Electrode substrate / hof-on-mof photosensitive gate, preparation method, sensing electrode, and organic photoelectrochemical transistor biosensor and application thereof

Abstract

The application belongs to the technical field of biosensing, and discloses an electrode substrate / HOF-on-MOF light-sensitive gate, a preparation method, a sensing electrode, an organic photoelectrochemical transistor biosensor and an application thereof. The light-sensitive gate comprises a titanium-based MOF layer chemically bonded on the surface of an electrode substrate and a HOF layer chemically bonded on the surface of the titanium-based MOF layer by forming an amide bond, wherein the titanium-based MOF is a metal organic framework material with a titanium metal center and a surface rich in amino groups, and the HOF is a hydrogen-bonded organic framework material with a surface rich in carboxyl groups. On the one hand, the interface compatibility and directional assembly of HOF and MOF are realized at the molecular scale, and structural collapse is avoided; on the other hand, the energy band matching and charge transport path of the heterojunction realize efficient photoelectric synergy; at the same time, the specific surface area can be effectively increased, rich functional groups can be introduced, and the mechanical strength and chemical stability of the composite material can be significantly improved, thereby producing a synergistic enhancement effect.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to an electrode substrate / HOF-on-MOF photosensitive gate, its preparation method, sensing electrode, and an organic photoelectrochemical transistor biosensor and its applications. Background Technology

[0002] Organic photoelectrochemical transistors (OPECTs), which combine photoelectrochemistry (PEC) with organic electrochemical transistors (OECTs), have recently developed into a multifunctional platform for studying complex light-matter-biological interactions. This technology combines the advantages of low background noise and signal self-amplification. By introducing photostimulation into the photosensitive gate, a weak photovoltage can be used to redistribute the potential drop at the gate-electrolyte and channel-electrolyte interfaces, thereby achieving efficient control of the transistor's response. This characteristic allows OPECTs to achieve transconductance enhancement and significant improvement in electronic sensing performance even under zero gate voltage conditions. Compared to OECTs, OPECTs, by introducing photoresponse characteristics, achieve non-invasive, energy-free light-controlled modulation, providing a new dimension for device behavior regulation and expanding its application scenarios in fields such as biosensing and biomimetic systems. For example, Inal et al. developed an OPECT based on an n-type semiconductor gate for highly sensitive photoplethysmography (PPG) monitoring; Santoro's team developed an OPECT based on an azobenzene-thiazole-PEDOT:PSS photoisomerized gate, successfully simulating the visual neural pathway; and Zhao's research group designed an addressable photogate OPECT array and applied it to optogenetics, retinal morphological synapses, and multichannel biosensing. These studies demonstrate that the structural design and functionalization of the photogate are crucial to the performance and functionality of OPECTs. In recent years, covalent organic framework materials, metal-organic framework materials, and heterostructures have been widely used in OPECT. For example, the article reported by Professor Gaochao Fan of Qingdao University of Science and Technology and Professor Weiwei Zhao of Nanjing University (Adv. Funct. Mater. 2024, 2404497) discusses the importance of achieving a high on / off ratio in OPECT and proposes a method to achieve an Ag / AgCl-like optical gating effect through a hybrid ligand MOF modified with photosensitive COF-LZU1 (COF-on-MOF). Under appropriate illumination, this COF-on-MOF heterostructure can significantly modulate the physical properties of the device and optimize IT. On / I OffCompared to other technologies, this enhances signal transduction. For example, Chinese patent document (application number 202311311330.8) discloses a method for constructing an integrated organic photoelectrochemical transistor sensor for detecting the pesticide methamidophos. This method integrates the gate, source, and drain electrodes of the organic photoelectrochemical transistor onto a single fluorine-doped tin oxide conductive glass chip using laser etching technology. To improve the activity and stability of the photosensitive gate material, a method of encapsulating Pd NPs within a Cu-MOF and then pyrolyzing them was employed to form Pd NPs / p-Cu-MOF. Hydrogen-bonded organic frameworks (HOFs) are a class of crystalline porous materials formed through hydrogen bond self-assembly. They have attracted widespread attention due to their well-defined structure and tunable function, but their relatively weak chemical stability limits their practical applications. Summary of the Invention

[0003] In view of this, the present invention proposes an electrode substrate / HOF-on-MOF photosensitive gate, a preparation method, a sensing electrode, and an organic photoelectrochemical transistor biosensor and its application, which not only overcomes the chemical stability problem of HOF materials, but also achieves a better photoelectric response in the electrode substrate / HOF-on-MOF photosensitive gate.

