An antibiotic sensor based on high-performance graphene transistor and its preparation method and application

Abstract

The present invention relates to an antibiotic sensor based on a high-performance graphene transistor and a preparation method and application thereof, comprising: a graphene transistor; a polylysine coupling agent fixed on the graphene surface of the graphene transistor channel; an antibiotic aptamer fixed on the polylysine coupling agent; and a microfluidic chip placed on the graphene transistor. The present invention uses an easily removable camphor-rosin double-layer structure as a support layer to transfer a single-layer graphene grown on a copper foil to a substrate, obtains a high-performance graphene transistor through a micro-nano process, and obtains a highly sensitive antibiotic sensor by fixing an antibiotic-specific aptamer probe on the transistor channel and combining it with a microfluidic chip. Thereby solving the problem of complex antibiotic detection process and low detection sensitivity. The present invention greatly improves the sensitivity of the antibiotic sensor, simplifies the detection cycle of the antibiotic, and optimizes the detection process to achieve rapid and highly sensitive antibiotic detection and analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to an antibiotic detection sensor and a preparation method and application thereof. Background Art

[0002] Antibiotics have made important contributions to the environment, medical care and food safety by inhibiting the growth of pathogens. However, the abuse of antibiotics has led to many problems, such as pathogen tolerance, endangering human life and health, and ecological environment pollution. Therefore, the monitoring of antibiotics in water has become one of the important issues of concern and has attracted much attention. The traditional methods for detecting antibiotics in water mainly include liquid chromatography, electrochemical method, Raman spectroscopy, etc. At the same time, electrochemical sensors based on fluorescence, transistors, etc. have achieved certain results, but low detection limit, convenient and efficient antibiotic detection methods are still to be developed.

[0003] Graphene field effect transistors are widely used in sensing research due to their good electrical properties. Obtaining high-quality graphene materials is a key strategy to improve sensor performance, and designing and selecting biological probes is the core step in building specific sensors. At present, graphene field effect transistor sensors have achieved successful detection of some antibiotics. For example, Chinese patent document CN108956742B discloses a graphene field effect transistor array biosensor and its preparation method and detection method. This technology uses amino-functionalized graphene aqueous solution to prepare graphene channel materials; in order to improve monitoring sensitivity, Chinese patent document CN108956743B also discloses gold nano-ion enhanced graphene transistor sensors and monitoring methods; however, due to the quality of materials used to prepare graphene transistors, such as the use of nano-graphene oxide sheets, impurities remain on the transferred graphene, and the performance of the prepared graphene transistors is not ideal. In addition, due to the differences in the surface functionalization of the antibiotic transistor sensor channel, the types and fixation methods of the antibiotic detection probes, and the sensing mechanisms, the sensitivity of the antibiotic transistor sensor is also different. At present, most antibiotic transistor sensors either use transistors with low performance, or use transistor channel surface functionalization and antibiotic probe fixation methods that cannot achieve high-density probe fixation, making the existing graphene transistor sensors have low sensitivity in detecting antibiotics. Therefore, the research goal is to study sensors with lower detection limits and higher sensitivity. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an antibiotic sensor based on a high-performance graphene transistor; the present invention also provides a method for preparing the above-mentioned antibiotic sensor based on a high-performance graphene transistor and a method for detecting antibiotics; so as to solve the problem of low sensitivity of the graphene transistor sensor in detecting antibiotics mentioned in the background technology.

[0005] Terminology explanation:

[0006] 1. PMMA: polymethyl methacrylate;

[0007] 2. PLL: polylysine;

[0008] 3. CPT-FET: conventional PMMA transferred graphene and its transistor;

[0009] 4. CRCT-FET: Novel camphor-rosin double support layer transfer clean graphene and its transistor;

[0010] 5. Room temperature: generally refers to 25 degrees Celsius;

[0011] 6. V ds : source-drain voltage;

[0012] 7.I ds : source-drain current;

[0013] 8. V gs : gate voltage;

[0014] 9. V dirac : Dirac point;

[0015] 10. PDMS, polydimethylsiloxane;

[0016] 11. STR: streptomycin;

[0017] 12. CIP: ciprofloxacin;

[0018] 13. KAN: kanamycin;

[0019] 14. TOB: Tobramycin;

[0020] 15.TET: Tetracycline.

