Preparation method of graphene field effect transistor biosensor for unmarked multiple detection of cytokine storm

Abstract

The invention belongs to the technical field of biomedical detection, and discloses a preparation method of a microfluidic integrated graphene field effect transistor biosensor for rapid diagnosis of cytokine storm. The micro-fluidic integrated graphene field effect transistor biosensor is composed of a GFET sensing chip and a PMMA micro-fluidic module. Specific aptamers of cell factors such as TNF-alpha and IL-6 are fixed on the surface of the chip through the AL-BSA connecting layer, and the AL-BSA connecting layer serves as an anti-pollution layer, so that the anti-interference capability is remarkably improved. The microfluidic system is bonded on the sensing chip. According to the invention, synchronous, real-time and label-free detection of two cell factors is realized, the detection limit of TNF-alpha reaches 0.017 pg / mL, and the detection limit of IL-6 reaches 0.0158 pg / mL. The sensor has the advantages of high specificity, simplicity and convenience in operation and the like, and provides a reliable platform for bedside diagnosis of cytokine storm.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection and in vitro diagnostic technology, and relates to a method for preparing a graphene field-effect transistor biosensor for label-free multiplex detection of cytokine storms. Background Technology

[0002] Cytokine storm (CRS) is a life-threatening systemic inflammatory response commonly seen after severe infections, autoimmune diseases, and CAR-T cell immunotherapy. The pathological characteristic of a cytokine storm is the rapid and massive release of various pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-1β). Rapid and accurate monitoring of the dynamic concentration changes of these cytokines is crucial for the early diagnosis, disease assessment, and therapeutic intervention of CRS, and represents a major challenge in the field of biomedical testing. Currently, the gold standard for clinical cytokine detection mainly relies on enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay. While these methods offer good specificity, they suffer from insurmountable limitations: First, their procedures are cumbersome, involving multiple incubation and washing steps, resulting in long processing times that cannot meet the minute-level detection speed requirements in the treatment of critically ill patients with CRS. Second, they are typically single-detection methods, meaning a single reaction can only detect one indicator, making it difficult to simultaneously reflect the co-release of multiple cytokines, leading to low efficiency and an inability to provide comprehensive inflammatory information. Furthermore, these methods rely on enzyme or fluorescent labeling, increasing the complexity and cost of detection. The labeling process may also affect the activity of biomolecules, leading to false positives. Finally, techniques such as enzyme-linked immunosorbent assay (ELISA) have limited sensitivity for detecting low concentrations of cytokines, potentially delaying early diagnosis.

[0003] To overcome the limitations of traditional methods, biosensing technologies based on novel principles have been extensively explored. Among them, graphene field-effect transistor (GFET) biosensors have shown great potential due to their unique advantages. When target proteins (such as cytokines) bind to specific recognition elements (such as aptamers or antibodies) modified on the graphene surface, changes in the channel surface charge occur, directly affecting the conductivity of graphene. This change can be read in real time and label-free by monitoring the shift in the Dirac point voltage or changes in the source-drain current in the transfer characteristic curve. However, pushing GFET technology into actual clinical diagnosis of cytokine storms still faces a series of severe challenges. First, the Debye shielding effect: In complex samples such as buffer solutions or serum with near-physiological ionic strength, high concentrations of ions in the solution can shield the charges carried by biomolecules, greatly weakening the sensor signal and leading to a significant decrease in sensitivity. Second, the problem of non-specific adsorption: Real pathological samples contain a large number of high concentrations of impurity proteins, which are easily adsorbed onto the graphene surface in a non-specific manner, generating huge background noise, causing specific biorecognition signals to be submerged, resulting in detection signal failure. Although some studies have attempted to use single antifouling layers such as bovine serum albumin (BSA), their antifouling effect is often unsatisfactory in complex real-world samples. Third, the long-term stability of the sensor in liquid environments: the baseline of GFET devices (such as the Dirac point voltage) is prone to drift in liquid environments. Achieving ultra-low noise and a stable baseline is crucial for obtaining reliable data. Fourth, multiplexing capabilities and integration: Simultaneous detection of two or more cytokines requires integrating multiple sensing units on the same chip and ensuring that the signals from each unit do not interfere with each other (i.e., avoiding cross-reactions). Simultaneously, integrating the sensing chip with an efficient sample delivery interface (such as a microfluidic system) is essential for achieving automated and controllable detection processes and ensuring the uniformity and reproducibility of the interaction between the sample and the sensing interface.