[0004] One objective of this invention is to provide an electrode substrate / HOF-on-MOF photosensitive gate with high light absorption performance. Given the relatively weak chemical stability of HOFs, this invention proposes to precisely anchor HOFs onto the surface of MOFs to construct a stable HOF-on-MOF heterostructure. However, HOFs are typically connected by weak hydrogen bonds, while MOFs are connected by strong coordination bonds. The crystal structure, thermal stability, and chemical stability of the two at the interface differ significantly, making it difficult to form a high-quality heterojunction interface. Therefore, a two-step method of interfacial chemical bonding and amide coupling is proposed to construct the electrode substrate / HOF-on-MOF heterojunction photosensitive gate (the first two aspects are also the technical bottlenecks faced in constructing HOF-on-MOF heterojunctions): on the one hand, achieving interfacial compatibility and directional assembly of HOF and MOF at the molecular scale to avoid structural collapse; on the other hand, achieving efficient photoelectric synergy through band matching and charge transport paths of the heterojunction; simultaneously, effectively increasing the specific surface area, introducing abundant functional groups, and significantly improving the mechanical strength and chemical stability of the composite material, resulting in a synergistic enhancement effect. To achieve the above objectives, the present invention adopts the following technical solution:

[0005] An electrode substrate / HOF-on-MOF photosensitive gate includes an electrode substrate, a titanium-based MOF layer chemically bonded to the surface of the electrode substrate, and an HOF layer chemically bonded to the surface of the titanium-based MOF layer by forming amide bonds. The titanium-based MOF is a metal-organic framework material with titanium as the metal center and an amino-rich surface, and the HOF is a hydrogen-bonded organic framework material with carboxyl groups rich in the surface.

[0006] To further enhance the intensity and stability of high photoelectric signals, HOF-100 was further optimized as the HOF material.

[0007] The fabrication method of the aforementioned electrode substrate / HOF-on-MOF photosensitive gate includes the following steps: A titanium-based MOF with an amino-rich surface is coated onto the surface of a hydroxyl-activated electrode substrate (ITO or FTO), and chemically bonded to obtain a photoelectric active electrode substrate / MOF electrode. Subsequently, a HOF material with a carboxyl-rich surface is selected, and its carboxyl groups are activated using EDC / NHS. This activated carboxyl groups are then coated onto the surface of the aforementioned electrode substrate / MOF electrode. Covalent anchoring of the HOF on the MOF is achieved through the formation of amide bonds, ultimately constructing a HOF-on-MOF composite photosensitive layer with Type-II heterojunction characteristics and strong interfacial coupling, thereby obtaining a high photoelectric response electrode substrate / HOF-on-MOF photosensitive gate.

[0008] Specifically, the electrode substrate is first cleaned to enrich its surface with hydroxyl groups (hydroxylation methods commonly used in the chemical industry can also be used, such as solution etching and electrochemical anodizing; however, this invention prefers plasma activation for better signal amplification). Then, a titanium-based MOF solution is coated onto the electrode substrate, where the titanium centers condense with the hydroxyl groups on the electrode substrate surface to form Ti-OM (M is In or Sn) interfacial chemical bonds, thus fixing the titanium-based MOF layer on the conductive substrate. The carboxyl groups on the HOF material surface are activated using a system of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then coated onto the amino-rich titanium-based MOF layer. This covalent anchoring of the HOF on the MOF surface is achieved through amide bond formation, thereby constructing a structurally stable electrode substrate / HOF-on-MOF photosensitive gate with strong interfacial coupling.

[0009] It is worth noting that photosensitive HOF-on-MOF heterojunction materials can significantly improve light absorption efficiency, promote hot electron generation, and suppress carrier recombination, exhibiting excellent photoelectric conversion characteristics. However, how to efficiently integrate such photoactive heterojunctions into semiconductor device interfaces and achieve stable and sensitive responses in photoelectric sensing systems still faces multiple technical bottlenecks, including material-device compatibility, dynamic control of interface charge, and long-term operational stability.

[0010] Another objective of this invention is to provide a sensing electrode comprising the aforementioned high light absorption electrode substrate / HOF-on-MOF photogrid electrode, the surface of which is immobilized with a DNA-functionalized specific oxidase complex structure (RCA DES, also known as a DNA-enzyme superstructure with multiple G-quadruplex sequences) constructed based on rolling circle amplification technology.

[0011] The above-mentioned method for preparing the sensing electrode includes the following steps:

[0012] The captured DNA is immobilized onto the electrode substrate / HOF-on-MOF photosensitive grid. Then, through DNA complementation reaction, the complex of specific oxidase and primer and template DNA are sequentially linked to the photosensitive grid. Subsequently, through T4 DNA ligation reaction and rolling circle amplification reaction, a complex structure of specific oxidase with multiple G-quadruplex sequences (RCA DES, also known as DNA-enzyme superstructure with multiple G-quadruplex sequences) is constructed. After binding with heme, the sensing electrode is obtained, referred to as the electrode substrate / HOF-on-MOF / RCA DES sensing electrode.