[0021] The technical solution of the present invention is:

[0022] An antibiotic sensor based on high-performance graphene transistor, comprising:

[0023] Graphene transistors;

[0024] A polylysine coupling agent fixed on the graphene surface of the graphene transistor channel;

[0025] Antibiotic aptamers immobilized on polylysine coupling agents;

[0026] A microfluidic chip placed on top of a graphene transistor.

[0027] Further preferably, the microfluidic chip is a PDMS microfluidic chip.

[0028] Further preferably, the microfluidic chip includes a microfluidic controller, and the microfluidic controller is respectively connected to an inlet and an outlet.

[0029] The method for preparing the antibiotic sensor based on high-performance graphene transistor comprises:

[0030] (1) Preparation of single-layer graphene:

[0031] (2) preparing source and drain electrodes on the single-layer graphene to obtain a graphene transistor;

[0032] (3) preparing a microfluidic chip; the microfluidic chip includes a microfluidic controller, and the microfluidic controller is respectively connected to an inlet and an outlet;

[0033] (4) aligning the flow channel of the prepared microfluidic chip with the channel of the graphene transistor, and bonding the microfluidic chip to the substrate of the single-layer graphene;

[0034] (5) adding a polylysine coupling agent to the injection port of the bonded microfluidic chip so that the polylysine coupling agent fills the entire flow channel, and incubating the polylysine coupling agent to fully adhere to the graphene surface of the channel, and flushing the flow channel;

[0035] (6) adding the antibiotic aptamer to the injection port of the microfluidic chip so that the antibiotic aptamer fills the entire flow channel, incubating the antibiotic aptamer to allow the antibiotic aptamer to fully bind to the polylysine coupling agent fixed in the channel and then be fixed in the channel, and flushing the flow channel;

[0036] (7) After chemically blocking the sites of the unfixed antibiotic aptamers with ethanolamine, the flow channel is flushed to complete the preparation of the antibiotic sensor based on the high-performance graphene transistor and store it for future use.

[0037] Preferably, according to the present invention, in step (1), preparing a single-layer graphene comprises:

[0038] Growing graphene on copper foil to obtain copper-based graphene;

[0039] A camphor-rosin double-layer support layer is coated on the copper-based graphene, and the copper-based graphene is transferred to a substrate through the camphor-rosin double-layer support layer; and the camphor-rosin double-layer support layer is completely removed to obtain a single-layer graphene.

[0040] Preferably according to the present invention, the width of the channel of the microfluidic chip is smaller than the distance between the source and drain electrodes.

[0041] Preferably, according to the present invention, the following steps are performed before step (6): the antibiotic aptamer is annealed at 80-98° C. and then cooled to room temperature within 5 minutes.

[0042] Further preferably, the antibiotic aptamer is annealed at 95°C.

[0043] Preferably according to the present invention, in step (5), the incubation time of the polylysine coupling agent is more than 10 min.

[0044] Preferably according to the present invention, in step (6), the incubation time of the antibiotic aptamer is more than 30 minutes.

[0045] A method for detecting antibiotics using the antibiotic sensor based on high-performance graphene transistors, comprising:

[0046] ①Fix the reference electrode to the sample outlet of the microfluidic chip of the antibiotic sensor, and connect the source electrode, drain electrode and reference electrode of the antibiotic sensor to the source meter respectively;

[0047] ② Add the sample to be tested to the inlet of the microfluidic chip, so that the sample to be tested fills the entire flow channel and incubates, then extract the sample to be tested and rinse away the uncaptured antibiotic molecules and other molecular impurities with PBS solution;

[0048] ③ Add PBS solution to the inlet of the microfluidic chip and fill the entire flow channel and outlet;

[0049] ④ Use the source meter to scan the transfer curve of the antibiotic sensor and read the Dirac point position of the antibiotic sensor;

[0050] ⑤ Compare the measured Dirac point position of the antibiotic sensor with the Dirac point position when the antibiotic sensor does not detect the sample to be detected, and obtain the offset;

[0051] ⑥Compare the offset obtained in step ⑤ with the Dirac point offset of the antibiotic sensor and the standard curve of antibiotic concentration to obtain the antibiotic concentration of the sample to be tested.

