A chitosan-modified graphene field effect transistor biosensor and its application

Abstract

The invention discloses a chitosan-modified graphene field effect transistor biosensor and application thereof. The invention uses a silicon dioxide / silicon dioxide (Si / SiO2) substrate modified with a source and a drain as a substrate, transfers graphene as a sensing channel to the substrate surface to form a GFET, uses CTS as a linker and is electrostatically adsorbed on the sensing channel surface, and uses an anti-procalcitonin antibody (anti-PCT) as a probe to be modified on the CTS surface through glutaraldehyde cross-linking to specifically capture PCT; the invention can detect the procalcitonin in a manner of causing a voltage shift of the CTS-GFET Dirac point after PCT specifically binds to the PCT antibody on the CTS-GFET channel surface, has high detection sensitivity and specific selectivity, and its detection sensitivity for procalcitonin can reach 0.82 ag / mL, which is 3 times higher than the detection limit of a traditional GFET.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to a chitosan-modified graphene field effect transistor biosensor and applications thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by bacterial infection. Therefore, early and sensitive diagnosis of sepsis is of great significance to prevent the occurrence of septic shock and improve survival rate. A series of biomarkers have been developed to detect sepsis, such as C-reactive protein (CRP), interleukin 6 (IL-6) and procalcitonin (PCT). Compared with them, PCT can more specifically indicate bacterial infection or infection severity. Generally, under normal physiological conditions, PCT levels are below 0.05 ng / ml, while serum concentrations in sepsis patients increase by at least 1000 times within 6-12 hours. Therefore, a variety of methods have been developed, such as enzyme-linked immunosorbent (ELISA), fluorescent immunoassay, electrochemiluminescence immunoassay and colorimetric immunoassay, for the qualitative and quantitative detection of PCT. However, these methods are limited by long analysis time, high cost and the need for well-trained personnel and sophisticated instruments.

[0003] Biosensors have been widely used in the field of biochemical molecules and detection due to their advantages such as sensitivity, speed, simple operation and ease of automation. Among various biosensing platforms, graphene-based field effect transistors (GFETs) are ideal for creating simple and reliable biosensors due to their fast, sensitive, specific and low-cost detection characteristics. Graphene is a single-layer carbon-based material that is an ideal sensing material for biomolecules due to its large surface area and good electrical properties. Field effect transistors (FETs) are three-terminal transistors that use electric fields to control current. For GFET sensing performance, specificity and action are entirely dependent on the coupling of effective recognition components on the surface of the sensing channel. Among the many coupling methods, non-covalent interactions have become the most important coupling method in GFET sensing because they do not destroy the graphene lattice or reduce its electronic properties. Some bifunctional non-covalent linkers, such as 1-pyrenebutyric acid succinimidyl ester, N-hydroxysuccinimide ester (NHS), pyrenebutyric acid, and bovine serum albumin (BSA) have been successfully used to construct the biointerface of GFETs for the detection of single point mutations (SNPs), exosomes, and biotin.

[0004] Chitosan (CTS) is a non-toxic and biocompatible amine-rich linear polysaccharide synthesized by diacylation of chitin. Generally, CTS can be dissolved in acidic media and form a layer structure. The film produced has high permeability and mechanical strength, good adhesion and sensitivity to chemical modification. In addition, protons and counterions will be close to the CTS chain and produce high-performance dielectrics, which will improve the conductivity and sensitivity of bioanalysis. Therefore, CTS has been widely used to manufacture various electronic devices such as electrochemical sensors, synaptic devices and organic thin film transistors (OTFTs). Therefore, the present invention combines chitosan with GFET sensor performance to develop a graphene field effect transistor biosensor for detecting PCT with more sensitive and higher accuracy, so as to provide a new direction for better detection of sepsis. Summary of the invention

[0005] Therefore, in view of the above background, the present invention provides a chitosan-modified graphene field effect transistor biosensor and its application, which is based on a non-covalently modified, label-free CTS-GFET immunosensor, with a source and drain modified silicon dioxide / silicon dioxide (Si / SiO2) substrate as a substrate, graphene as a sensing channel is transferred to the substrate surface to form a GFET, CTS is used as a linker and is electrostatically adsorbed on the surface of the sensing channel, and anti-procalcitonin antibody is used as a probe to be modified on the CTS surface by glutaraldehyde cross-linking to specifically capture PCT.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a chitosan-modified graphene field effect transistor biosensor comprises the following steps:

[0008] S1: Graphene sheets are spin-coated with polymethyl methacrylate and then cut into graphene / PMMA sheets;

[0009] S2: placing the graphene / PMMA flakes in an ammonium persulfate solution to remove the surface copper substrate; after the copper at the bottom of the graphene / PMMA flakes is oxidized, transferring them to deionized water to remove the ammonium persulfate molecules remaining on the graphene surface;

[0010] S3: transferring and adsorbing the graphene / PMMA flakes onto a silicon dioxide / silicon substrate and performing a drying process;

[0011] S4: placing the dried sample in an acetone solution to remove polymethyl methacrylate on the surface of the graphene; then soaking the sample in an isopropanol solution and drying it to obtain a graphene / SiO2 / Si sample;

[0012] S5: placing the dried graphene / SiO2 / Si sample in a nitrogen / hydrogen mixture with a volume ratio of 1:1 and heating it to further remove the polymethyl methacrylate on the graphene surface;

[0013] S6: After heating is completed and the temperature drops to room temperature, silver electrodes are modified on both ends of the graphene. After drying, insulating glue is modified around the graphene as a gating device to obtain a GFET sensor, which is dried for later use.

[0014] S7: Modify chitosan onto the graphene surface of the GFET sensor and wash it with PBS solution;

[0015] S8: Modify the chitosan on the graphene with glutaraldehyde, wash it with PBS solution, and then test the electrical properties;

[0016] S9: The probe is modified onto chitosan by glutaraldehyde, washed with PBS solution, and then modified and blocked with bovine serum albumin to prepare a chitosan-modified graphene field effect transistor biosensor.

[0017] Preferably, the probe in step S8 is a procalcitonin antibody.

[0018] Preferably, the operation steps of modifying the graphene surface with chitosan in step S7 are as follows:

[0019] A 5% chitosan solution was dropped onto the surface of the GFET sensing channel and electrostatically adsorbed for 2 h.

[0020] Preferably, the operation steps of modifying chitosan with glutaraldehyde in step S8 are as follows:

[0021] A 15% glutaraldehyde solution was added dropwise to the surface of chitosan and allowed to stand for 1 h.

[0022] Preferably, the steps of modifying the bioantibody onto chitosan in step S9 are as follows:

[0023] The procalcitonin antibody solution was added dropwise to the surface of the GFET sensing channel and allowed to stand for 1 h.

[0024] The present invention also provides a chitosan-modified graphene field effect transistor biosensor, which is prepared by using the above-mentioned method for preparing a chitosan-modified graphene field effect transistor biosensor.

[0025] The present invention also provides a method for detecting procalcitonin, which uses a biosensor prepared by the above-mentioned method for preparing a chitosan-modified graphene field effect transistor biosensor for detection.

[0026] The present invention also provides an application of a biosensor prepared by the method for preparing a chitosan-modified graphene field effect transistor biosensor, which can be used to prepare a product for detecting sepsis.

[0027] The beneficial effects achieved by the present invention are as follows:

[0028] (1) The present invention uses a silicon dioxide / silicon dioxide (Si / SiO2) substrate modified with a source and a drain as a substrate, transfers graphene as a sensing channel to the substrate surface to form a GFET, uses CTS as a linker and is electrostatically adsorbed on the surface of the sensing channel, and uses an anti-procalcitonin antibody (anti-PCT) as a probe to be modified on the CTS surface by glutaraldehyde cross-linking to specifically capture PCT; it can detect PCT by causing a voltage shift at the Dirac point of the CTS-GFET after PCT specifically binds to the PCT antibody on the surface of the CTS-GFET channel, and has high detection sensitivity and specific selectivity. Its detection sensitivity for procalcitonin can reach 0.82 ag / mL, which is 3 times higher than the detection limit of traditional GFET;

[0029] (2) And after experimental verification, its detection in spiked serum samples proved its practical application ability, indicating its reliable potential use in clinical applications;

[0030] (3) The GTS-GFET sensor of the present invention has good reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention.