[0013] Specifically, taking glucose as an example, DNA is immobilized and captured on the HOF-on-MOF gate surface. Glucose oxidase-primer complex (GOx-pDNA, also referred to as GOx-pDNA conjugate in this invention) and template DNA and T4 DNA ligase are sequentially introduced through DNA complementarity. Rolling circle amplification is carried out under the catalysis of phi29 DNA polymerase, and a DNA-enzyme superstructure with multiple G-quadruplex sequences is generated in situ.

[0014] The G-quadruplex sequence generated by rolling circle amplification exhibits horseradish peroxidase-like catalytic activity in the presence of heme. By using GOx to catalyze the production of hydrogen peroxide from glucose, which is then used to catalyze the precipitation of the horseradish peroxidase-like enzyme, steric hindrance is created, enabling the organic photoelectrochemical transistor biosensor to detect significant changes in the signal and sensitively detect the target glucose.

[0015] Another objective of this invention is to provide an organic photoelectrochemical transistor biosensor, comprising a gate, wherein the gate is the sensing electrode after incubation with heme, and the incubation refers to incubating the high light absorption sensing electrode with heme at room temperature to induce multiple G-quadruplex sequences to exhibit horseradish peroxidase-like catalytic properties.

[0016] Furthermore, the small molecule metabolite to be tested is incubated for detection. The incubation method includes the following steps: immersing the sensing electrode in a PBS buffer solution containing the small molecule metabolite to be tested and 4-chloro-1-naphthol, and incubating at room temperature.

[0017] The application of the above-mentioned organic photoelectrochemical transistor biosensor for detecting small molecule metabolites includes the following steps: assembling the above-mentioned gate and the prefabricated organic photoelectrochemical transistor source and drain electrodes into a complete OPECT device; placing the device in an electrolytic cell containing PBS buffer (pH 7.4, 0.1 M) containing triethanolamine (0.1 M); illuminating the device with an LED light source at a wavelength of 420 nm under zero gate voltage conditions; and recording the channel current response signal under zero gate voltage conditions to achieve quantitative detection of small molecule metabolites such as glucose at different concentrations.

[0018] A multi-stage signal amplification mechanism is established, involving "photovoltaic voltage regulation - enzyme cascade catalysis - precipitation-mediated interface change": The target analyte (e.g., glucose) triggers a specific oxidase reaction to generate H₂O₂, which in turn activates the horseradish peroxidase activity of the G-quadruplex / heme DNAzyme, catalyzing the precipitation of 4-chloro-1-naphthol and forming an insulating layer at the gate interface. This precipitation layer dynamically regulates the efficiency of the grating voltage in controlling the channel current, ultimately achieving high-gain signal output via OPECT. This signal amplification mechanism combines the synergistic effect of optoelectronic device regulation and enzyme-catalyzed precipitation, exhibiting excellent signal amplification characteristics. Its system integration and application in glucose detection are the innovative aspects of this application.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0020] 1) The organic photoelectrochemical transistor biosensor based on a novel high-light-absorption HOF-on-MOF heterojunction material provided by this invention utilizes its functional interface to connect an RCA DES structure. The heterojunction generated by the HOF-MOF composite exhibits a high photocurrent response, enabling high-performance detection of small molecule metabolites such as glucose by the organic photoelectrochemical transistor biosensor, and demonstrating potential practical applications in the detection of small molecule metabolites such as glucose.

[0021] 2) The high-performance organic photoelectrochemical transistor biosensor for detecting small molecule metabolites such as glucose provided by this invention can detect small molecule metabolites such as glucose without purification, which is convenient and fast. It is a high-performance biosensor that integrates the signal amplification advantages of photoelectrochemical biosensors and organic electrochemical transistor technology. The signal amplification effect is more than five times better than that of existing technologies using inorganic materials. It not only provides an efficient sensing mode for organic photoelectrochemical transistor biosensors, but also effectively improves the accuracy of glucose detection in actual biological samples. It has profound significance for the judgment of physiological state.

[0022] 3) The method for preparing the high-performance organic photoelectrochemical transistor biosensor for glucose detection provided by the present invention has simple and efficient preparation steps, mild conditions, simple operation, and low cost. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 This is a scanning electron microscope image of the MOF in Embodiment 1 of the present invention.

[0025] Figure 2 This is a scanning electron microscope image of HOF in Embodiment 1 of the present invention.

[0026] Figure 3 This is a scanning electron microscope image of HOF-on-MOF in Embodiment 1 of the present invention.

[0027] Figure 4 This is the X-ray diffraction pattern of HOF-on-MOF in Embodiment 1 of the present invention.