[0004] Therefore, developing a GFET biosensor platform that can effectively solve Debye shielding, achieve excellent anti-fouling performance, ensure baseline stability, and successfully integrate multiple detection functions with a simple microfluidic sample delivery interface has significant clinical value and practical significance for promoting rapid and accurate diagnosis of cytokine storms. Summary of the Invention

[0005] The present invention aims to provide a graphene field-effect transistor (GFET) biosensor integrating microfluidic technology, which not only has high sensitivity and multiple detection capabilities, but also ensures high repeatability and reliability of the detection process by means of a stable microfluidic sample introduction interface, making it suitable for rapid diagnosis of cytokine storms. The fabrication method of the GFET biosensor includes the following steps: (1) using AutoCAD software to draw a field-effect transistor structure design with the source, drain and gate coplanar; (2) coating a uniform, continuous and stable graphene oxide (GO) film on a clean PET flexible substrate; (3) reducing the GO film to a reduced graphene oxide (RGO) film by hydrazine vapor reduction combined with air thermal annealing process to improve its conductivity and bipolar electrical characteristics; (4) patterning the RGO film by photolithography and dry etching process, and then combining it with physical vapor deposition process to prepare the source, drain and gate electrodes of the GFET; (5) modifying amyloid-like bovine serum albumin (AL-BSA) on the surface of the GFET channel, and activating its surface carboxyl groups by EDC / NHS to fix the IL-6 nucleic acid aptamer or TNF-α nucleic acid aptamer by covalent bond; (6) integrating a microfluidic solution transport system on the GFET chip to form a biosensor that can be used for high-sensitivity detection of IL-6 protein and TNF-α protein.

[0006] The technical solution of this invention:

[0007] A method for fabricating a graphene field-effect transistor biosensor for label-free multiplex detection of cytokine storms, comprising:

[0008] GFET sensing chip: Based on graphene field-effect transistor array, each sensing unit is modified with specific recognition molecules (such as nucleic acid aptamers) corresponding to different cytokines, and AL-BSA is used as an anti-fouling coating.

[0009] Integrated microfluidic sample delivery interface: It includes a microchannel structure that is hermetically integrated with the GFET sensing chip, which is used to deliver the sample to be tested to each sensing unit in a controllable and stable manner.

[0010] The specific steps are as follows:

[0011] Step 1: Design the graphene field-effect transistor structure with the source, drain, and gate arranged in the same plane;

[0012] Step 2: Based on the designed circuit layout and referring to the preparation method disclosed in the patent document (publication number: CN 119959318 A), a reduced graphene oxide field-effect transistor is fabricated on a PET flexible substrate; wherein, the passivation layer is prepared using S1805 positive photoresist to replace the SU-8 2002 negative photoresist used in the patent.

[0013] Step 3: Mix 50 mM tris(2-carboxyethyl)phosphine hydrochloride solution (pH 4.5-5.5) with an equal volume of bovine serum albumin (BSA) aqueous solution (1.5-2.5 mg / mL), and apply the mixture uniformly to the device channel region. React at room temperature for 2-3 hours. After the reaction, gently rinse the surface with deionized water and dry with nitrogen to form a dense amyloid and BSA modification layer on the channel surface. Next, activate the carboxyl groups on the surface of the modification layer with a 2:1 mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Then, drop a 0.25-0.35 μM PBS solution of IL-6 aptamer or TNF-α aptamer onto the activated surface and incubate at 4°C for 12 hours to achieve covalent fixation of the aptamer. Finally, rinse 3-5 times with PBS buffer to remove non-specifically adsorbed molecules, resulting in the modified GFET device.