[0052] The Dirac point shift of the antibiotic sensor refers to the change in the electrical signal of the antibiotic sensor after detecting the sample to be detected;

[0053] The method for obtaining the standard curve of antibiotic concentration is as follows: using an antibiotic sensor to measure antibiotic samples of known different concentrations, obtaining the changes in the antibiotic sensor electrical signals of the antibiotic samples of different concentrations, and plotting the relationship between the different concentrations of the antibiotics and the changes in the antibiotic sensor electrical signals to obtain the standard curve of antibiotic concentration;

[0054] Preferably according to the present invention, in step ②, the incubation time of the sample to be detected is more than 10 minutes.

[0055] The beneficial effects of the present invention are:

[0056] The present invention uses an easily removable camphor-rosin double-layer structure as a supporting layer to transfer the monolayer graphene grown on the copper foil to a substrate, obtains a high-performance graphene transistor through a micro-nano process, and obtains a highly sensitive antibiotic sensor by fixing an antibiotic-specific aptamer probe on the transistor channel and combining it with a microfluidic chip. Thus, the problem of complex antibiotic detection process and low detection sensitivity is solved. The antibiotic sensor based on the graphene transistor prepared by this method causes the structure of the aptamer to change by combining the antibiotic with the specific aptamer fixed on the sensor channel, so that more charges of the aptamer fall into the Debye region close to the transistor surface, thereby causing a change in the sensor electrical signal. The large change amplitude of the sensor electrical signal is due to the high-performance graphene device. Compared with the device prepared by the ordinary method, the sensitivity of the antibiotic sensor is increased by nearly 3 times, and the detection cycle of the antibiotic is simplified, and the detection process is optimized to achieve rapid and highly sensitive antibiotic detection and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0058] Figure 1 The antibiotic sensor based on high-performance graphene transistor in the embodiment of the present invention

[0059] Figure 2 Schematic diagram of the graphene wet transfer steps in an embodiment of the present invention.

[0060] Figure 3 Schematic diagram of the preparation process of a graphene transistor in an embodiment of the present invention.

[0061] Figure 4 Schematic diagram of the preparation process of the antibiotic sensor of the graphene transistor in an embodiment of the present invention.

[0062] Figure 5 This is the Raman and optical characterization diagram of CPT graphene in an embodiment of the present invention.

[0063] Figure 6 This is a SEM characterization image of CPT graphene in an embodiment of the present invention.

[0064] Figure 7 This is a CPT graphene Raman uniformity test diagram in an embodiment of the present invention.

[0065] Figure 8 These are the Raman and optical characterization images of CRCT graphene in an embodiment of the present invention.

[0066] Fig. 9 This is a SEM characterization image of CRCT graphene in an embodiment of the present invention.

[0067] Fig.10 This is a CRCT graphene Raman uniformity test diagram in an embodiment of the present invention.

[0068] Fig.11 Schematic diagram of output characteristic curves of CPT and CRCT graphene transistors in an embodiment of the present invention.

[0069] Fig.12 Schematic diagram of transfer characteristic curves of CPT and CRCT graphene transistors in an embodiment of the present invention.

[0070] Fig.13 Schematic diagram of the stability test results of CPT and CRCT graphene transistors in an embodiment of the present invention.

[0071] Fig.14 This is a Raman test diagram of PLL-functionalized graphene in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0072] Fig.15 This is a test diagram of the Raman uniformity of PLL-functionalized graphene in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0073] Fig.16 This is an AFM test image of PLL / Gr in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0074] Fig.17 This is an AFM test image of the aptamer / PLL / Gr in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0075] Fig.18 Schematic diagram of the transfer curve of the construction process of the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0076] Fig.19 This is a Dirac point shift diagram of the graphene field transistor antibiotic sensor construction process in an embodiment of the present invention.

[0077] Fig. 20 Schematic diagram of the stability test results of the graphene field transistor antibiotic sensor in different liquid environments according to an embodiment of the present invention.

[0078] Fig.21 This is the molecular structure of tetracycline in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0079] Fig. 22 This is a predicted diagram of the three-dimensional conformation of the aptamer in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0080] Fig.23Schematic diagram of the electrostatic gating mechanism before antibiotic capture in the graphene field transistor antibiotic sensor in an embodiment of the present invention.