[0028] Figure 5 This is the Fourier transform infrared spectrum of the HOF-on-MOF in Embodiment 1 of the present invention.

[0029] Figure 6 This is a photocurrent signal diagram of HOF-on-MOF in Embodiment 1 of the present invention.

[0030] Figure 7 This is a diagram showing the channel current and gate current signals of the ITO / HOF-on-MOF / RCA DES sensing gate in Embodiment 2 of the present invention.

[0031] Figure 8 This is a gain diagram of the ITO / HOF-on-MOF / RCA DES sensing gate in Embodiment 2 of the present invention.

[0032] Figure 9 This is a diagram of the channel current signal for glucose detection based on an ITO / HOF-on-MOF / RCA DES sensor in Embodiment 3 of the present invention.

[0033] Figure 10 This is a standard curve diagram of glucose detection based on the ITO / HOF-on-MOF / RCA DES sensor in Embodiment 3 of the present invention.

[0034] Figure 11This is a graph showing experimental data of selective identification based on the ITO / HOF-on-MOF / RCA DES sensor in Embodiment 4 of the present invention. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the embodiments.

[0036] This invention discloses a high-performance organic photoelectrochemical transistor biosensor with high sensitivity and accuracy, constructed using a high-light-absorbing HOF-on-MOF gate-bound nucleic acid-functionalized enzyme composite structure.

[0037] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0038] In some embodiments, an electrode substrate / HOF-on-MOF photosensitive gate is provided, including an electrode substrate, a titanium-based MOF layer chemically bonded to the surface of the electrode substrate, and an HOF layer chemically bonded to the surface of the titanium-based MOF layer by forming amide bonds. The titanium-based MOF is a metal-organic framework material with titanium as the metal center and an amino-rich surface, and the HOF is a hydrogen-bonded organic framework material with carboxyl groups rich in the surface. Precise anchoring can be achieved on the titanium-based MOF.

[0039] In some preferred embodiments, HOF-100 is combined with a titanium-based MOF. HOF-100 can be more precisely anchored on the titanium-based MOF, thereby further improving the high photoelectric signal intensity and stability of the constructed electrode substrate / HOF-on-MOF.

[0040] In some embodiments, a method for fabricating the electrode substrate / HOF-on-MOF photosensitive gate is also provided, comprising the following steps: coating a titanium-based MOF with an amino-rich surface onto a hydroxyl-activated electrode substrate surface (ITO or FTO), and obtaining a photoelectric active electrode substrate / MOF electrode through chemical bonding. Subsequently, a HOF material with a carboxyl-rich surface is selected, its carboxyl groups are activated using EDC / NHS, and then coated onto the surface of the aforementioned electrode substrate / MOF electrode. Covalent anchoring of the HOF on the MOF is achieved by forming amide bonds, ultimately constructing a HOF-on-MOF composite photosensitive layer with Type-II heterojunction characteristics and strong interfacial coupling, thereby obtaining an electrode substrate / HOF-on-MOF photosensitive gate with high photoelectric response.

[0041] In some preferred embodiments, plasma activation is used to enrich the surface with hydroxyl groups, which can achieve better signal amplification.

[0042] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0043] First, it should be noted that the current signal response in the experiments of this invention was tested on an organic photoelectrochemical transistor system, as detailed below:

[0044] An LED with an emission wavelength of 425 nm was used as the excitation source, and it was turned on and off every 10 s. Channel current was recorded using an electrochemical workstation. When testing the channel signal response, a photoelectrode with a modified area of ​​0.5 cm × 0.5 cm was used as the gate electrode, and the transistor source and drain were connected to the corresponding electrodes, respectively. The system operated at zero gate voltage, without any external voltage applied, and was self-powered.

[0045] For the fabrication of the channel portion of the organic photoelectrochemical transistor, the glass substrate was ultrasonically cleaned with acetone, ethanol, and deionized water for 10 min each, and then dried at 60 °C. The substrate was then further treated with plasma. Subsequently, the substrate was shielded with a template with a channel length of 6.0 mm and a width of 0.2 mm, and coated with a 10 nm chromium and a 100 nm gold target by magnetron sputtering. Next, the obtained substrate was further cleaned with plasma, and then spin-coated onto the electrodes with a mixture of 1.5 mL of PEDOT:PSS solution and 7.5 μL of 5% (v / v) dimethyl sulfoxide at 3500 rpm to form a uniform PEDOT:PSS film. Finally, the substrate was annealed at 180 °C for 1 h in an inert atmosphere to ensure a more robust adhesion of the PEDOT:PSS to the electrodes, thus obtaining the channel portion of the OPECT device.

[0046] Example 1

[0047] Materials and reagents:

[0048] The metal-organic frameworks and hydrogen-bonded organic frameworks used in this embodiment were all prepared in the laboratory.

