A proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid-gated graphene transistor

Abstract

This invention discloses a proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid-gate graphene transistor, belonging to the field of analytical detection technology. The sensor includes: a photoelectrochemical liquid-gate graphene field-effect transistor with a monolayer graphene channel between its source and drain; a molecularly imprinted polymer layer modified on the surface of the photoelectrochemical gate for specific recognition of proline; and a colorimetric reaction system containing ninhydrin. After recognizing proline, the molecularly imprinted polymer reacts with ninhydrin to generate a colored product. This product produces a light-blocking effect under illumination, modulating the photoelectric response of the photoelectrochemical gate, and thus modulating the carrier concentration in the graphene channel. Quantitative detection of proline is achieved through changes in the source-drain current. This invention combines the high selectivity of molecular imprinting, the simplicity of ninhydrin colorimetric reaction, and the high sensitivity of the photoelectrochemical transistor, offering advantages such as high sensitivity, good selectivity, and ease of operation. It can be applied to the quantitative analysis of proline in plant tissues.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid gate graphene transistor, its preparation method, and its application. Background Technology

[0002] Proline (Pro) is a cyclic amino acid widely found in both plants and animals. In animals, proline participates in protein synthesis and the metabolism of arginine, polyamines, and glutamate. In plants, proline maintains the integrity of cell membrane structures, scavenge reactive oxygen species (ROS), and stabilizes enzyme and protein functions, providing important protection against abiotic stresses. Studies have shown that the proline content in plant tissues increases significantly by several times or even hundreds of times under drought, salinity, or other abiotic stresses. Therefore, proline content is often used as an important physiological indicator for evaluating the strength of plant stress resistance. Accurate determination of proline content is of significant research importance.

[0003] Currently, the main methods for detecting proline include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and spectrophotometry. However, these methods generally suffer from limitations such as cumbersome sample pretreatment, long processing times, and reliance on large instruments, making it difficult to meet the needs of rapid on-site detection.

[0004] Colorimetric methods are another common method for the quantitative detection of amino acids. The principle is based on the characteristic colorimetric reaction between proline and a specific reagent (such as acidic ninhydrin), where the intensity of the resulting colored product is positively correlated with the concentration of the target analyte. Colorimetric methods have advantages such as low cost and ease of operation. However, routine colorimetric quantitative analysis still requires the use of spectroscopic instruments such as UV-Vis spectrophotometers. These instruments suffer from poor portability and high cost, limiting their application in on-site detection.

[0005] In recent years, graphene transistor sensors have attracted widespread attention due to their advantages such as high sensitivity, fast response, and ease of miniaturization. For example, Chinese patent CN115718131A discloses a molecularly imprinted electrochemical transistor sensor, which uses acetaminophen as the target molecule and modifies the gate surface with a molecularly imprinted polymer. Electron transfer is generated through the electrochemical oxidation reaction of the target molecule on the gate to achieve the detection of the target molecule. However, this sensor still adopts a conventional electrochemical mode, and both its excitation and detection signals are electrical signals, resulting in high background noise. At the same time, its signal generation depends on the electrochemical activity of the target molecule itself, making it difficult to apply to target molecules with weak or no electrochemical activity (such as proline). In addition, although existing photoelectrochemical liquid-gate graphene field-effect transistor (PEC-SGGT) sensors have the advantages of low background signal and high sensitivity due to the separation of the excitation source (light) and detection signal (electricity), their selective recognition ability for specific target molecules still needs to be improved, especially the detection of proline, which has not yet been reported.

[0006] How to combine the high selectivity of molecularly imprinted polymers, the simplicity of ninhydrin colorimetry, and the high sensitivity of PEC-SGGT signal amplification characteristics to construct a sensing platform suitable for the highly sensitive and selective detection of proline is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the problems of cumbersome sample pretreatment, reliance on large instruments, insufficient sensitivity, poor selectivity of existing electrochemical transistor sensors, and difficulty in applying them to electrochemically inert target molecules, this invention provides a proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid-gate graphene transistor.