[0081] Fig.24 Schematic diagram of the electrostatic gating mechanism after antibiotic capture in the graphene field transistor antibiotic sensor according to an embodiment of the present invention.

[0082] Fig.25 Schematic diagram of the transfer curve of antibiotic concentration gradient detection in the CPT-FET antibiotic sensor in an embodiment of the present invention.

[0083] Fig.26 Schematic diagram of the transfer curve of antibiotic concentration gradient detection in the CRCT-FET antibiotic sensor in an embodiment of the present invention.

[0084] Fig. 27 Schematic diagram of the antibiotic concentration gradient quantitative curve of the CPT-FET and CRCT-FET antibiotic sensors in the embodiments of the present invention.

[0085] Fig.28 Schematic diagram of the transfer curve of the antibiotic specificity test of the CRCT-FET antibiotic sensor in an embodiment of the present invention.

[0086] Fig.29 Schematic diagram of the antibiotic specificity test results of the CRCT-FET antibiotic sensor in an embodiment of the present invention.

[0087] Fig.30 Schematic diagram of the sensing stability test results of CPT-FET and CRCT-FET antibiotic sensors in an embodiment of the present invention.

[0088] Fig.31 It is a schematic diagram of the quantitative curve of the antibiotic concentration gradient test sensed by the CPT-FET and CRCT-FET antibiotic sensors in lake water and sea water in an embodiment of the present invention.

[0089] Fig.32 Schematic diagram of the signal response of the CRCT-FET antibiotic sensor in an embodiment of the present invention to actual samples in lake water and sea water.

[0090] Fig.33 This is a schematic diagram of the signal response of the CRCT-FET antibiotic sensor to actual samples from the Bohai Bay in an embodiment of the present invention. DETAILED DESCRIPTION

[0091] The present invention will be further defined below in conjunction with the accompanying drawings and embodiments, but is not limited thereto.

[0092] Example 1

[0093] An antibiotic sensor based on high-performance graphene transistors, where high performance refers to the high mobility of the device, which can make the prepared antibiotic sensor more sensitive, such as Figure 1 As shown, including:

[0094] Graphene transistors;

[0095] A polylysine coupling agent fixed on the graphene surface of the graphene transistor channel;

[0096] Antibiotic aptamers immobilized on polylysine coupling agents;

[0097] A microfluidic chip placed on top of a graphene transistor.

[0098] The microfluidic chip is a PDMS microfluidic chip.

[0099] The microfluidic chip includes a microfluidic controller, which is respectively connected to an inlet and an outlet.

[0100] Example 2

[0101] The method for preparing the antibiotic sensor based on the high-performance graphene transistor described in Example 1 comprises:

[0102] (1) Preparation of single-layer graphene: including:

[0103] Growing graphene on copper foil to obtain copper-based graphene;

[0104] A camphor-rosin double-layer support layer is coated on the copper-based graphene, and the copper-based graphene is transferred to a substrate through the camphor-rosin double-layer support layer; the camphor-rosin double-layer support layer is completely removed by an organic solvent and heating to obtain an ultra-clean single-layer graphene.

[0105] (2) preparing source and drain electrodes on the transferred clean single-layer graphene through patterning and metal deposition processes to obtain a graphene transistor;

[0106] (3) preparing a microfluidic chip by using a template; the microfluidic chip includes a microfluidic controller, which is respectively connected to an inlet and an outlet;

[0107] (4) The flow channel of the prepared microfluidic chip is aligned with the channel of the graphene transistor, and the microfluidic chip is bonded to a substrate with a transferred clean single-layer graphene; the width of the channel of the microfluidic chip is smaller than the distance between the source and drain electrodes.

[0108] (5) adding a polylysine coupling agent to the injection port of the bonded microfluidic chip so that the polylysine coupling agent fills the entire flow channel, incubating the polylysine coupling agent to fully adhere to the graphene surface of the channel, and washing the flow channel with a PBS solution;

[0109] (6) adding the antibiotic aptamer to the injection port of the microfluidic chip so that the antibiotic aptamer fills the entire channel, and incubating the antibiotic aptamer to allow the antibiotic aptamer to fully bind to the polylysine coupling agent fixed in the channel and then be fixed in the channel, and then washing the channel with a PBS solution;

[0110] (7) After chemically blocking the sites of the unfixed antibiotic aptamers using ethanolamine, the flow channel was flushed with PBS solution to complete the preparation of the antibiotic sensor based on high-performance graphene transistors.