[0049] 1. Preparation of MOF (Ti-MOF) materials based on titanium metal centers

[0050] Weigh 0.20 g of 2-aminoterephthalic acid and dissolve it in 10 mL of a 7:3 mixture of methanol and N,N-dimethylformamide. While stirring continuously, slowly add 0.3 mL of tetrabutyl titanate to the solution and sonicate at room temperature for 5 min to ensure thorough mixing. Transfer the resulting mixture to a 30 mL high-pressure reactor lined with polytetrafluoroethylene (PTFE) and react in an oven at 150 °C for 16 h. After the reaction, allow it to cool naturally to room temperature, and collect the resulting yellow precipitate by centrifugation. Wash the precipitate five times each with N,N-dimethylformamide and methanol, and finally dry it under vacuum at 80 °C for 6 h to obtain MOF powder.

[0051] 2. Preparation of HOF-100

[0052] Weigh 20 mg of 1,3,6,8-tetracarboxylic acid pyrene and add 1 mL of N,N-dimethylformamide. Dissolve the solution by stirring in an oil bath at 120 °C for 40 min to obtain a clear solution. After the solution has cooled naturally to room temperature, it is slowly added dropwise to 16 mL of acetone under magnetic stirring. After the addition is complete, stirring is continued at room temperature for 12 h to allow the crystals to grow fully. The precipitated solid product is collected by centrifugation, washed three times with acetone to remove residual solvent, and finally dried under vacuum to obtain HOF-100 powder.

[0053] 3. Fabrication of ITO / HOF-on-MOF photogate

[0054] ITO substrate pretreatment: ITO conductive glass was ultrasonically cleaned in acetone, anhydrous ethanol and deionized water for 15 min each, dried with high-purity nitrogen, and then treated with plasma for 5 min to enhance the degree of surface hydroxylation and wettability.

[0055] Preparation of MOF modified layer: 1.0 mg of self-made MOF powder was dispersed in 1.0 mL of deionized water and sonicated for 30 minutes to form a uniform 1.0 mg / mL suspension. 20.0 μL of this suspension was precisely drop-coated onto the defined active area of ​​the pretreated ITO electrode and placed horizontally at room temperature to allow the solvent to evaporate naturally, thus preparing the ITO / MOF electrode.

[0056] Covalent anchoring of the HOF layer: An activation solution containing 5.0 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 2.0 mg / mL N-hydroxysuccinimide (NHS) was prepared. 20.0 μL of this activation solution was mixed with 20 μL of a 1.0 mg / mL HOF-100 dispersion and incubated at room temperature for 15-30 min to fully activate the carboxyl groups on the HOF-101 surface. Subsequently, 20 μL of the above mixture was drop-coated onto the surface of the ITO / MOF electrode. During this process, the activated carboxyl groups on the HOF-100 surface and the amino groups on the MOF surface underwent an amidation reaction. The reaction was carried out at room temperature for 12 h to form amide bonds, thereby achieving stable anchoring of the HOF in the MOF layer, ultimately yielding an ITO / HOF-on-MOF photosensitive gate.

[0057] Scanning electron microscope images are attached. Figure 1 As shown in the figure, the MOF exhibits a disk-like morphology with an average diameter of approximately 400 nm; the scanning electron microscope image is attached. Figure 2 As shown in the figure, HOF exhibits a rod-like nanostructure. Scanning electron microscope images are attached. Figure 3 As shown in the figure, the surface of the MOF disk is covered with a layer of HOF nanorods, proving that the HOF-on-MOF photosensitive gate has been successfully fabricated.

[0058] The X-ray diffraction pattern of the HOF-on-MOF photogate is as follows: Figure 4 As shown in the figure, the diffraction peaks at 6.8°, 9.8°, and 11.6° correspond to the characteristic diffraction peaks of MOF, while 7.1° corresponds to the characteristic diffraction peak of HOF. This confirms the successful fabrication of the HOF-on-MOF photogate. The infrared spectrum of the HOF-on-MOF photogate electrode is attached. Figure 5 As shown, MOF at 500 cm -1 -800cm -1 Vibrational absorption peaks of Ti-O-Ti clusters appear at 1386 cm⁻¹, and also at 1386 cm⁻¹. -1 1623 cm -1 and 3435 cm -1 Stretching vibration peaks of CN, C=O, and NH appear at 1660 cm⁻¹, respectively. Correspondingly, HOF shows peaks at 1660 cm⁻¹. -1 and 3240 cm -1 The presence of stretching vibration peaks at C=O and OH at the locations proves the successful fabrication of the HOF-on-MOF photogate.