[0008] This invention combines the high selectivity of molecularly imprinted polymers, the simplicity of ninhydrin colorimetric development, and the high sensitivity of photoelectrochemical liquid-gate graphene transistors, proposing for the first time a multi-stage signal amplification strategy of "recognition-color development-light blocking-photoelectric modulation-current output." The sensor utilizes the specific recognition of proline by the molecularly imprinted polymer, which reacts with ninhydrin to generate a colored product. This colored product produces a light-blocking effect under specific wavelength illumination, modulating the photoelectric response characteristics of the photoelectrochemical gate, thereby modulating the carrier concentration in the graphene channel. Finally, highly sensitive and selective detection of proline is achieved through quantitative changes in the source-drain current. Experimental results show that this sensor exhibits excellent sensitivity, selectivity, and stability in proline detection, enabling quantitative analysis of proline in plant tissues.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid-gate graphene transistor, comprising: A photoelectrochemical liquid-gate graphene field-effect transistor includes a source, a drain, a graphene channel connecting the source and drain, and a photoelectrochemical gate; the source and drain are made of a gold / chromium bilayer metal film; the graphene channel is a single layer of graphene; the photoelectrochemical gate includes a conductive substrate and a photosensitive material layer loaded thereon. A molecularly imprinted polymer layer, modified on the surface of the photosensitive material layer, is used for the specific recognition of proline; the molecularly imprinted polymer layer is a molecularly imprinted polymer with spatially matched imprinted cavities corresponding to proline, prepared by gelation or polymerization using proline as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a crosslinking agent, and formed by elution treatment; the molecularly imprinted polymer layer is formed by crosslinking the molecularly imprinted polymer with a crosslinking agent, the crosslinking agent including agarose; and A colorimetric reaction system containing ninhydrin, wherein the ninhydrin is prepared by dissolving ninhydrin in acetic acid and phosphoric acid; In this process, the molecularly imprinted polymer layer recognizes proline and reacts with ninhydrin under heating conditions to generate a colored product. The color intensity of this colored product is positively correlated with the proline concentration and can produce a light-blocking effect under illumination, modulating the photoelectric response characteristics of the photoelectrochemical gate, thereby modulating the carrier concentration of the graphene channel. Finally, proline detection is achieved through quantitative changes in the source and drain current.

[0010] Furthermore, the photosensitive material is a semiconductor material with photoelectric response in the visible light region, selected from at least one of silver halide, CdS, CdSe, organic photoconductive materials, perovskite materials, TiO2, g-C3N4, quantum dots and dye sensitizing materials, or a composite thereof.

[0011] Furthermore, in the molecularly imprinted polymer layer, the crosslinking agent is selected from at least one of Nafion and PEGDA; the crosslinking method includes direct crosslinking, gel crosslinking or photoinitiated polymerization crosslinking.

[0012] Furthermore, the colored product is a red product, and the wavelength of the light irradiation is 390nm-560nm.

[0013] Furthermore, the sensor also includes a supporting electrolyte, which is a phosphate buffer containing a sacrificial agent; the sacrificial agent is selected from at least one of ascorbic acid, triethanolamine, triethylamine, diethanolamine, monoethanolamine, hydrogen peroxide, and sulfite; the concentration of the sacrificial agent is 0.01-1M, the concentration of the phosphate buffer is 0.01-1M, and the pH value is 6.5-8.0.

[0014] Furthermore, the photosensitive material layer is formed on the surface of the conductive substrate through a self-assembly process, which uses poly(diallyldimethylammonium chloride) as a medium and is formed through multiple alternating depositions.

[0015] Secondly, the present invention provides a method for constructing the above-mentioned proline detection sensor, comprising the following steps: (1) Preparation of liquid gate graphene field-effect transistor: After cleaning the SiO2 glass substrate, a gold / chromium bilayer metal film was deposited on the substrate surface by thermal evaporation through a mask to form the source and drain. A polymethyl methacrylate protective layer was formed on the surface of copper-based monolayer graphene by spin casting. After curing, the layer was cut and etched by immersing in ferric chloride solution to transfer the graphene to the substrate. Before the transfer, the substrate was treated with oxygen plasma. After the transfer, the substrate was dried and annealed. Finally, the polymethyl methacrylate layer was removed by immersion in acetone. (2) Preparation of photoelectrochemical gate: FTO glass was treated with alkaline solution and acid solution in sequence and then dried; CdS quantum dots were modified onto the surface of FTO electrode using poly(diallyldimethylammonium chloride): FTO electrode was immersed in poly(diallyldimethylammonium chloride) solution, washed and then immersed in CdS quantum dot solution, and the deposition process was repeated multiple times to obtain CdS / FTO electrode; proline was used as template molecule, methacrylic acid as functional monomer and ethylene glycol dimethacrylate as crosslinking agent, and polymerized under heating conditions. The template molecule was eluted with methanol by Soxhlet extraction and dried to obtain molecularly imprinted polymer; agarose was dissolved in water, and the molecularly imprinted polymer aqueous solution was added. After stirring, it was dropped onto the surface of CdS / FTO electrode and allowed to stand to obtain CdS / molecularly imprinted polymer / FTO electrode; (3) The CdS / molecularly imprinted polymer / FTO electrode obtained in step (2) is used as a gate and assembled with the graphene transistor in step (1) to form the sensor.