[0111] (8) Store the prepared high-performance single-layer graphene transistor-based antibiotic sensor at 25°C (refrigerator) for future use.

[0112] Example 3

[0113] The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to Example 2 is different in that:

[0114] Before step (6), perform the following steps: anneal the antibiotic aptamer at 80-98°C and then cool it to room temperature within 5 minutes to ensure that the antibiotic aptamer does not form a secondary structure;

[0115] In step (5), the incubation time of the polylysine coupling agent is more than 10 minutes.

[0116] In step (6), the incubation time of the antibiotic aptamer is more than 30 minutes.

[0117] Example 4

[0118] The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to Example 3 is different in that:

[0119] The antibiotic aptamers were annealed at 95 °C.

[0120] Example 5

[0121] The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to Example 2 is different in that:

[0122] The specific implementation steps of step (1) include:

[0123] First, a 20 wt.% hydrogenated rosin ethyl lactate solution was spin-coated on the copper-based graphene at 500 rpm for 10 s, 1200 rpm for 60 s, and cured at 40 °C for 2 h;

[0124] Then, an ethanol solution with 50 wt. % camphor was spin-coated at 500 rpm for 10 s, at 800 rpm for 60 s, and cured at 30° C. for 20 min.

[0125] Again, copper was etched in FeCl3 (0.02 M) solution for 5 h to obtain a camphor / rosin / graphene stack floating on the solution.

[0126] Finally, the cleaned camphor / rosin / graphene stack was transferred to the target Si substrate.

[0127] In this embodiment, in order to ensure that the camphor / rosin / graphene stack remains intact and maintains complete contact between the rosin and the graphene and the substrate, first, the camphor / rosin / graphene stack is stacked on a hot stage and maintained at 40°C for 30 minutes; then, the temperature is increased to 100°C for 20 minutes to completely remove water vapor and camphor; finally, Si / Gr / Rosin is immersed in acetone and isoamyl acetate solutions for 30 minutes, repeated 3 times, and the camphor-rosin double-layer supporting layer is completely removed, and high-purity nitrogen is blown dry to obtain silicon-based clean graphene, i.e., ultra-clean single-layer graphene. Figure 2 The basic graphene transfer process route is demonstrated.

[0128] Example 6

[0129] The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to Example 2 is different in that:

[0130] The specific implementation steps of step (2) include: in order to construct a FET device, 10nm Ti and 50nm Au are deposited on silicon-based graphene using a shadow mask and electron beam evaporation deposition method to prepare the source and drain.

[0131] The specific implementation steps of step (3) include: using photolithography and inversion process to prepare a 8×10×1mm 3 , Microfluidic channel volume is 6000×250×40μm 3 PDMS microfluidic chip;

[0132] Figure 3 The process of preparing transistors by transferring graphene using conventional PMMA and camphor-rosin support layers and the properties of the prepared transistors are demonstrated. ds is the source-drain voltage, I ds is the source-drain current, V gs is the gate voltage, from Figure 3 It can be seen that the performance of the transistor prepared by transferring graphene using a camphor-rosin support layer (CRCT-FET) is much higher than that of the transistor prepared by transferring graphene using a conventional PMMA support layer (CPT-FET). Figure 5 Raman and optical characterization images of CPT graphene. Figure 6 This is the SEM characterization image of CPT graphene. Figure 7 This is the CPT graphene Raman uniformity test diagram. Figure 8 This is the Raman and optical characterization image of CRCT graphene. Fig. 9 This is the SEM characterization image of CRCT graphene. Fig.10 This is the CRCT graphene Raman uniformity test diagram. Figure 5-10 The graphene transferred by the two methods were characterized and analyzed respectively. The analysis results showed that the performance of graphene transferred by camphor-rosin support layer was much higher than that of graphene transferred by PMMA support layer.

[0133] Example 7

[0134] The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to Example 6 is different in that:

[0135] In this embodiment, Figure 4 The preparation process of the antibiotic sensor is shown.

[0136] In step (5), a 1 mg / mL PLL solution was incubated in the microfluidic channel for 20 min to form a probe interaction enhancement layer, and the microfluidic channel was rinsed with a 1x PBS solution to remove residues.