[0032] Attached Figure 1 Schematic diagram of the preparation process of the GTS-GFET sensor of the present invention;

[0033] Attached Figure 2 This is a comparison chart of the detection sensitivity of the GTS-GFET sensor and the GFET sensor of the present invention for procalcitonin; wherein Figure 2 A is the schematic diagram of GFET and CTS-GFET biosensors for PCT detection; Figure 2 B is the comparison of PCT detection results between GFET and CTS-GFET biosensors;

[0034] Attached Figure 3 The SEM image of the sensing channel of the CTS-GFET biosensor preparation process of the present invention is shown in FIG. Figure 3A is the SEM image of the GFET sensor channel surface; Figure 3 B is the SEM image after modification of CTS; Figure 3 C is the SEM image of the modified Ab (procalcitonin antibody);

[0035] Attached Figure 4 The XPS diagram of the preparation process of the CTS-GFET biosensor of the present invention is shown in FIG. Figure 4 A is the XPS measurement spectrum of the GFET sensor before modification; Figure 4 B and Figure 4 C is the XPS measurement spectrum of the biosensor modified with chitosan;

[0036] Attached Figure 5 The intrinsic curve and resistance change of the CTS-GFET biosensor preparation process of the present invention; wherein Figure 5 A is the IV curve change diagram during the CTS-GFET modification process (G in the figure: single GFET; CTS: GFET modified with chitosan; BSA: CTS-GFET blocked with BSA)); Figure 5 B is the resistance (R) change diagram during the CTS-GFET modification process;

[0037] Attached Figure 6 The CTS-GFET biosensor of the present invention detects PCT at different concentrations; Figure 6 A is the transfer characteristics of CTS-GFET biosensor at different PCT concentrations. Figure 6 B is the ΔVdirac shift and fitted linear equation of the PCT concentration-dependent CTS-GFET biosensor; Figure 6 C is the transfer characteristics of the GFET biosensor at different PCT concentrations;

[0038] Attached Figure 7 The present invention is to study the sensing enhancement mechanism model of the CTS-GFET biosensor mechanism performance, wherein Figure 7 A is a schematic diagram of the sensing model of the CTS-GFET biosensor; Figure 7 B is the change of graphene contact angle before and after graphene PLL functionalization; Figure 7 C is the UV absorption spectrum of the remaining antibody after CTS-GFET and GFET modification;

[0039] Attached Figure 8 is the specific detection result of the CTS-GFET biosensor of the present invention, wherein Figure 8 A is the specific detection result of CTS-GFET; Figure 8 B is the transfer characteristics of the CTS-GFET biosensor at different PCT concentrations in serum samples; Figure 8C is the PCT concentration-dependent V of the CTS-GFET biosensor Dirac displacement and fitting linear equations;

[0040] Attached Fig. 9 The CTS-GFET biosensor of the present invention repeatedly detects the results, wherein Fig. 9 A is the transfer characteristics of CTS-GFET biosensor at different PCT concentrations. Fig. 9 B is the PCT concentration-dependent V of the CTS-GFET biosensor. Dirac Displacement and fitting linear equations. DETAILED DESCRIPTION

[0041] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0042] Therefore, it is intended that the present invention covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It should be understood by those of ordinary skill in the art that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention. The present invention is further described below in conjunction with examples.

[0043] The technical solution of the present invention is:

[0044] A method for preparing a chitosan-modified graphene field effect transistor biosensor comprises the following steps:

[0045] S1: The graphene sheet is spin-coated with polymethyl methacrylate and then cut into graphene / PMMA small sheets. The specific spin-coating thickness is 500nm.