[0016] Further, the cleaning in step (1) includes ultrasonic cleaning with acetone, ethanol, and deionized water in sequence; the annealing is carried out under an inert atmosphere; the oxygen plasma treatment time is 1-5 minutes; the alkaline treatment in step (2) is to immerse the FTO glass in a potassium hydroxide isopropanol solution and boil it; the acid treatment is to place the FTO glass in a mixed solution of concentrated sulfuric acid and hydrogen peroxide for cleaning; the number of repeated depositions is 3-6 times.

[0017] Thirdly, the present invention provides an application of the above-mentioned sensor for quantitative analysis of proline in plant tissues for non-diagnostic and therapeutic purposes, comprising the following steps: (1) Immerse the photoelectrochemical gate of the sensor into the sample solution to make proline specifically recognized by the molecularly imprinted polymer layer; (2) Immerse the identified photochemical grid in ninhydrin colorimetric reagent and react for 5-20 minutes under heating conditions of 60-90℃ to carry out the colorimetric reaction; (3) The color-developed photoelectrochemical gate is integrated with the sensor, and the source and leakage current changes are measured under 390nm-560nm wavelength illumination and in the supporting electrolyte. (4) The concentration of proline in the sample is obtained based on the predetermined quantitative relationship between the proline concentration and the source-leakage current.

[0018] Furthermore, the supporting electrolyte is a 0.01-1M phosphate buffer containing 0.01-1M sacrificial agent, with a pH of 6.5-8.0; the sacrificial agent is selected from at least one of ascorbic acid, triethanolamine, triethylamine, diethanolamine, monoethanolamine, hydrogen peroxide, and sulfite.

[0019] Compared with the prior art, the present invention has the following outstanding advantages: 1. High Sensitivity: This invention is the first to use a photoelectrochemical liquid-gate graphene field-effect transistor (PEC-SGGT) for proline detection. By completely separating the excitation source (light) and the detection signal (electricity), extremely low background signal and extremely high sensitivity are achieved. Simultaneously, a multi-stage signal amplification strategy involving "recognition-color development-light blocking-photoelectric modulation-current output" further enhances the detection sensitivity. Experimental results show that the detection platform of this invention can achieve sensitive detection of trace amounts of proline.

[0020] 2. High Selectivity: This invention modifies the surface of the photochemical gate electrode with a molecularly imprinted polymer layer using proline as a template molecule. This layer possesses "imprinted holes" that perfectly match proline in spatial configuration, functional group sites, and molecular size, enabling extremely high specificity for proline recognition. Experimental results show that even in the presence of various interfering molecules (such as malic acid, glycine, vitamin B5, xylose, and glucose), the sensor of this invention still exhibits a significant specific response to proline.

[0021] 3. Simple operation and low cost: This invention does not require complicated sample pretreatment, nor does it rely on large instruments such as high performance liquid chromatography and mass spectrometry. It only requires a simple colorimetric reaction and electrical signal reading to achieve quantitative detection of proline, which significantly reduces the time cost and technical threshold of detection and is suitable for rapid on-site detection.

[0022] 4. Good stability: Compared with natural recognition elements such as antibodies and aptamers, the molecularly imprinted polymer used in this invention has higher chemical and thermal stability, can be reused, and is easy to store and transport.

[0023] 5. Wide Applicability: This invention uses proline as the model target molecule to verify the feasibility of the detection platform. Due to the universality of molecular imprinting technology, by changing the template molecule, the sensor platform of this invention can be extended to the detection of other amino acids, small molecule metabolites, and even proteins, demonstrating good versatility and promotional value.

[0024] In summary, the proline detection platform based on molecularly imprinted colorimetric photoelectrochemical liquid-gate graphene transistor constructed in this invention has the advantages of high sensitivity, high selectivity, simple operation, low cost and good stability, providing a brand-new technical means for proline detection in plant stress physiology research, agricultural quality evaluation and related fields. Attached Figure Description