[0137] In step (6), the ssDNA aptamer was diluted to 1 μM in a buffer and denatured at 95°C for 5 min, and then rapidly cooled in an ice bath to obtain a single-stranded aptamer. The aptamer was injected into the channel and incubated at 4°C for 24 h to ensure that the aptamer was fully fixed in the graphene channel, and the microfluidic channel was rinsed with 1x PBS solution.

[0138] In step (7), 100 mM EA and 3% BSA are used to block the nonspecific reaction sites in the channel. At this point, the antibiotic sensor is ready to detect antibiotics. When antibiotics are present in the solution, the Dirac point V dirac Move left ( Figure 4 ). Otherwise, the Dirac point V dirac will remain in the same position ( Figure 4 ).

[0139] Fig.11 Schematic diagram of the output characteristic curves of CPT and CRCT graphene transistors. Fig.12 Schematic diagram of the transfer characteristic curves of CPT and CRCT graphene transistors. Fig.13 Schematic diagram of the stability test results of CPT and CRCT graphene transistors. Figure 11-13The results of analyzing the transistors prepared by the two methods respectively show that the performance of the transistors prepared by using graphene transferred from a camphor-rosin support layer is better than that of the transistors prepared by using graphene transferred from a PMMA support layer in terms of electrical performance and stability.

[0140] Fig.14 Figure 2. Raman test of PLL-functionalized graphene in graphene field transistor antibiotic sensor. Fig.15 This is a test diagram of the Raman uniformity of PLL-functionalized graphene in the graphene field transistor antibiotic sensor. Fig.16 AFM test image of PLL / Gr in graphene field transistor antibiotic sensor. Fig.17 AFM test image of aptamer / PLL / Gr in graphene field transistor antibiotic sensor. Fig.18 Schematic diagram of the transfer curve of the graphene field transistor antibiotic sensor construction process. Fig.19 Dirac point shift diagram for the construction process of graphene field transistor antibiotic sensor. Fig. 20 Schematic diagram of the stability test results of graphene field transistor antibiotic sensor in different liquid environments. Figure 14-20 The constructed antibiotic transistor sensors were analyzed and verified Figure 4 The antibiotic transistor sensor prepared by the process shown was successful.

[0141] Fig.21 Molecular structure of tetracycline in graphene field transistor antibiotic sensor. Fig. 22 Prediction of the three-dimensional conformation of the aptamer in the graphene field transistor antibiotic sensor. Figure 21-22 Schematic diagrams of the structures of the polylysine and antibiotic aptamers used are given respectively.

[0142] Fig.23 Schematic diagram of the electrostatic gating mechanism before antibiotic capture in graphene field transistor antibiotic sensor. Fig.24 Schematic diagram of the electrostatic gating mechanism after antibiotic capture in the graphene field transistor antibiotic sensor. Figure 23-24 The sensing mechanism of the prepared transistor sensor was analyzed respectively. Since the antibiotic aptamer fixed on the sensor did not form a secondary structure, all bases fell within the Debye length region of the transistor sensor, such as Fig.23 ,When antibiotics are present, the antibiotics will react with the aptamers to form secondary structures, making most of the aptamer bases exceed the Debye length region of the transistor sensor, e.g. Fig.24 , causing the electrical signal of the transistor sensor to change.

[0143] Fig.25 Schematic diagram of the transfer curve of antibiotic concentration gradient detection in CPT-FET antibiotic sensor. Fig.26 Schematic diagram of the transfer curve of antibiotic concentration gradient detection in CRCT-FET antibiotic sensor. Fig. 27 Schematic diagram of the antibiotic concentration gradient quantification curve of CPT-FET and CRCT-FET antibiotic sensors. Figure 25-27 The sensitivity of transistor sensors prepared by the two methods was characterized and analyzed respectively. The results showed that the sensitivity of the transistor sensor prepared by graphene transferred from a camphor-rosin support layer was nearly three times that of the transistor sensor prepared by graphene transferred from a PMMA support layer, and the detection limit was reduced by two orders of magnitude.