[0014] Step 4: Microfluidic chip fabrication is performed using a 0.25-0.5 cm thick polymethyl methacrylate (PMMA) substrate. First, a 3M double-sided adhesive layer is bonded to the back of the PMMA substrate for subsequent encapsulation and integration. Microchannels, 500-600 μm wide and 51 mm long, are then laser-cut onto the substrate. The cut PMMA microchannel substrate is precisely aligned and bonded to the surface of the modified GFET device using the adhesive layer, thereby achieving effective integration of the microfluidic channels and sensor channels to form a complete biosensor chip capable of fluid control.

[0015] The beneficial effects of this invention are:

[0016] (1) Significantly improved detection repeatability and reliability:

[0017] (2) The integrated microfluidic interface ensures the uniformity and controllability of the interaction between the sample and the sensing interface, effectively reducing manual operation errors and obtaining a more stable detection baseline.

[0018] (3) Potential for automation and portability: The introduction of microfluidics lays the foundation for its integration with automated sample delivery systems, making it more conducive to the development of rapid point-of-care testing equipment. It retains the core sensing advantages:

[0019] (4) This system fully retains the high sensitivity, multiple detection and fast response capabilities of the GFET sensor, and enhances its potential applicability in practical applications through microfluidic integration. Attached Figure Description

[0020] Figure 1These are optical characterization images of the GFET array prepared in this invention, wherein a is an optical microscopic image of the uniform and continuous RGO film and its GFET array on a PET substrate; b is an overall optical photograph of the GFET array; and c is an optical microscopic image of the GFET with an S1805 passivation layer.

[0021] Figure 2 The results represent the characterization of AL-BSA formation in solution, where a and b are transmission electron microscope images of AL-BSA before and after transformation in solution; c and d are circular dichroism chromatograms of AL-BSA in solution before and after transformation; and e is the infrared spectrum of AL-BSA before and after transformation in solution.

[0022] Figure 3 The surface characterization of GFET during the modification process is shown in Figures 1-3. a, b, c, and d represent the XPS spectra corresponding to the modification stages of PET substrate, PET+RGO, PET+RGO+AL-BSA, and PET+RGO+AL-BSA+aptamer, respectively. e and f represent the Raman spectra before and after GO reduction, respectively. g and h represent the atomic force microscopy images of the RGO film and the AL-BSA modified film, respectively. i and j represent the surface contact angle images before and after AL-BSA modification, respectively.

[0023] Figure 4 The electrical properties of the GFET used to detect two cytokines are characterized. Here, a and b are the transfer characteristic curves of the modified device when detecting the two cytokines; c and d are the quantitative calibration curves of the two proteins obtained by extracting the relationship between ΔV and concentration from the transfer curves; e and f are the selectivity test results of the modified device for the target analytes.

[0024] Figure 5 This is a flowchart illustrating the fabrication method of the graphene field-effect transistor biosensor for label-free multiplex detection of cytokine storms according to the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0026] This embodiment provides a method for fabricating an integrated microfluidic graphene field-effect transistor biosensor under specific modification conditions. The specific steps are as follows:

[0027] 1) First, a reduced graphene oxide field-effect transistor (e.g., ...) was prepared according to existing patented methods. Figure 1 a, b, c and Figure 2 (as shown in e and f).