[0025] Figure 1 A schematic diagram of the construction process of the MIP-PEC-SGGT sensor for proline (Pro) detection; Figure 2 (a) Transfer characteristic curves of the MIP-PEC-SGGT device under dark and light conditions; (b) MIP-PEC-SGGT device under illumination at different V values. G Output characteristic curve under the given conditions; Figure 3 (a) The modulation of the transfer characteristics of MIP-PEC-SGGT under darkness and illumination using three electrodes as gates: a proline-pre-intercalated but not eluted photoelectrochemical electrode (CdS / MIP(Pro)), a molecularly imprinted functionalized photoelectrochemical electrode obtained by eluting proline pre-intercalation (CdS / MIP), and a photoelectrochemical electrode obtained by capturing proline and developing it with ninhydrin (CdS / MIP+Pro+Ninhydrin); (b) The modulation of the output characteristics of MIP-PEC-SGGT under darkness and illumination using CdS / MIP(Pro), CdS / MIP, and CdS / MIP+Pro+Ninhydrin gates. Figure 4 Optimization of experimental parameters: (a) optimization of the ratio of functional monomer to crosslinking agent, (b) elution time, (c) heating time, (d) heating temperature, (e) pH; Figure 5 (a) I in MIP-PEC-SGGT device at different Pro concentrations DS Curve showing the change over time, (b) ΔI DS / I 0 DS With logC Pro(a) Linear correlation fitting curve (n = 3), (c) IDS of MIP-PEC-SGGT device at different Pro concentrations over time without the addition of ninhydrin, (d) Selectivity test results of MIP-PEC-SGGT and NIP-PEC-SGGT against 0.1 μM Pro and 10-fold excess of various interfering substances (n = 3) (a: malic acid, b: glycine, c: vitamin B5, d: xylose, e: glucose, f: proline). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. (Note: The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.) Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0027] The overall structure and construction process of the sensor are as follows: Figure 1 As shown.

[0028] CdS quantum dot preparation: Under nitrogen protection, 50 mL of a 1.0 mM cadmium chloride (CdCl2) aqueous solution was magnetically stirred for 15 minutes. Then, 250 μL of mercaptoacetic acid (TGA) was rapidly added, and the pH of the system was immediately adjusted to 11 with 2.0 M sodium hydroxide (NaOH) solution. After reacting for 30 min, 5.0 mL of a 100 mM Na2S·9H2O solution was added dropwise, and the reaction was refluxed at 110°C for 4 hours (under nitrogen protection with continuous stirring throughout). The resulting CdS QDs were diluted 1:1 (v / v) with ultrapure water and stored at 4 °C for later use.

[0029] Poly(diallyldimethylammonium chloride) (PDDA, 20%, w / w in water) was purchased from Sigma-Aldrich; proline (≥99.5%) was purchased from Sigma-Aldrich; FTO conductive glass (e.g., sheet resistance <15 Ω / sq, transmittance >83%), and other chemical reagents such as potassium hydroxide, sodium chloride, acetic acid, phosphoric acid, and ferric chloride were all commercially available analytical grade.

[0030] Example 1 Fabrication of liquid-gate graphene field-effect transistors (SGGTs).

[0031] (I) Preparation process A 1 cm × 1 cm SiO2 glass substrate was ultrasonically cleaned sequentially with acetone, ethanol, and deionized water for 30 minutes each. An 80 nm gold / 20 nm chromium bilayer metal film was deposited on the substrate surface using a thermal evaporation method through a mask to form the source and drain electrodes, with a channel size of 6 mm in length and 0.2 mm in width. A wet etching-transfer process was used to transfer monolayer graphene: a polymethyl methacrylate (PMMA) protective layer was formed on the surface of copper-based monolayer graphene by spin spinning and cured at 130°C for 30 minutes; the PMMA / graphene composite film was cut into 3×3 mm cubes, immersed in a 1 M ferric chloride (FeCl3) solution to etch copper foil, thoroughly rinsed with deionized water, and then transferred to a SiO2 substrate; before transfer, the substrate was subjected to oxygen plasma treatment for 3 minutes to enhance surface hydrophilicity; the transferred graphene was air-dried for 12 hours, annealed at 90°C for 30 minutes, and finally immersed in acetone at 60°C for 6 hours (the acetone was replaced every 2 hours) to completely remove the PMMA layer, thus obtaining the SGGT device. This device serves as the signal conversion and amplification element of the MIP-PEC-SGGT sensor.

[0032] (II) Device Performance Verification The electrical performance of the fabricated SGGT device was tested. For example... Figure 2 As shown in (a), the transfer characteristic curves of the device exhibit typical graphene bipolar behavior under both dark and illuminated conditions, with a distinct Dirac point. Compared to the dark condition, the transfer characteristic curves of the device under illumination shift overall towards the negative gate voltage direction (the Dirac point shifts to the left), indicating that illumination introduces a positive gate bias effect in the graphene channel, confirming that the fabricated SGGT device possesses photoelectric sensing capabilities. Figure 2 (b) Shows different gate voltages (V) G The output characteristic curves of the device under illumination show that the device maintains good transistor performance under illumination and operates in the variable resistance region (linear region), providing a stable signal readout platform for subsequent proline detection.