[0046] S2: placing the graphene / PMMA flakes in an ammonium persulfate solution to remove the surface copper substrate; after the copper at the bottom of the graphene / PMMA flakes is oxidized, transferring them to deionized water to remove the ammonium persulfate molecules remaining on the graphene surface;

[0047] S3: transferring and adsorbing the graphene / PMMA flakes onto a silicon dioxide / silicon substrate and performing a drying process;

[0048] In this step, the graphene sheet / PMMA floats on the surface of the water. The graphene sheet / PMMA can be dragged out of the water using a silicon dioxide / silicon substrate. Since graphene is hydrophobic and the surface of silicon dioxide is also hydrophobic, the graphene and silicon dioxide are bonded together by hydrophobic forces.

[0049] S4: placing the dried sample in an acetone solution to remove PMMA on the graphene surface; then soaking the sample in an isopropanol solution and drying it to obtain a graphene / SiO2 / Si sample;

[0050] S5: placing the dried graphene / SiO2 / Si sample in a nitrogen / hydrogen mixture with a volume ratio of 1:1 and heating it to further remove the polymethyl methacrylate on the graphene surface;

[0051] S6: After heating is completed and the temperature drops to room temperature, conductive silver glue is used to modify the upper silver electrodes at both ends of the graphene. After drying, insulating glue is modified around the graphene as a gating device to obtain a GFET sensor, which is dried for later use.

[0052] S7: drop a 5% chitosan solution onto the surface of the GFET sensing channel, and after electrostatic adsorption for 2 h, chitosan is modified onto the graphene surface of the GFET sensor, and then washed with PBS solution;

[0053] S8: dropping a 15% glutaraldehyde solution onto the surface of chitosan and letting it stand for 1 hour to modify the chitosan on the graphene with glutaraldehyde, and then washing with PBS solution and conducting an electrochemical performance test;

[0054] The electrochemical performance test includes: determination of Ids-Vgs curve and resistance to observe V Driac The offset and resistance value change, V Driac The shift and increase in resistance value indicate that the glutaraldehyde modification is successful.

[0055] S9: Add the procalcitonin antibody solution to the surface of the GFET sensing channel and let it stand for 1 hour. Then, modify the probe onto chitosan using glutaraldehyde and wash it with PBS solution. Add 2% BSA solution to the surface of the GFET sensing channel and let it stand for 1 hour. Then, modify and seal it with bovine serum albumin to prepare a chitosan-modified graphene field effect transistor biosensor.

[0056] In addition to S8, steps S7 and S9 also require corresponding Ids-Vgs curve and resistance measurement after modification to determine whether the modification is successful.

[0057] In order to better understand the present invention, the following is a related test verification and description of the present invention in combination with specific embodiments.

[0058] The experimental materials used in the following examples are:

[0059] PBS buffer and BSA were purchased from Solarbio Life Sciences (Beijing Solarbio Technology Co., Ltd., Beijing, China);

[0060] PCT, interleukin-6 (IL-6), C-reactive protein (CRP), and procalcitonin antibody (Ab) (CSB-DA131DmN②) were purchased from Cusabio Biotech Co. (Wuhan, China);

[0061] Chitosan, phosphate buffered saline (PBST), and glutaraldehyde were purchased from Tansloo (Shanghai, China), and ethanol base (EA) and silver were purchased from Sigma-Aldrich;

[0062] Graphene is grown by CVD.

[0063] Example 1: A method for preparing a chitosan-modified graphene field effect transistor biosensor, comprising the following steps:

[0064] S1: Graphene sheets were spin-coated with polymethyl methacrylate (PMMA) and then cut into 1 mm × 1 mm graphene / PMMA sheets, with a spin-coating thickness of 500 nm;

[0065] S2: Place the graphene / PMMA flakes in a 1 mM ammonium persulfate (AP) solution to remove the copper substrate on the surface; after the copper on the bottom of the graphene / PMMA flakes is oxidized, transfer them to deionized water and soak them overnight to remove the ammonium persulfate molecules remaining on the graphene surface;

[0066] S3: Transfer and adsorb the graphene / PMMA flakes onto a 1 cm×1 cm cut silicon dioxide / silicon substrate (SiO2 / Si) and dry overnight;

[0067] S4: The dried sample is placed in an acetone solution for 6 h to remove the polymethyl methacrylate (PMMA) on the graphene surface; the sample is then immersed in an isopropanol solution for 3 h and air-dried to obtain a graphene / SiO2 / Si sample;

[0068] S5: The dried graphene / SiO2 / Si sample was placed in a nitrogen / hydrogen mixture with a volume ratio of 1:1 and heated at 400 °C for 2 h to further remove the polymethyl methacrylate on the graphene surface;

[0069] S6: After heating, the temperature is lowered to room temperature. At room temperature, silver (Ag) electrodes are modified on both ends of the graphene. After drying, insulating glue is modified around the graphene as a gating device to obtain a GFET sensor, which is dried for later use.