[0059] The photocurrent signal diagram of the HOF-on-MOF is shown below. Figure 6As shown, compared with the lower signals generated by the original MOF and HOF, the HOF-on-MOF heterojunction exhibits a significantly enhanced photocurrent signal. This result indicates that the heterojunction structure can effectively promote the separation and interface transport of photogenerated carriers, thereby effectively improving photoelectric performance.

[0060] Example 2

[0061] Fabrication of ITO / HOF-on-MOF / RCA DES sensing electrode

[0062] 20 μL of capture DNA (cDNA) was dropped onto the ITO / HOF-on-MOF photosensitive gate prepared in Example 1 and incubated at 4 °C for 12 h. Then, 20 μL of a 0.2 μM GOx-pDNA conjugate solution was added, and the reaction was carried out at room temperature for 10 min, immobilizing the conjugate on the electrode surface through DNA complementation. Next, 1 μM template was added to the above solution, and the reaction was carried out at 37 °C for 2 h. Then, 5 U / μL of T4 DNA ligase was added to the electrode, and the reaction was carried out at 37 °C for 30 min, followed by inactivation of the T4 DNA ligase at 65 °C for 10 min. Next, 5 μL of P29 DNA polymerase (2 U / μL) and 4 μL of dNTP mixture (5 mM) were added to the electrode surface, and the reaction was carried out at 30 °C for 60 min to complete the rolling circle amplification reaction, generating sequences rich in G-quadruplexes. This resulted in the in-situ construction of a DNA-enzyme superstructure (RCADES) based on multiple G-quadruplex sequences generated by rolling circle amplification on the electrode surface. This yielded a composite electrode modified with RCA DES, denoted as the "ITO / HOF-on-MOF / RCA DES electrode". Subsequently, the ITO / HOF-on-MOF / RCA DES electrode was placed in PBS buffer solution (10 mM, pH 7.4) containing 0.5 μM heme and incubated overnight at 4 °C, allowing the heme to intercalate into the G-quadruplex sequences in the RCA product, forming a DNAzyme with horseradish peroxidase-like activity. Through this step, an ITO / HOF-on-MOF / RCA DES sensing electrode that can be used for detection is finally obtained.

[0063] Preparation of GOx-pDNA conjugates

[0064] 30 μM glucose oxidase and 1.5 mM bifunctional cross-linking agent 15-azido-4,7,10,13-tetraoxopentadecanoic acid-N-succinimide ester (NHS ester) were reacted in 10 mM PBS buffer (pH 7.4) at 25 °C with gentle stirring at 300 rpm for 1 h, allowing the amino groups on the GOx surface to react with the NHS ester to form GOx-azides. After the reaction, the product was purified using a 50 kDa ultrafiltration centrifuge tube and washed six times with 10 mM PBS buffer (pH 7.4) to remove excess cross-linking agent. Subsequently, the obtained GOx-azides were mixed with dibenzocyclooctynyl-pDNA (DBCO-pDNA) at a concentration of 10 μM:100 μM and incubated at 25 °C for 24 h, forming GOx-pDNA conjugates through click chemistry. Finally, the GOx-pDNA conjugate was purified again using a 50kDa ultrafiltration centrifuge tube, and washed 6 times with 10 mM PBS buffer to remove unreacted DBCO-pDNA. The obtained GOx-pDNA conjugate was stored at 4 °C for later use.

[0065] Nucleic acid sequence design and synthesis

[0066] All nucleic acid sequences used in this invention were synthesized by Shanghai Sangon Biotech Co., Ltd. Their design and function are as follows:

[0067] cDNA sequence (5'-3'): NH2-ATC CCT ATA GTG AGT CGT ATT A; Function: The amino group at the 5' end is used for chemical covalent fixation to the surface of the ITO / HOF-on-MOF electrode via EDC / NHS.

[0068] Dibenzocyclooctyne-pDNA (DBCO-pDNA) sequence (5'-3'): DBCO-TTT TTT AAT ACGACT CAC TAT AGG GAT; Function: DBCO links to GOx, and the sequence portion is complementary to cDNA.

[0069] Template sequence (5'-3'): PO4-AGT CGT ATT AAA AAA CCA CCA CCA CCA CAA CCA CCACCA CCT TTT TTA TCC CTA TAG T; Function: To form a circular template after circularization by T4 DNA ligase.

[0070] The channel current of the prepared organic photochemical transistor biosensor system was measured as follows: Figure 7As shown, at zero gate voltage and under 425 nm illumination, the gate current is in the nanoampere range, while the channel current is in the microampere range. This phenomenon is due to the photovoltage generated at the gate-electrolyte interface inducing ion implantation into the channel, thereby effectively modulating the conductivity of the polymer channel. The corresponding current gain is calculated as follows: Figure 8 As shown, the gain exceeds 10. 3 .