[0028] 2) To verify that bovine serum albumin can be reduced by tris(2-carboxyethyl)phosphine to form β-sheet-rich amyloid-like bovine serum albumin, the product was characterized in solution by infrared spectroscopy, circular dichroism spectroscopy, and transmission electron microscopy (e.g., Figure 2 (As shown in a, b, c, d, and e). A 50 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution with a pH of 4.5 was mixed with an equal volume of 1.5 mg / mL bovine serum albumin aqueous solution and uniformly dropped onto the channel region of the device. After reacting for 2 hours, the surface was gently rinsed with deionized water, and the rinsing was repeated 3-5 times. The surface was then dried with nitrogen gas, thus forming an amyloid-like bovine serum albumin film on the channel surface. To confirm successful modification, the surface before and after modification was characterized by contact angle and atomic force microscopy (e.g., ...). Figure 3 (as shown in g, h, i, j).

[0029] 3) Next, the carboxyl groups on the modified membrane were activated using a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at a mass ratio of 2:1. Then, a PBS solution of 0.25 μM IL-6 or TNF-α aptamer was dropped onto the activated surface and incubated at 4 °C for 12 hours. After that, the membrane was washed with PBS 3-5 times to remove non-specifically adsorbed molecules. The resulting device was used for subsequent detection.

[0030] 4) To confirm the successful execution of each modification step, X-ray photoelectron spectroscopy was performed on the surface after each modification step to analyze changes in elemental composition (e.g., ...). Figure 3 (as shown in a, b, c, and d).

[0031] 5) The transfer characteristic curves of the device were tested in 1×PBS solution until they basically overlapped, indicating that the device had reached a stable state. Then, the PBS solution was removed, the channel region was dried with nitrogen, and 200 μL of 1×PBS solution containing 0.1 pg / mL IL-6 and TNF-α was added. After incubation at room temperature for 4 minutes, the transfer curves were tested and the data were saved. The above process was repeated, testing solutions containing 1, 10, 100, 1000, 2000, 5000, 7500, and 10000 pg / mL IL-6 and TNF-α sequentially. Based on the changes in the transfer curves corresponding to different concentrations, the voltage offset ΔV was extracted and linearly fitted with the logarithm of the concentration to obtain the detection limit and linear range of the sensor. The detection limits of the prepared GFET sensor for IL-6 and TNF-α were 0.0158 pg / mL and 0.017 pg / mL, respectively, and the linear range was 0.1~1000 pg / mL (e.g., ...). Figure 4 (as shown in a, b, c, and d).

[0032] 6) Subsequently, in the presence of the target substance, an interfering substance with a concentration 10 times that of the target substance was added, and a selectivity test was performed (e.g., Figure 4 (as shown in e and f).

[0033] 7) Finally, a 0.25 cm thick polymethyl methacrylate (PMMA) substrate was selected for microfluidic chip fabrication. A 3M double-sided adhesive layer was bonded to the back of the substrate, and microchannels with a width of 500 μm and a length of 51 mm were processed using laser cutting technology. The PMMA microchannel plate was then precisely aligned and bonded to the modified sensor surface using the double-sided adhesive layer, achieving the integration of the microchannels and sensor channels to form a complete biosensor chip that can be used for fluid control.

[0034] Example 2

[0035] This embodiment modifies specific conditions in the fabrication and modification process based on Example 1 as follows: In step 2, a tris(2-carboxyethyl)phosphine hydrochloride buffer with a pH of 5.0 is used, the bovine serum albumin concentration is 2.0 mg / mL, and the reaction time is adjusted to 2.5 h. In step 3, the aptamer concentration is 0.28 μM. In step 7, a PMMA board with a thickness of 0.35 cm and a microchannel width of 550 μm are used. The remaining steps, processes, and detection methods in this embodiment are consistent with those in Example 1.

[0036] Example 3

[0037] This embodiment modifies specific conditions in the fabrication and modification process based on Example 1 as follows: In step 2, a tris(2-carboxyethyl)phosphine hydrochloride buffer with a pH of 4.8 is used, the bovine serum albumin concentration is 1.8 mg / mL, and the reaction time is adjusted to 2.2 h. In step 3, the aptamer concentration is 0.30 μM. In step 7, a PMMA board with a thickness of 0.40 cm and a microchannel width of 520 μm are used. The remaining steps, processes, and detection methods in this embodiment are consistent with those in Example 1.