[0070] S7: The graphene surface of the GFET sensor was modified with 30 μL 5% chitosan solution for 2 h and then washed three times with 1× PBS solution;

[0071] S8: After the chitosan on the graphene was modified with 30 μL 15% glutaraldehyde solution for 1 h, it was washed three times with 1× PBS solution and the electrochemical performance was tested;

[0072] S9: After modification with 30 μL 14 μg / mL procalcitonin antibody for 1 h, the probe was modified onto chitosan using glutaraldehyde and washed three times with 1× PBS solution, and then modified and blocked with 30 μL 2% bovine serum albumin (BSA) to prepare a chitosan-modified graphene field effect transistor biosensor.

[0073] The GFET sensor was prepared. Compared with the present embodiment, the GFET sensor was modified in steps S8 to S9. 30 mL of 5 mM 1-pyrenebutyric acid succinimidyl ester (PBASE) was dripped on the surface of the graphene sensing channel. After standing for 2 hours, it was washed three times with dimethylformamide solution (DMF) and deionized water respectively. The intrinsic electrical curve (IV curve) of the graphene field effect transistor was measured. According to the Dirac point voltage (V Dirac ) to determine whether the PBASE modification was successful. Then, 30 μL of 14 μg / mL procalcitonin antibody (Ab) was dripped onto the surface of the graphene sensing channel. After standing for 2 hours, it was washed three times with 1´ PBS. The intrinsic electrical curve (IV curve) of the modified graphene field effect transistor was measured. According to the Dirac point voltage (V Dirac ) to determine whether the Ab modification is successful or not. 30μL 2% bovine serum albumin (BSA) was dripped on the surface of the graphene sensing channel for modification and sealing. After standing for 3 hours, it was washed 3 times with 1´ PBS. The GFET sensors involved below were all made by this method. The preparation process of the CTS-GFET sensor prepared in this example was characterized by SEM and XPS and its electrical properties. The characterization results are shown in Figures 3 to 5 .

[0074] In this example, the morphology of the CTS-GFET biosensor was characterized by scanning electron microscopy. Figure 3A shows the signal layer of graphene transferred to Si / SiO2 substrate. The wrinkles, gaps and cracks that occurred during this process can be clearly seen. After chitosan modification, the graphene surface forms a layered structure and produces some cavities ( Figure 3 B). When the antibody (Ab) was linked to the chitosan surface by glutaraldehyde, the SEM image showed that the chitosan-modified particles were scattered on the surface of the sensing channel ( Figure 3 C). Clearly, the sensing surface of the CTS-GFET biosensor is well constructed.

[0075] And the chitosan functionalization on the graphene surface was also confirmed by XPS. Figure 4 A shows the XPS measurement spectra of the GFET sensor before and after CTS modification. Graphene is mainly composed of C and CTS is mainly composed of C, N and O. The O peak detected in graphene before CTS modification is mainly due to the adsorption of O2 and H2O on the surface. After chitosan modification, the appearance of the N 1s peak and the increase in the intensity of the O 1s peak indicate the presence of CTS (Table 1). The N1s spectrum of CTS shows two binding energy peaks at 398.9 eV and 396.7 eV, representing the presence of –NH and C=N bonds, respectively ( Figure 4 B). For the O 1s spectrum, the binding energies are located at 532.6 eV and 530.9 eV, which are attributed to the presence of C=O and –OH bonds, respectively ( Figure 4 C). XPS spectrum results further confirmed that chitosan has been successfully covered on the graphene surface.