[0144] Fig.28 Schematic diagram of the transfer curve of the antibiotic specificity test of the CRCT-FET antibiotic sensor. STR refers to streptomycin, CIP refers to ciprofloxacin, KAN refers to kanamycin, TOB refers to tobramycin, and TET refers to tetracycline; Fig.29 Schematic diagram of the antibiotic specificity test results of CRCT-FET antibiotic sensor. Figure 28-29 The selectivity of the transistor sensor prepared by graphene transferred from camphor-rosin support layer for antibiotic detection is given. The results show that the transistor sensor prepared by graphene transferred from camphor-rosin support layer has good selectivity.

[0145] Fig.30 Schematic diagram of the sensing stability test results of CPT-FET and CRCT-FET antibiotic sensors. Fig.30 The sensitivity and stability of the sensors prepared by the two methods were compared. The results showed that the sensitivity and stability of the transistor sensor prepared by graphene transferred from a camphor-rosin support layer were better than those of the transistor sensor prepared by graphene transferred from a PMMA support layer.

[0146] Example 8

[0147] A method for detecting antibiotics using the antibiotic sensor based on the high-performance graphene transistor described in Example 1, comprising:

[0148] ①Fix the reference electrode to the sample outlet of the microfluidic chip of the antibiotic sensor, and connect the source electrode, drain electrode and reference electrode of the antibiotic sensor to the source meter respectively;

[0149] ② Add the sample to be tested to the injection port of the microfluidic chip, so that the sample to be tested fills the entire flow channel and incubates, then extract the sample to be tested and rinse away the uncaptured antibiotic molecules and other molecular impurities with PBS solution; the incubation time of the sample to be tested is more than 10 minutes.

[0150] ③ Add PBS solution to the inlet of the microfluidic chip and fill the entire flow channel and outlet; ensure that the reference electrode is in repeated contact with the PBS solution in the flow channel of the microfluidic chip;

[0151] ④ Use the source meter to scan the transfer curve of the antibiotic sensor and read the Dirac point position of the antibiotic sensor;

[0152] ⑤ Compare the measured Dirac point position of the antibiotic sensor with the Dirac point position when the antibiotic sensor does not detect the sample to be detected, and obtain the offset;

[0153] ⑥Compare the offset obtained in step ⑤ with the Dirac point offset of the antibiotic sensor and the standard curve of antibiotic concentration to obtain the antibiotic concentration of the sample to be tested.

[0154] The Dirac point shift of the antibiotic sensor refers to the change in the electrical signal of the antibiotic sensor after detecting the sample to be detected;

[0155] The method for obtaining the standard curve of antibiotic concentration is as follows: using an antibiotic sensor to measure antibiotic samples of known different concentrations, obtaining the change in the antibiotic sensor electrical signal of the antibiotic samples of different concentrations, and plotting the relationship between the different concentrations of the antibiotic and the change in the antibiotic sensor electrical signal to obtain the standard curve of antibiotic concentration; Fig. 27 The curve in .

[0156] To be closer to actual detection applications, Fig.31 Schematic diagram of the quantitative curve of CPT-FET and CRCT-FET antibiotic sensors sensing antibiotic concentration gradient tests in lake water and sea water. Fig.31 The application of transistor sensors prepared by graphene transferred from a camphor-rosin support layer in seawater and freshwater was analyzed, including concentration gradient, quasi-real samples, and real samples. The results showed that the prepared sensors had good performance in both seawater and freshwater samples, with a slightly better performance in the detection of seawater samples.

[0157] Fig.32 Schematic diagram of the signal response of the CRCT-FET antibiotic sensor sensing real samples in lake water and sea water. Fig.32 The detection of simulated samples with antibiotics added into seawater and fresh water is given, and the results show that the device has good detection effect.

[0158] Fig.33 Schematic diagram of the signal response of CRCT-FET antibiotic sensor sensing actual samples from Bohai Bay. Fig.33The results of testing actual samples using transistor sensors prepared with graphene transferred from a camphor-rosin support layer are presented and compared with the results of mass spectrometry. The results show that the detection results of actual samples are very consistent with those of mass spectrometry, indicating that the prepared antibiotic sensor can be applied to the detection of actual samples.