[0071] Example 3

[0072] Detection of glucose using an organic photochemical transistor biosensor based on a high-absorption HOF-on-MOF gate:

[0073] The ITO / HOF-on-MOF / RCA DES sensing electrode prepared in Example 2 was transferred to 5 mL of 10 mM PBS buffer (pH 7.4), which contained a specific concentration of glucose and 10 mM 4-chloro-1-naphthol, and incubated at room temperature for 10 min. After incubation, the electrode was removed and the surface was gently rinsed with PBS buffer. The sensing electrode incubated with the target substance served as the gate electrode and was assembled with the pre-fabricated organic electrochemical transistor source and drain electrodes to form a complete OPECT device. The device was placed in an electrolytic cell containing PBS buffer (pH 7.4, 0.1 M) containing triethanolamine (0.1 M), and illuminated with an LED light source at a wavelength of 420 nm under zero gate voltage conditions. The channel current response signal was recorded under zero gate voltage conditions to achieve quantitative detection of glucose at different concentrations.

[0074] The detection results show that as the concentration of the target analyte glucose increases, the corresponding channel current signal gradually decreases, indicating that more target glucose is catalyzed by GOx to generate hydrogen peroxide, which participates in the horseradish peroxidase-like catalytic reaction. The resulting precipitation increases the steric hindrance effect, hindering charge transfer, as shown in the attached figure. Figure 9 As shown in the attached figure. Furthermore, within the target glucose concentration range of 1 µM to 10 mM, the channel current change rate exhibits a linear relationship with the logarithm of the glucose concentration, as shown in the attached figure. Figure 10 As shown, the linear fitting equation is ΔI / I0 = 0.8045 + 0.1161logC (M), the linear correlation coefficient is 0.9987, and the experimental limit of detection is 0.34 µM. This indicates that the organic photoelectrochemical transistor biosensor based on a high light absorption HOF-on-MOF gate prepared by the present invention has high sensitivity for glucose detection.

[0075] Methods for detecting glucose of unknown concentration:

[0076] An unknown concentration of glucose solution was diluted in 5 mL of 10 mM PBS buffer (pH 7.4) containing 10 mM 4-chloro-1-naphthol and incubated at room temperature for 10 minutes. After incubation, the electrode was removed from the solution and its surface was gently rinsed with PBS buffer. The glucose-incubated sensing electrode served as the gate and was assembled with the pre-fabricated source-drain electrodes of an organic electrochemical transistor to form a complete OPECT device. The device was then placed in an electrolytic cell containing 0.1 M triethanolamine in PBS buffer (pH 7.4, 0.1 M) and illuminated with a 420 nm LED light source under zero gate voltage conditions to collect the channel current response signal. Substituting this into the linear fitting equation described above, the diluted glucose concentration could be calculated. Finally, the diluted concentration was multiplied by the corresponding dilution factor to obtain the concentration of the unknown glucose.

[0077] This method can accurately detect glucose solutions of unknown concentrations and exhibits good quantitative ability through linearity, making it suitable for the determination of glucose concentration in biological samples.

[0078] Glucose, as an essential energy source for life activities, is a key monosaccharide for maintaining metabolic homeostasis. Its main physiological functions include cellular energy supply and basic metabolic processes such as the synthesis of biomolecules. Simultaneously, glucose plays a crucial role in maintaining the normal function of the central nervous system and regulating metabolic balance. Studies have shown that various disease states, such as diabetes, hypoglycemia, and metabolic syndrome, are closely related to abnormal glucose metabolism. Therefore, accurate detection of glucose content in biological systems is of great significance for related metabolic research and health monitoring. Based on this, this invention selects glucose as a model detection target to verify the feasibility and detection performance of the constructed sensing system in the detection of small biomolecules. The sensor described in this invention is a modular biosensing platform, and its detection target is not limited to glucose. By replacing DNA-functionalized glucose oxidase with other specific oxidases, the detection of various small molecule metabolites can be achieved. For example, replacing glucose oxidase with lactate oxidase allows for lactate detection; replacing it with cholesterol oxidase allows for cholesterol detection; and replacing it with uric acid oxidase allows for uric acid detection. These oxidases (specific oxidases) can all catalyze the production of hydrogen peroxide from their respective substrates, thus reusing the rolling circle amplification and G-quadruplex / heme catalytic precipitation signal amplification system and OPECT signal readout mechanism established in this invention. This demonstrates the versatility and scalability of the sensing platform of this invention in both design and application.