[0076] Table 1: Changes in the contents of C, N, and O elements before and after chitosan modification

[0077]

[0078] In addition, the process of sensor surface modification was also identified by the change of the sensor intrinsic curve. After CTS modification, the Dirac point voltage (VDirac) of GFET shifted to the left by 50mV (0.65mV to 0.60mV) ( Figure 5 A), which is mainly due to the negatively charged CTS-induced n-doping of graphene. After Ab immobilization, the transfer curve further shifted to the left by 150 mV on the CTS-GFET. In addition, the resistance of the sensor gradually increased as the modification process proceeded ( Figure 5 B). These results confirm the successful construction of CTS-GFET and GFET biosensors.

[0079] Example 2: PCT detection

[0080] The CTS-GFET sensor and GFET prepared in Example 1 were used for the detection of PCT.

[0081] The CTS-GFET sensor of the present invention detects PCT mainly by causing a voltage shift of the Dirac point of the CTS-GFET after PCT specifically binds to the PCT antibody on the surface of the CTS-GFET channel.

[0082] The present invention uses CTS-GFE sensor for PCT detection operation. First, PBS is used to prepare PCT solutions of different concentrations. Then, PCT of different concentrations is combined with antibodies on the surface of CTS-GFET and incubated for 120 minutes. Then, the surface of the sensor channel is washed with 1×PBS to remove free PCT on the surface of the sensor channel. The following Ids-Vgs curve is measured on a probe station using a Keithley 4200A source meter. Figure 2 A shows the principle of using GFET and CTS-GFET for PCT detection. And the detection results show that CTS-GFET exhibits relatively higher sensitivity than GFET ( Figure 2 B, C).

[0083] Example 3: Sensitivity of CTS-GFET sensor for PCT detection

[0084] In order to evaluate the sensing performance of CTS-GFET, this example tested PBS buffer solutions with different PCT concentrations.

[0085] The blank test was measured with 30 L of PBS buffer solution.

[0086] PBS buffers with different PCT concentrations were prepared. The PCT concentrations were 1 ag / mL, 10 ag / mL, 100 ag / mL, 1 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, and 10 pg / mL, respectively. The PBS buffer for each PCT concentration was 30 L.

[0087] Then, PBS buffer solutions of different concentrations were added to the sensing channel. The specific operation was as follows: 30 L of PBS containing different PCT concentrations was directly dripped onto the surface of the sensing channel using a pipette.

[0088] Figure 6 A shows the IV curve changes with the change of PCT concentration. The VDirac of the biosensor gradually shifts to the right as the PCT concentration increases from 1 ag / mL to 10 pg / mL, and shows a good linear relationship in this range ( Figure 6B) The right shift of VDirac indicates that PCT combined with CTS-GFET biosensor induces the p-doping effect of graphene. Generally, the minimum detection limit of the sensor is calculated as 3 times the standard deviation of the sensor response to the control solution. The results show that the CTS-GFET sensor exhibits an ultra-low detection limit of about 0.82 ag / mL.

[0089] As a comparison, this embodiment uses a GFET sensor (, as above, to detect different concentrations of PCT in PBS buffer. In the range of 6 ag / mL to 10 pg / mL, the V Dirac As PCT concentration increases, it shifts to the right ( Figure 6 C), LOD was 2.47 ag / mL. Chitosan functionalization achieved a 3-fold sensitivity enhancement.

[0090] Example 4: Mechanism of CTS-GFET Biosensor

[0091] In order to study the performance of CTS-GFET biosensor, an electric double layer (EDL) model was established in this example. Figure 7 As shown in Figure A, after applying the gate voltage, the EDL will form at the electrode / electrolyte / graphene interface. When the recognition probe is anchored on the graphene surface, the total gate capacitance (C) of the sensor device is composed of four parallel plate capacitors (C G1 , C G2 , C G3 and C Q )composition.