[0033] Example 2 Fabrication of photoelectrochemical gate (CdS / FTO).

[0034] (I) Preparation process The photoelectrode was fabricated using a self-assembly process. 4.488 g of potassium hydroxide (KOH) was dissolved in 40 mL of isopropanol, and the cut FTO conductive glass was immersed in this solution and boiled for 15 minutes. The treated FTO glass was then repeatedly rinsed with copious amounts of distilled water. Subsequently, the alkaline-washed FTO glass was placed in a 7:3 mixture of concentrated sulfuric acid and hydrogen peroxide (volume ratio) for approximately 6 seconds, rinsed thoroughly with copious amounts of distilled water, and dried under a nitrogen atmosphere for later use.

[0035] CdS quantum dots were modified onto the surface of an FTO electrode using poly(diallyldimethylammonium chloride) (PDDA). The specific steps were as follows: 2.922 g of sodium chloride (NaCl) was dissolved in 97 mL of distilled water, and 3 mL of PDDA was added and stirred for 5 hours. The acid-washed and dried FTO electrode was immersed in the PDDA solution for 10 minutes, then removed and repeatedly rinsed with distilled water. Subsequently, the electrode was immersed in a CdS quantum dot solution for 10 minutes, and again repeatedly rinsed with distilled water. This deposition process was repeated four times to finally obtain the desired CdS / FTO electrode, which served as the photosensitive material substrate for the photoelectrochemical gate.

[0036] (II) Photoelectric response verification The prepared CdS / FTO electrode was used as a gate and assembled with the SGGT device of Example 1, and its signal modulation capability under illumination was tested. The results show that the CdS / FTO electrode can generate a stable photocurrent under illumination and effectively modulate the carrier concentration of the graphene channel, laying the foundation for subsequent molecular imprinting functionalization.

[0037] Example 3 Preparation of proline molecularly imprinted polymers (MIPs).

[0038] (I) Preparation process 57 mg (0.5 mmol) of proline (Pro) was dispersed in 20 mL of acetonitrile at 55 °C. 6 mL of ethanol was gradually added with constant stirring to ensure complete dissolution of Pro. Subsequently, 1.5 mmol of a functional monomer (methacrylic acid, MAA) and 6 mmol of a crosslinking agent (ethylene glycol dimethacrylate, EGDMA) were introduced into the system. The resulting solution was purged with nitrogen for 3 minutes to remove oxygen. The container was sealed and incubated in a 60 °C water bath for 12 hours to initiate the polymerization reaction. After polymerization, the polymer was extracted with methanol using a Soxhlet extraction method for 12 hours to remove template molecules and unreacted monomers, yielding a molecularly imprinted polymer (MIP). Finally, the polymer was dried in a 70 °C oven for 12 hours for later use. This MIP was used to subsequently modify a photoelectrochemical gate as a specific recognition unit for proline.

[0039] (II) Verification of elution effect To verify the elution effect of template molecules in the MIP, three gate electrodes were fabricated: a CdS / MIP(Pro) electrode with proline pre-intercalation but not yet eluted, a CdS / MIP electrode with molecularly imprinted holes obtained after elution, and a CdS / MIP+Pro+Ninhydrin electrode after capturing proline with a molecularly imprinted functionalized electrode and developing with ninhydrin. Using these three electrodes as gate electrodes, their modulation effect on SGGT transfer characteristics under darkness and illumination was tested. Figure 3As shown in (a), the CdS / MIP(Pro) electrode has a weaker light-blocking effect due to the template molecules filling the MIP channels, resulting in a smaller shift of the Dirac point under illumination. However, the eluted CdS / MIP electrode has a looser structure due to the formation of vacancies, leading to a slightly enhanced light response. When the CdS / MIP electrode recaptures proline and develops color (CdS / MIP+Pro+Ninhydrin), the light-blocking effect of the red product causes a significant leftward shift of the Dirac point. Figure 3 (b) The output characteristic curve also shows a consistent pattern. This result proves the rationality and effectiveness of the "recognition-color development-blocking-electrical signal modulation" strategy, and also shows that the MIP-PEC-SGGT has a rapid photosensitive response, can quickly output a stable signal and achieve amplification.