[0079] Example 4:

[0080] Selectivity performance testing of organic photochemical transistor biosensors based on high-performance HOF-on-MOF gates:

[0081] To demonstrate the excellent selectivity of the organic photochemical transistor biosensor based on the high light absorption performance HOF-on-MOF gate, common small molecules—fructose (Fru), sucrose (Suc), lactic acid (LA), uric acid (UA), L-cysteine ​​(L-Cys), and dopamine (DA)—were selected as typical interfering agents for verification experiments. The specific procedures are as follows:

[0082] 1 mM Glu, 10 mM Fru, Suc, LA, UA, L-Cys, and DA were added separately or in combination to the detection solution, and the organic photoelectrochemical transistor biosensor prepared in this invention was used to detect them according to the above method. The channel current signal change rate is shown in the attached figure. Figure 11 As shown in the figure, only the target molecule Glu and the mixture can produce a significant photocurrent response. This demonstrates that the organic photoelectrochemical transistor biosensor prepared in this invention has excellent selective recognition ability for the target molecule glucose, unaffected by interfering molecules.

Claims

1. An electrode substrate / HOF-on-MOF photosensitive gate, characterized in that, It includes an electrode substrate, a titanium-based MOF layer chemically bonded to the surface of the electrode substrate, and an HOF layer chemically bonded to the surface of the titanium-based MOF layer by forming amide bonds. The titanium-based MOF is a metal-organic framework material with titanium as the metal center and an amino-rich surface, and the HOF is a hydrogen-bonded organic framework material with carboxyl groups rich in the surface.

2. The electrode substrate / HOF-on-MOF photogate according to claim 1, characterized in that, The HOF material is HOF-100.

3. A method for fabricating the electrode substrate / HOF-on-MOF photosensitive gate as described in claim 1 or 2, characterized in that, The process includes the following steps: coating a titanium-based MOF rich in amino groups onto the surface of a hydroxyl-activated electrode substrate, and obtaining a photoelectric active electrode substrate / MOF electrode through chemical bonding; subsequently, selecting a HOF material rich in carboxyl groups, activating its carboxyl groups using EDC / NHS, and coating it onto the surface of the aforementioned electrode substrate / MOF electrode, thereby achieving covalent anchoring of the HOF on the MOF through the formation of amide bonds, and obtaining a high photoelectric response electrode substrate / HOF-on-MOF photosensitive gate.

4. The method for fabricating the electrode substrate / HOF-on-MOF photosensitive gate according to claim 3, characterized in that, The process also includes the following steps: First, the electrode substrate is cleaned to enrich its surface with hydroxyl groups; then, a titanium-based MOF solution is coated onto the electrode substrate, where the titanium centers condense with the hydroxyl groups on the electrode substrate surface to form Ti-OM interfacial chemical bonds, where M is In or Sn; the carboxyl groups on the surface of the HOF material are activated using a system of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then coated onto a titanium-based MOF layer rich in amino groups.

5. A sensing electrode, characterized in that, The electrode substrate / HOF-on-MOF photosensitive gate according to claim 1 or 2 has a DNA-functionalized specific oxidase complex structure RCA DES constructed based on rolling circle amplification technology immobilized on its surface.

6. A method for preparing a sensing electrode as described in claim 5, characterized in that, The process includes the following steps: capturing DNA is immobilized onto the electrode substrate / HOF-on-MOF photosensitive grid, and then the specific oxidase-primer complex and template DNA are sequentially linked to the photosensitive grid through a DNA complementation reaction. After that, an RCA DES is constructed through a T4 DNase ligation reaction and a rolling circle amplification reaction. Finally, after binding heme, the sensing electrode is obtained.

7. An organic photoelectrochemical transistor biosensor, characterized in that, The sensing electrode of the sensor is the sensing electrode as described in claim 6.

8. A method for detecting small molecule metabolites, characterized in that: Based on the organic photoelectrochemical transistor biosensor as described in claim 7.

9. The method for detecting small molecule metabolites according to claim 8, characterized in that, Includes the following steps: 1) Prepare PBS buffer solutions with pH 7.4 containing different concentrations of small molecule metabolites, and add 4-chloro-1-naphthol. Immerse the sensing electrodes in the buffer solution and incubate at room temperature. After incubation, remove the sensing electrodes and gently rinse the surface with PBS buffer solution. Assemble them with the source and drain electrodes of the pre-fabricated organic electrochemical transistor to form a complete OPECT device. Place the device in an electrolytic cell containing PBS buffer solution with pH 7.4 containing triethanolamine. Irradiate with an LED light source with a wavelength of 420 nm under zero gate voltage conditions. Record the channel current response signal under zero gate voltage conditions and construct a linear equation between the channel current change rate and the logarithm of the small molecule metabolite. 2) Prepare a PBS buffer solution with pH 7.4 containing the small molecule metabolite to be tested, and add 4-chloro-1-naphthol. Immerse the sensing electrode in the buffer solution and incubate at room temperature. After incubation, repeat the steps in step 1) to obtain the channel current. Calculate the concentration of the small molecule metabolite in the solution containing the small molecule metabolite to be tested by substituting it into the linear equation.

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