[0092]

[0093] Among them C G1 , C G2 and C G3 Represent the capacitance of graphene solution, Ab solution and reference electrode solution respectively. C Q Represents the quantum capacitance of graphene, which is due to the Pauli principle. The immune recognition reaction of Ab-PCT on the graphene surface of the CTS-GFET biosensor changes the charge (Dq) at the solution-channel surface interface, resulting in a change in electrostatic potential, which ultimately causes the Dirac point to shift, and the relationship is as follows:

[0094]

[0095] Generally speaking, Dq can be expressed as kakbqaS, where ka is the enhancement coefficient of PCT on the graphene surface, kb is the binding efficiency of PCT and Ab probe, qa is the charge contributed by the unit surface density of PCT binding on the surface of the CTS-GFET biosensor, and S is the effective channel area. So eq2 can be written as

[0096]

[0097] From eq3, it can be seen that under the same conditions, the higher sensitivity of CTS-GFET can be attributed to the higher PCT capture ability. In general, the PCT capture ability is positively correlated with the amount of modified antibodies on the detection channel surface. First, the contact angles of different channel surfaces were studied. Figure 7 As shown in B, for graphene, the contact angle is 106°, and after chitosan modification, the contact angle is reduced to 50°. Previous studies have shown that a reduction in the contact angle means better contact between the molecules in the solution and the sensor channel, which is conducive to the binding of charged molecules to the surface. We chose to use a UV spectrophotometer to identify the remaining antibody concentration after the channel surface was modified. Figure 7 C It can be seen that the absorbance (A) of GFET is lower than that of CTS-GFET, which means that more Ab will be anchored on the channel surface modified with PBSAE. This result is inconsistent with our suspicion, so the PCT capture ability is not the main reason for improving the detection sensitivity.

[0098] From eq2, the total gate capacitance is another important factor affecting the detection performance of the sensor. Previous studies have shown that when CTS is modified on the surface of the sensing channel, the protons and counterions near the chitosan chain will generate dielectrics and form EDL after applying a bias. The formation of EDL leads to higher surface capacitance values ​​than traditional dielectrics (such as SiO2) and ultimately exhibits a high degree of detection performance. Therefore, with GFET In comparison, the higher sensing performance of CTS-GFET is mainly due to the higher areal capacitance value of CTS.

[0099] Example 5: Detection of sensor-specific selectivity

[0100] In the performance evaluation of biosensors, not only the sensitivity of the sensor is considered, but also its specificity is evaluated. In fact, due to the complex composition of biosensors, selectivity has become an important parameter in sensor applications. Therefore, this example further measures the specific selectivity of the CTS-GFET sensor for PCT by detecting interleukin-6 (IL-6), C-reactive protein (CRP) and their mixtures.

[0101] The specific operation process of this embodiment is as follows: interleukin-6 (IL-6), C-reactive protein (CRP), PCT and a mixture of the three proteins in equal proportions at a concentration of 10 pg / mL are detected by CTS-GFET, and the detection selectivity of the CTS-GFET sensor for PCT is determined by comparing the shift trend of VDriac caused by the above proteins. The results show that the CTS-GFET sensor has a significantly higher response to PCT or a sensor containing PCT than IL-6, CRP and a mixture thereof in 1´PBS buffer at pH 7.0, with a final concentration of 10 pg / mL.

[0102] like Figure 8 As shown in A, the excellent specificity of the sensor is mainly due to the high affinity between PCT and PCT antibody.

[0103] In addition, the CTS-FET biosensor was also tested in human serum. Figure 8 As shown in B, with the increase of PCT concentration, the V Dirac Gradually move left, DV Dirac and PCT concentration showed a good linear relationship ( Figure 8 C), indicating that it exhibits excellent sensing performance in human serum. It is worth noting that with the increase of PCT concentration, the DV in PBS buffer and human serum Dirac The main reason for this phenomenon is that PCT in human serum is negatively charged compared with PBS buffer, which leads to V Dirac Move left.

[0104] Example 6: Reproducibility and reusability of CTS-GFET biosensor

[0105] For biosensors, reproducibility and reusability are also very important. 20 CTS-GFET immunosensors were prepared using the present invention, and reproducibility tests were performed on the 20 CTS-GFET sensors prepared. These immunosensors were independently produced by the same procedure. The RSD (relative standard deviation) value was 0.24%. The low RSD value indicates the reliability of the immunosensor production process.