[0040] Example 4 Fabrication of molecularly imprinted functionalized gates (CdS / MIP / FTO).

[0041] (I) Preparation process Add 0.04 g agarose to 2 mL of distilled water, heat in a water bath and stir for 10 minutes to ensure complete dissolution. Then add 1 mL of a 1 mg / mL solution. -1 The MIP aqueous solution (prepared in Example 3) was stirred for another 3 minutes to obtain a homogeneous mixture. 20 μL of this mixture was drop-coated onto the surface of the CdS / FTO electrode prepared in Example 2 and allowed to stand at room temperature for 1 h to obtain the CdS / MIP / FTO gate. This gate is used for subsequent proline recognition, color development, and sensor assembly.

[0042] (II) Gate Structure Verification The prepared CdS / MIP / FTO gate was characterized by morphology and electrochemical properties, confirming that the molecularly imprinted polymer layer had been successfully modified onto the surface of the CdS / FTO electrode.

[0043] Example 5 Colorimetric detection of proline and sensor performance evaluation.

[0044] (a) Identification and colorimetric reaction The CdS / MIP / FTO gates prepared in Example 4 were immersed in proline solutions of different concentrations and incubated at room temperature for 30 minutes for specific recognition. The gates were then removed and the surface was washed with deionized water. The colorimetric reagent was prepared by dissolving 125 mg of ninhydrin in 3 mL of acetic acid and 2 mL of 6 mol·L⁻¹ solution. -1 Dilute the phosphoric acid solution 10 times with water for later use. Immerse the cleaned gate electrode in the above colorimetric reagent and heat at 70°C for 10 minutes to induce a colorimetric reaction.

[0045] (II) Sensor Assembly The CdS / MIP / FTO gate after color development was assembled with the SGGT device prepared in Example 1. The gate was used as a liquid gate to form a complete proline detection sensor (MIP-PEC-SGGT) based on molecularly imprinted colorimetric photoelectrochemical liquid gate graphene transistor.

[0046] The sensor fabrication of this invention is now complete.

[0047] (III) Optimization of detection parameters To obtain optimal detection performance, key parameters in the sensor fabrication and detection processes were systematically optimized, and the results are as follows: Figure 4 As shown.

[0048] like Figure 4 As shown in (a), when the molar ratio of functional monomer (MAA) to crosslinking agent (EGDMA) is 1:4 (i.e., the ratio in Example 3), the sensor has the largest response current to proline, indicating that the imprinted vacancies formed under this ratio are the most ideal. Figure 4 (b) shows that after elution for 12 hours (i.e., the elution time in Example 3), the template molecules were basically completely removed, and further extending the elution time had limited effect on signal enhancement. Figure 4 (c) and 4(d) show that the optimal colorimetric reaction conditions are heating at 70°C for 10 minutes, at which point the colorimetric reaction is complete and the background signal is low. Figure 4 (e) indicates that the optimal pH of the supporting electrolyte is 7.4, which is close to physiological conditions and beneficial for the recognition of proline and the stability of the chromogenic product. The following detection experiments all used the optimized parameters described above.

[0049] (iv) Quantitative detection performance The assembled MIP-PEC-SGGT sensor was used to measure the source-leakage current (IL) under 390-560 nm wavelength illumination and in a supporting electrolyte (0.1 M phosphate buffer containing 0.1 M ascorbic acid, pH=7.4). DS ) changes. For example... Figure 5 As shown in (a), as the proline concentration increased from 0.1 nM to 100 μM, I DS As time gradually increases, the response reaches equilibrium in approximately 200 seconds. The change in current at equilibrium (ΔI) is then calculated. DS ) and initial current (I 0 DS Plot the ratio of the two groups against the logarithm of the proline concentration, as shown in the figure. Figure 5 As shown in (b), ΔI DS / I 0 DS With logC Pro It exhibits a good linear relationship in the range of 0.1 nM to 100 μM (R 2=0.993), the linear equation is ΔI DS / I 0 DS = 0.132 logC Pro +0.425. Based on a signal-to-noise ratio of 3, the detection limit is as low as 0.03 nM. This indicates that sensitive quantitative detection of proline can be achieved by monitoring changes in source leakage current.

[0050] (v) Verification of the shading effect mechanism To demonstrate that the signal source of this invention is the "light-shielding effect" of the chromogenic product rather than the charge or electrochemical reaction of the target molecule itself, a control experiment was conducted without the addition of ninhydrin. Figure 5 As shown in (c), even when the CdS / MIP / FTO gate is immersed in a high-concentration proline solution, the current response of the device is extremely weak due to the lack of a colorimetric reaction. This clearly confirms that the signal generation depends on the light-blocking effect of the ninhydrin colorimetric product.

