability and biocompatibility, making it suitable for use as an electrode material for biosensors.

[0027] (4) PEG surface modification: After the gold layer is formed and solidified, polyethylene glycol (PEG) is used to modify the surface of carbon nanotubes.

[0028] PEG modification can improve the compatibility between carbon nanotubes and antibodies. Through the self-assembly of PEG's thiol functionalized derivatives and the gold surface, a stable chemical bond is formed to increase the stability of the sensor and reduce nonspecific adsorption, ensuring good binding between carbon nanotubes and antibodies. PEG modification not only increases the functional groups on the surface of carbon nanotubes, but also improves the water solubility and biocompatibility of carbon nanotubes, making it easier for antibodies to bind to the surface of carbon nanotubes, thereby improving the sensitivity and stability of the sensor.

[0029] The shielding effect formed by PEG modification can effectively reduce nonspecific adsorption and improve the selectivity and specificity of the sensor. Subsequent BSA treatment can block the redundant binding sites on the sensor surface, prevent nonspecific adsorption, and ensure that only p53 antigen can bind to the fixed antibody, thereby improving the signal-to-noise ratio and specificity of the sensor.

[0030] 3. Antibody fixation: (1) Activating the surface of carbon nanotubes using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups so that they can form stable amide bonds with the amine groups; Specifically: first disperse the PEG-modified carbon nanotubes in PBS buffer; then add EDC and NHS (final concentrations of 10 mM and 10 mM, respectively) and react at room temperature for 30 minutes; finally, wash the carbon nanotubes with PBS buffer to remove excess EDC and NHS.

[0031] The X-ray photoelectron spectroscopy (XPS) test results of the prepared sensor surface are as follows: Figure 3 As shown, Figure 3 The presence of N element was shown in the figure, indicating that the EDC / NHS treatment was successful.

[0032] The advantages of activation treatment are: Precise control of activation sites: Through EDC / NHS activation treatment, the carboxyl groups on the surface of carbon nanotubes can be precisely activated to form stable amide bonds with the amino groups on the antibodies, ensuring that the antibodies can be accurately fixed on the sensor surface, avoiding nonspecific adsorption and improving the specificity of the sensor. At the same time, EDC / NHS activation treatment can ensure that the spatial conformation and activity of the antibodies are not affected, thereby ensuring the effective binding of the antibodies to the p53 antigen.

[0033] Enhanced binding strength: Through EDC / NHS activation treatment, the binding strength between the antibody and the carbon nanotube surface is significantly enhanced, which helps to improve the sensitivity and stability of the sensor. Strong binding can ensure that the antibody will not fall off during the detection process, thereby providing more reliable detection results.

[0034] (2) Add p53 antibody, the carbon nanotube-activated surface reacts with the amine groups on the antibody to form a covalent bond, thereby fixing the antibody on the sensor surface; Specifically: first disperse the activated carbon nanotubes in PBS buffer, add p53 antibody (final concentration is 1 μg / mL), and react at room temperature for 2 hours; then wash the carbon nanotubes with PBS buffer to remove unbound antibodies.

[0035] (3) The sensor was treated with BSA (bovine serum albumin) to prevent nonspecific binding.

[0036] Specifically: first disperse the antibody-immobilized carbon nanotubes in PBS buffer, add BSA (final concentration of 1%), and react at room temperature for 1 hour; then use PBS buffer to wash the carbon nanotubes to remove excess BSA.

[0037] The sensor surface is covered with a layer of protein to reduce the unreacted activation sites that may remain after the antibody is fixed. This layer of protein can prevent other molecules from directly contacting the sensor surface, thereby reducing nonspecific adsorption. This helps to improve the signal-to-noise ratio of the sensor, making it more sensitive and specific.

[0038] Thus, a carbon nanotube FET biosensor for detecting lung cancer marker p53 was prepared. Figure 1 shown.

[0039] The sensors prepared above are calibrated, tested and verified as follows: 1. Calibration process The sensor was calibrated using a p53 standard of known concentration to ensure that the sensor response was proportional to the p53 concentration.