[0106] Finally, to verify the reusability, the sensing channel was washed with 1× PBST after PCT was captured by CTS-GFET. Then, the procalcitonin antibody was re-modified and blocked with bovine serum albumin, and the sensing performance of the sensor was tested again. Fig. 9 As shown, the sensing channel of procalcitonin antibody showed good sensing performance again, and DV DiracA good linear relationship was shown between the TEM and PCT concentration, indicating that the CTS-GFET has good reusability.

[0107] Therefore, the present invention has developed an ultrasensitive, CTS-modified GFET biosensor for the selective detection of PCT. This CTS-GFET biosensor provides a wider detection range (1ag / ml-10 pg / mL) and a minimum detection value (LOD) of 0.82 ag / ml in PBS buffer, which is more than 3 times more sensitive than the GFET biosensor. CTS-GFET was also used to detect spiked PCT samples in human serum, indicating the potential application of the sensor in clinical diagnosis.

[0108] Matters not covered in the present invention are all conventional techniques in the art and will not be elaborated here.

[0109] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A method for preparing a chitosan-modified graphene field effect transistor biosensor, characterized in that: It includes the following steps: S1: Graphene sheets are spin-coated with polymethyl methacrylate (PMMA) and then cut into graphene / PMMA sheets; S2: placing the graphene / PMMA flakes in an ammonium persulfate solution to remove the surface copper substrate; after the copper at the bottom of the graphene / PMMA flakes is oxidized, transferring them to deionized water to remove the ammonium persulfate molecules remaining on the graphene surface; S3: transferring and adsorbing the graphene / PMMA flakes onto a silicon dioxide / silicon substrate and performing a drying process; S4: placing the dried sample in an acetone solution to remove PMMA from the graphene surface; Then the sample was immersed in an isopropanol solution and dried to obtain a graphene / SiO2 / Si sample; S5: placing the dried graphene / SiO2 / Si sample in a nitrogen / hydrogen mixture and heating it to further remove PMMA on the graphene surface; S6: After heating is completed and the temperature drops to room temperature, silver electrodes are modified on both ends of the graphene. After drying, insulating glue is modified around the graphene as a gating device to obtain a graphene field effect transistor biosensor, which is dried for later use; S7: modifying chitosan onto the graphene surface of the graphene field effect transistor biosensor and washing with PBS solution; S8: Modify the chitosan on the graphene with glutaraldehyde, wash it with PBS solution, and then test the electrical properties; S9: The probe is modified onto chitosan by glutaraldehyde, and after being washed with a PBS solution, it is modified and blocked with bovine serum albumin to prepare a chitosan-modified graphene field effect transistor biosensor; The probe is an anti-procalcitonin antibody.

2. The method for preparing a chitosan-modified graphene field effect transistor biosensor according to claim 1, characterized in that: The operation steps of modifying the graphene surface with chitosan in step S7 are as follows: A chitosan solution with a concentration of 5% was dropped onto the surface of the sensing channel of the graphene field effect transistor biosensor and electrostatically adsorbed for 2 h.

3. The method for preparing a chitosan-modified graphene field effect transistor biosensor according to claim 1, characterized in that: The operation steps of modifying chitosan with glutaraldehyde in step S8 are as follows: A 15% glutaraldehyde solution was added dropwise to the surface of chitosan and allowed to stand for 1 h.

4. The method for preparing a chitosan-modified graphene field effect transistor biosensor according to claim 2, characterized in that: The operation steps of modifying the probe onto chitosan in step S9 are as follows: The procalcitonin antibody solution was added dropwise to the surface of the sensing channel of the graphene field effect transistor biosensor and allowed to stand for 1 hour.

5. A chitosan-modified graphene field effect transistor biosensor, characterized in that: The biosensor is prepared by the method for preparing a chitosan-modified graphene field effect transistor biosensor according to any one of claims 1 to 4.

6. A method for detecting procalcitonin, characterized in that: The biosensor prepared by the method for preparing a chitosan-modified graphene field effect transistor biosensor according to claim 1 is used for detection.

7. Use of the chitosan-modified graphene field effect transistor biosensor according to claim 5 in the preparation of a product for detecting sepsis.

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