[0051] (vi) Selective assessment The MIP-PEC-SGGT sensor was exposed to 0.1 μM proline and 10-fold excess of interfering substances (malic acid, glycine, vitamin B5, xylose, and glucose), and the current responses were recorded. Simultaneously, a non-molecularly imprinted (NIP) control device (without the addition of the template molecule proline during fabrication) was prepared for parallel testing. Figure 5 As shown in (d), the MIP-PEC-SGGT sensor exhibits a significantly higher response to proline than other interfering substances, while the NIP control device shows low responses to both proline and other interfering substances with no significant difference. These results demonstrate that the molecularly imprinted layer constructed in this invention endows the sensor with excellent proline selectivity and anti-interference capabilities.

[0052] (vii) Application of actual samples Fresh vegetable samples (cauliflower, cabbage, garlic) of edible portion were washed with deionized water, dried, and 5.0 g were weighed and placed in a mortar for thorough homogenization. 10 mL of phosphate buffered saline (PBS, 0.1 M, pH=7.4) was added to extract proline. The mixture was vortexed for 5 minutes and centrifuged at 4000 rpm for 10 minutes. The supernatant was collected for later use. The CdS / MIP / FTO gate prepared in Example 4 was immersed in the supernatant and incubated at room temperature for 30 minutes for specific recognition. The gate was removed, and the surface was cleaned with deionized water. The sensor was assembled according to step (II), and the source-leakage current change was measured under the detection conditions in step (IV) (390-560 nm wavelength light, 0.1 M PBS containing 0.1 M ascorbic acid, pH=7.4). The obtained ΔI was used to measure the source-leakage current change. DS / I 0 DSSubstitute the standard curve equation (ΔI) from step (iv) DS / I 0 DS = 0.132 logC Pro The proline content was calculated using +0.425. Each sample was measured in triplicate, and the results are shown in Table 1. Simultaneously, the supernatant was analyzed in parallel using high-performance liquid chromatography (HPLC) to verify the accuracy of the sensor's detection results.

[0053] Table 1. Pro analysis results in actual samples Comparing the Pro content measured by this sensor with the results obtained by HPLC in Table 1, the relative deviations between the two methods were between 3.7% and 4.2% (cauliflower: 4.1%, cabbage: 3.7%, garlic: 4.2%), all within ±5%, indicating that this sensor has good accuracy and reliability and can be used for rapid quantitative analysis of proline in actual vegetable samples. Furthermore, compared to HPLC, this sensor requires no complex sample pretreatment or large instruments, is simple to operate, and has a short detection time, showing good application potential for rapid on-site detection of proline in plant tissues.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A proline detection sensor based on a molecularly imprinted colorimetric photoelectrochemical liquid-gate graphene transistor, characterized in that, include: A photoelectrochemical liquid-gate graphene field-effect transistor includes a source, a drain, a graphene channel connecting the source and the drain, and a photoelectrochemical gate. The source and drain electrodes are made of gold / chromium bilayer metal films; the graphene channel is made of single-layer graphene; the photoelectrochemical gate includes a conductive substrate and a photosensitive material layer loaded thereon. A molecularly imprinted polymer layer, modified on the surface of the photosensitive material layer, is used for the specific recognition of proline; the molecularly imprinted polymer layer is a molecularly imprinted polymer with spatially matched imprinted cavities corresponding to proline, prepared by gelation or polymerization using proline as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a crosslinking agent, and formed by elution treatment; the molecularly imprinted polymer layer is formed by crosslinking the molecularly imprinted polymer with a crosslinking agent, the crosslinking agent including agarose; and A colorimetric reaction system containing ninhydrin, wherein the ninhydrin is prepared by dissolving ninhydrin in acetic acid and phosphoric acid; In this process, the molecularly imprinted polymer layer recognizes proline and reacts with ninhydrin under heating conditions to generate a colored product. The color intensity of this colored product is positively correlated with the proline concentration and can produce a light-blocking effect under illumination, modulating the photoelectric response characteristics of the photoelectrochemical gate, thereby modulating the carrier concentration of the graphene channel. Finally, proline detection is achieved through quantitative changes in the source and drain current.

2. The sensor according to claim 1, characterized in that, The photosensitive material is a semiconductor material with photoelectric response in the visible light region, selected from at least one of silver halide, CdS, CdSe, organic photoconductive materials, perovskite materials, TiO2, g-C3N4, quantum dots and dye sensitizing materials, or a composite thereof.