[0040] 2. Testing process (1) Add the sample to be tested to the sensor surface and wait for a while to ensure that the p53 antigen is fully bound to the immobilized antibody. The waiting time is 30 minutes to 1 hour, which can achieve the maximum binding efficiency and ensure that most antigen-antibody pairs have enough time to bind. The sensor uses the specific binding between p53 antibody and p53 antigen. When the antigen binds to the sensor surface, it causes the electrical properties of the carbon nanotube FET to change, thereby realizing the detection of p53.

[0041] (2) After the sensor detects the p53 antigen, an energy dispersive spectroscopy (EDS) is used to observe and obtain the energy dispersive spectroscopy (EDS) characterization results, such as Figure 4 shown.

[0042] Depend on Figure 4 It can be seen that the C, O, and N elements are evenly distributed, indicating a good combination of the antigen and the antibody, and indicating that the sensor of the present invention is suitable for detecting the lung cancer marker p53.

[0043] (4) Prepare different concentrations of p53 antigen (1pM, 100fM, 10fM, 1fM, 100aM, all antibodies) and record the output signal of the sensor at different p53 antigen concentrations. (5) and plot the transfer characteristic curve of the sensor under different p53 antigen concentrations, such as Figure 5 shown.

[0044] Depend on Figure 5 It can be seen that as the concentration of p53 antigen increases, the response of the sensor of the present invention becomes more obvious. Under different p53 concentrations, the sensor can produce significant signal changes, with the lowest concentration response reaching 40% and the highest concentration response reaching 200%.

[0045] 3. Specificity verification (1) Separate comparative experiments Prepare the samples to be tested: Experimental group: solution containing p53 antigen diluted in phosphate buffered saline (PBS), denoted as p53; Control group 1: a solution containing CA125 protein (a non-target lung cancer marker) diluted in phosphate buffered saline (PBS), denoted as CA125; Control group 2: a solution containing KRAS protein (a gene associated with lung cancer but not a protein marker) diluted in PBS buffer, denoted as KRAS; Control group 3: only contains PBS buffer without any protein or biomolecule, denoted as Blank; serves as a negative control to evaluate the background signal of the sensor in the absence of target molecules.

[0046] The sensor prepared by the present invention was used to detect the experimental group and each control group. The results are as follows: Figure 6 shown.

[0047] Depend on Figure 6 It can be seen that the p53 curve is significantly higher than the other curves, while the CA125, KRAS, and Blank curves are all at a lower level, indicating that the sensor has little response to non-target lung cancer markers and non-protein molecules, but a significant response to p53, indicating that the sensor has good specificity.

[0048] (2) Mixed experiment The experiment was conducted using a mixed solution containing p53 antigen and other potential interfering substances to observe whether the sensor of the present invention responds only to p53. The other potential interfering substances may be at least one of BSA nonspecific protein, cancer antigen 125 and carcinoembryonic antigen tumor markers, and neuron-specific enolase (NSE) and cytokeratin fragment 21-1 (CYFRA 21-1) lung cancer markers.

[0049] It was observed that the sensor only responded significantly to p53, but had no response or very little response to other substances, which indicated that the sensor had good specificity.

[0050] In summary, the sensor is highly specific to p53 and can effectively distinguish p53 from other substances.

[0051] 4. Repeatability verification Experimental design: Select multiple identical sensors and test them using the same concentration of p53 standard.

[0052] Data statistics: Count the response values ​​of each sensor and draw a bar chart, such as Figure 7 As shown; calculate the standard deviation and evaluate the repeatability of the sensor.

[0053] Depend on Figure 7 It can be seen that the response values ​​of the sensor of the present invention are all between 0.07-0.075, and the standard deviation is small, indicating that the sensor has high repeatability.

[0054] Embodiment 2: This embodiment differs from Embodiment 1 in that: In this embodiment, the carbon nanotubes are prepared by growing multi-walled carbon nanotubes on a silicon dioxide insulating layer using chemical vapor deposition (CVD).