Claims

1. A method for preparing an antibiotic sensor based on high-performance graphene transistors, characterized in that: Antibiotic sensors include: Graphene transistors; A polylysine coupling agent fixed on the graphene surface of the graphene transistor channel; Antibiotic aptamers immobilized on polylysine coupling agents; A microfluidic chip placed on top of a graphene transistor; include: (1) Preparation of single-layer graphene: (2) preparing source and drain electrodes on the single-layer graphene to obtain a graphene transistor; (3) preparing a microfluidic chip; the microfluidic chip includes a microfluidic controller, and the microfluidic controller is respectively connected to an inlet and an outlet; (4) aligning the flow channel of the prepared microfluidic chip with the channel of the graphene transistor, and bonding the microfluidic chip to the substrate of the single-layer graphene; (5) adding a polylysine coupling agent to the injection port of the bonded microfluidic chip so that the polylysine coupling agent fills the entire flow channel, and incubating the polylysine coupling agent to fully adhere to the graphene surface of the channel, and flushing the flow channel; (6) adding the antibiotic aptamer to the injection port of the microfluidic chip so that the antibiotic aptamer fills the entire flow channel, incubating the antibiotic aptamer to allow the antibiotic aptamer to fully bind to the polylysine coupling agent fixed in the channel and then be fixed in the channel, and flushing the flow channel; (7) After chemically blocking the sites of the unfixed antibiotic aptamers with ethanolamine, the flow channel is flushed to complete the preparation of the antibiotic sensor based on the high-performance graphene transistor and store it for future use; In step (1), preparing a single-layer graphene comprises: Growing graphene on copper foil to obtain copper-based graphene; A camphor-rosin double-layer support layer is coated on the copper-based graphene, and the copper-based graphene is transferred to a substrate through the camphor-rosin double-layer support layer; and the camphor-rosin double-layer support layer is completely removed to obtain a single-layer graphene.

2. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: The microfluidic chip is a PDMS microfluidic chip.

3. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: The microfluidic chip includes a microfluidic controller, which is respectively connected to an inlet and an outlet.

4. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: The width of the channel of the microfluidic chip is smaller than the distance between the source and drain electrodes.

5. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: Before step (6), perform the following steps: anneal the antibiotic aptamer at 80-98°C and then cool it to room temperature within 5 minutes.

6. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 5, characterized in that: The antibiotic aptamers were annealed at 95 °C.

7. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: In step (5), the incubation time of the polylysine coupling agent is more than 10 minutes.

8. The method for preparing an antibiotic sensor based on a high-performance graphene transistor according to claim 1, characterized in that: In step (6), the incubation time of the antibiotic aptamer is more than 30 minutes.

9. A method for detecting antibiotics using an antibiotic sensor prepared by the method for preparing an antibiotic sensor based on a high-performance graphene transistor according to any one of claims 1 to 8, characterized in that: include: ①Fix the reference electrode to the sample outlet of the microfluidic chip of the antibiotic sensor, and connect the source electrode, drain electrode and reference electrode of the antibiotic sensor to the source meter respectively; ② Add the sample to be tested to the inlet of the microfluidic chip, so that the sample to be tested fills the entire flow channel and incubates, then extract the sample to be tested and rinse away the uncaptured antibiotic molecules and other molecular impurities with PBS solution; ③ Add PBS solution to the inlet of the microfluidic chip and fill the entire flow channel and outlet; ④ Use the source meter to scan the transfer curve of the antibiotic sensor and read the Dirac point position of the antibiotic sensor; ⑤ Compare the measured Dirac point position of the antibiotic sensor with the Dirac point position when the antibiotic sensor does not detect the sample to be detected, and obtain the offset; ⑥Compare the offset obtained in step ⑤ with the Dirac point offset of the antibiotic sensor and the standard curve of antibiotic concentration to obtain the antibiotic concentration of the sample to be tested; The Dirac point shift of the antibiotic sensor refers to the change in the electrical signal of the antibiotic sensor after detecting the sample to be detected; The method for obtaining the standard curve of antibiotic concentration is: using an antibiotic sensor to measure antibiotic samples of known different concentrations, obtaining the change in the antibiotic sensor electrical signal of antibiotic samples of different concentrations, and plotting the relationship between different antibiotic concentrations and the change in the antibiotic sensor electrical signal to obtain the standard curve of antibiotic concentration.

10. The method for detecting antibiotics using an antibiotic sensor according to claim 9 is characterized in that: In step ②, the incubation time of the sample to be tested is more than 10 minutes.

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