3. The sensor according to claim 1, characterized in that, In the molecularly imprinted polymer layer, the crosslinking agent is further selected from at least one of Nafion and PEGDA; the crosslinking method includes direct crosslinking, gel crosslinking or photoinitiated polymerization crosslinking.

4. The sensor according to claim 1, characterized in that, The colored product is red, and the wavelength of the light is 390nm-560nm.

5. The sensor according to claim 1, characterized in that, It also includes a supporting electrolyte, which is a phosphate buffer containing a sacrificial agent; the sacrificial agent is selected from at least one of ascorbic acid, triethanolamine, triethylamine, diethanolamine, monoethanolamine, hydrogen peroxide, and sulfite; the concentration of the sacrificial agent is 0.01-1M, the concentration of the phosphate buffer is 0.01-1M, and the pH value is 6.5-8.

0.

6. The sensor according to claim 1, characterized in that, The photosensitive material layer is formed on the surface of a conductive substrate through a self-assembly process, which uses poly(diallyldimethylammonium chloride) as a medium and is formed through multiple alternating depositions.

7. A method for constructing a proline detection sensor according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of liquid gate graphene field-effect transistor: After cleaning the SiO2 glass substrate, a gold / chromium bilayer metal film was deposited on the substrate surface by thermal evaporation through a mask to form the source and drain. A polymethyl methacrylate protective layer was formed on the surface of copper-based monolayer graphene by spin casting. After curing, the layer was cut and etched by immersing in ferric chloride solution to transfer the graphene to the substrate. Before the transfer, the substrate was treated with oxygen plasma. After the transfer, the substrate was dried and annealed. Finally, the polymethyl methacrylate layer was removed by immersion in acetone. (2) Preparation of photoelectrochemical gate: FTO glass was treated with alkaline solution and acid solution in sequence and then dried; CdS quantum dots were modified onto the surface of FTO electrode using poly(diallyldimethylammonium chloride): FTO electrode was immersed in poly(diallyldimethylammonium chloride) solution, washed and then immersed in CdS quantum dot solution, and the deposition process was repeated multiple times to obtain CdS / FTO electrode; proline was used as template molecule, methacrylic acid as functional monomer and ethylene glycol dimethacrylate as crosslinking agent, and polymerized under heating conditions. The template molecule was eluted with methanol by Soxhlet extraction and dried to obtain molecularly imprinted polymer; agarose was dissolved in water, and the molecularly imprinted polymer aqueous solution was added. After stirring, it was dropped onto the surface of CdS / FTO electrode and allowed to stand to obtain CdS / molecularly imprinted polymer / FTO electrode; (3) The CdS / molecularly imprinted polymer / FTO electrode obtained in step (2) is used as a gate and assembled with the graphene transistor in step (1) to form the sensor.

8. The construction method according to claim 7, characterized in that, The cleaning in step (1) includes ultrasonic cleaning with acetone, ethanol and deionized water in sequence; the annealing is carried out under an inert atmosphere; the oxygen plasma treatment time is 1-5 minutes; the alkaline treatment in step (2) is to immerse the FTO glass in a potassium hydroxide isopropanol solution and boil it; the acid treatment is to place the FTO glass in a mixed solution of concentrated sulfuric acid and hydrogen peroxide for cleaning; The number of repeated depositions is 3-6 times.

9. An application of the sensor described in any one of claims 1-6 for the quantitative analysis of proline in plant tissues for non-diagnostic and therapeutic purposes, characterized in that, Includes the following steps: (1) Immerse the photoelectrochemical gate of the sensor into the sample solution to make proline specifically recognized by the molecularly imprinted polymer layer; (2) Immerse the identified photoelectrochemical grid in ninhydrin colorimetric reagent and react for 5-20 minutes under heating conditions of 60-90℃ to carry out the colorimetric reaction; (3) The photoelectrochemical gate after color development is integrated with the sensor, and the source and leakage current changes are measured under 390nm-560nm wavelength illumination and in the supporting electrolyte. (4) The concentration of proline in the sample is obtained based on the predetermined quantitative relationship between the proline concentration and the source-leakage current.

10. The application according to claim 9, characterized in that, The supporting electrolyte is a 0.01-1M phosphate buffer containing 0.01-1M sacrificial agent, with a pH of 6.5-8.0; the sacrificial agent is selected from at least one of ascorbic acid, triethanolamine, triethylamine, diethanolamine, monoethanolamine, hydrogen peroxide, and sulfite.

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