[0038] Example 4

[0039] This embodiment modifies specific conditions in the fabrication and modification process based on Example 1 as follows: In step 2, a tris(2-carboxyethyl)phosphine hydrochloride buffer with a pH of 5.3 is used, the bovine serum albumin concentration is 2.2 mg / mL, and the reaction time is adjusted to 2.8 h. In step 3, the aptamer concentration is 0.32 μM. In step 7, a PMMA board with a thickness of 0.45 cm and a microchannel width of 580 μm are used. The remaining steps, processes, and detection methods in this embodiment are consistent with those in Example 1.

[0040] Example 5

[0041] This embodiment modifies specific conditions in the fabrication and modification process based on Example 1 as follows: In step 2, a tris(2-carboxyethyl)phosphine hydrochloride buffer with a pH of 5.5 is used, the bovine serum albumin concentration is 2.5 mg / mL, and the reaction time is adjusted to 3.0 h. In step 3, the aptamer concentration is 0.35 μM. In step 7, a PMMA board with a thickness of 0.50 cm and a microchannel width of 600 μm are used. The remaining steps, processes, and detection methods in this embodiment are consistent with those in Example 1.

[0042]

[0043] In summary, this invention effectively integrates microfluidic technology with a high-performance GFET biosensor chip to construct a stable and reliable rapid diagnostic platform for cytokine storms. Any equivalent substitutions or improvements based on the principles of this invention fall within the protection scope of this invention.

Claims

1. A method for fabricating a graphene field-effect transistor biosensor for label-free multiplex detection of cytokine storms, characterized in that, This graphene field-effect transistor biosensor includes: GFET sensing chip: Based on graphene field-effect transistor array, each sensing unit is modified with specific recognition molecules corresponding to different cytokines, and AL-BSA is used as an anti-fouling coating. Integrated microfluidic sample delivery interface: includes a microchannel structure hermetically integrated with the GFET sensing chip, used to deliver the sample to be tested to each sensing unit in a controllable and stable manner; The specific steps are as follows: Step 1: Design a graphene field-effect transistor structure with source, drain and gate arranged in the same plane, and fabricate a reduced graphene oxide field-effect transistor structure on a PET flexible substrate; wherein, the passivation layer is prepared using S1805 positive photoresist. Step 3: Mix 50 mM tris(2-carboxyethyl)phosphine hydrochloride solution (pH 4.5-5.5) with an equal volume of bovine serum albumin (BSA) aqueous solution (1.5-2.5 mg / mL), and apply the mixture uniformly to the channel region. React at room temperature for 2-3 hours. After the reaction, gently rinse the surface with deionized water and dry with nitrogen to form a dense amyloid and BSA modification layer on the channel surface. Next, activate the carboxyl groups on the surface of the modification layer with a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (2:1 mass ratio). Then, dropwise add a 0.25-0.35 μM PBS solution of IL-6 aptamer or TNF-α aptamer onto the activated modification layer surface and incubate at 4°C for 12 hours to achieve covalent fixation of the aptamer. Finally, wash 3-5 times with PBS buffer to remove non-specifically adsorbed molecules, resulting in the modified GFET device. Step 4: Select a polymethyl methacrylate (PMMA) substrate with a thickness of 0.25-0.5 cm for microfluidic chip fabrication; First, apply a 3M double-sided adhesive layer to the back of the PMMA substrate for subsequent encapsulation and integration; Use laser cutting technology to process microchannels on the PMMA substrate, align the cut PMMA microchannel board with the double-sided adhesive layer and attach it to the surface of the modified GFET device, thereby achieving effective integration of the microfluidic channel and sensor channel to form a complete biosensor chip capable of fluid control.

2. The method for fabricating a graphene field-effect transistor biosensor for label-free multiplex detection of cytokine storms according to claim 1, characterized in that, The microchannel has a width of 500-600 μm and a length of 51 mm.

Images