[0055] In this embodiment, the forming of the source and drain electrodes: During the evaporation process, the thickness of the gold layer is precisely controlled to be 100 nanometers.

[0056] In this embodiment, during the EDC and NHS activation treatment, after adding EDC and NHS, the reaction was carried out at room temperature for 60 minutes; after adding p53 antibody, the reaction was carried out at room temperature for 3 hours; after adding BSA, the reaction was carried out at room temperature for 3 hours.

[0057] The rest is the same as the first embodiment.

[0058] Embodiment 3: This embodiment differs from Embodiment 1 in that: In this embodiment, the carbon nanotubes are prepared by growing multi-walled carbon nanotubes on a silicon dioxide insulating layer using chemical vapor deposition (CVD).

[0059] In this embodiment, the forming of the source and drain electrodes: During the evaporation process, the thickness of the gold layer is precisely controlled to be 60 nanometers.

[0060] In this embodiment, during the EDC and NHS activation treatment, after adding EDC and NHS, the reaction was carried out at room temperature for 10 minutes; after adding p53 antibody, the reaction was carried out at room temperature for 1 hour; after adding BSA, the reaction was carried out at room temperature for 0.5 hour.

[0061] The rest is the same as the first embodiment.

[0062] The above is an exemplary description of the present invention in combination with the accompanying drawings. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned method. As long as such non-substantial improvements are made by adopting the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53, characterized in that: The steps include: S1, sequentially preparing an insulating layer, a carbon nanotube, a source electrode and a drain electrode on a substrate; S2, using polyethylene glycol to modify the surface of carbon nanotubes; S3, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the surface of carbon nanotubes; S4, adding p53 antibody, the surface activated by carbon nanotubes reacts with the amine groups on the antibody to form a covalent bond, thereby fixing the antibody on the sensor surface; S5. The sensor was treated with bovine serum albumin to prepare a carbon nanotube FET biosensor for detecting lung cancer marker p53.

2. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 1, characterized in that: Step S1 includes: S11, select a high-quality silicon wafer as a substrate, and deposit a layer of insulating material thereon as an insulating layer; S12, growing carbon nanotubes on the insulating layer using a chemical vapor deposition method; S13. Then, electron beam evaporation technology is used to deposit a layer of highly conductive material on both ends of the carbon nanotube to form a source and a drain.

3. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 2, characterized in that: In step S11, the insulating material is silicon dioxide; And / or, the highly conductive material is gold.

4. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 2, characterized in that: In step S13, the surface of the carbon nanotubes is first cleaned to ensure that there are no impurities, and then the source and drain electrodes are deposited.

5. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 2, characterized in that: In step S13, the thickness of the source electrode and the drain electrode are equal, both of which are 10-100 nanometers.

6. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 2, characterized in that: After the carbon nanotubes are formed, the Y2O3 layer is deposited: first, the Y2O3 powder is dissolved in deionized water to prepare a precursor solution; then the precursor solution is evenly deposited on the carbon nanotubes; finally, the sample with the Y2O3 layer deposited is annealed in an air atmosphere.

7. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 1, characterized in that: Step S3 includes: S31, dispersing the PEG-modified carbon nanotubes in PBS buffer; S32, add EDC and NHS, and react at room temperature for 10-60 minutes; S33. Wash the carbon nanotubes with PBS buffer to remove excess EDC and NHS.

8. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 1, characterized in that: Step S4 includes: S41, dispersing the activated carbon nanotubes in PBS buffer; S42, add p53 antibody and react at room temperature for 1-3 hours; S43. Wash the carbon nanotubes with PBS buffer to remove unbound antibodies.

9. The method for preparing a carbon nanotube FET biosensor for detecting lung cancer marker p53 according to claim 1, characterized in that: Step S5 includes: S51, dispersing the antibody-immobilized carbon nanotubes in PBS buffer; S52, add BSA, and react at room temperature for 0.5-3 hours; S53. Wash the carbon nanotubes with PBS buffer to remove excess BSA.

10. A carbon nanotube FET biosensor for detecting lung cancer marker p53, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 9.

Images