Biosensor, preparation method thereof and neurotransmitter detection system

Abstract

The invention provides a biosensor, a preparation method thereof and a neurotransmitter detection system. The biosensor comprises a substrate and a plurality of sensing units arranged on the substrate, wherein each sensing unit comprises an electrode pair, a boehmite functional layer, a functional organic molecule and an aptamer molecule. The electrode pair comprises a first electrode and a second electrode which are oppositely arranged to form a gap area. The boehmite functional layer is arranged in the gap area so as to connect the first electrode and the second electrode. The functional organic molecules are covalently connected with hydroxyl functional groups of the boehmite functional layer. And the aptamer molecule is covalently linked with the functional organic molecule. The aptamer molecules of different sensing units have different spatial configurations. According to the application, a plurality of sensing units are integrated and aptamer molecules of different sensing units are endowed with differential spatial configurations, so that the core problems of serious cross interference and incapability of synchronously detecting multiple targets caused by similar target molecular structures in multi-neurotransmitter detection of a traditional sensor are effectively solved.

Description

Technical Field

[0001] This application relates to the field of neurotransmitter detection technology, and more specifically, to a biosensor and its preparation method, and a neurotransmitter detection system. Background Technology

[0002] Accurate detection of neurotransmitters such as dopamine and serotonin is crucial for the early diagnosis of neurodegenerative diseases such as Parkinson's disease and depression. Clinical studies have confirmed that an abnormal dopamine to serotonin concentration ratio can serve as a highly sensitive biomarker for Parkinson's disease. However, practical detection faces three major challenges: First, neurological diseases require simultaneous monitoring of multiple neurotransmitters with highly similar structures (e.g., dopamine and serotonin differ by only one hydroxyl group), and single-indicator detection is prone to high misdiagnosis rates. Second, patients can provide very small amounts of cerebrospinal fluid or serum samples, usually less than 10 μL, requiring detection technologies with ultra-low sample consumption capabilities. Finally, neurotransmitter release is transient, with synaptic events lasting less than 5 seconds, and the detection response must be controlled within 3 seconds to capture key physiological signals. Current technologies struggle to meet these stringent requirements simultaneously.

[0003] To address these challenges, current mainstream technologies include electrochemical sensors, fluorescence detection, and high-performance liquid chromatography (HPLC), but all have fundamental drawbacks. While electrochemical sensors are low-cost and can achieve sensitivity up to 10 pM, they rely on redox reactions and require time-sequential detection of different neurotransmitters, resulting in sample consumption exceeding 50 µL per analysis, far exceeding clinically acceptable limits. Furthermore, the shielding layer formed by electrolyte ions on the electrode surface leads to excessively high cross-interference rates and long response times. Fluorescence detection, while offering high signal-to-noise ratios and strong specificity, requires different probes for multi-target detection, making it complex, time-consuming, and expensive, with sample consumption still reaching 20 to 30 µL. HPLC, while offering high resolution, suffers from excessively long sample pretreatment and chromatographic separation times, making it completely unsuitable for real-time monitoring. Additionally, it requires a large sample size per analysis, resulting in extremely low clinical applicability.

[0004] It is evident that while traditional techniques each have their advantages in single-index detection, they all possess certain limitations. Specifically, electrochemical sensors and fluorescence methods rely on external switching mechanisms and fail to establish synergy between molecular conformation and the detection interface. High-performance liquid chromatography (HPLC) simplifies multi-index detection to a physical separation process, failing to address signal confusion caused by similar neurotransmitter structures, resulting in high cross-interference rates and excessive sample consumption. Therefore, the core technical challenge urgently needing resolution in this field is: how to achieve specific parallel detection of multiple neurotransmitters using a single detection platform, i.e., a single sensor. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a biosensor and its preparation method, and a neurotransmitter detection system, so as to improve the technical problem that traditional neurotransmitter detection methods cannot achieve parallel and synchronous detection of multiple neurotransmitters using a single sensor.

[0006] To achieve the above objectives, the technical solutions adopted in the embodiments of this application are as follows: In a first aspect, this application provides a biosensor, including a substrate and a plurality of sensing units disposed on the substrate, each of the sensing units comprising: An electrode pair, comprising a first electrode and a second electrode disposed opposite to each other to form a gap region; A boehmite functional layer is disposed in the gap region to connect the first electrode and the second electrode; Functional organic molecules are covalently linked to the hydroxyl functional groups of the boehmite functional layer; The aptamer molecule is covalently linked to the aforementioned functional organic molecule; The aptamer molecules of different sensing units have different spatial configurations.

[0007] Optionally, the electrode pairs of the plurality of sensing units are arranged together in a comb-like layout, and in the plurality of sensing units, the plurality of first electrodes and the plurality of second electrodes are arranged alternately in parallel strips along a first direction, the plurality of first electrodes are arranged at intervals along a second direction, the plurality of second electrodes are arranged at intervals along the second direction, and the first direction and the second direction are perpendicular.

[0008] Optionally, the plurality of sensing units includes a first sensing unit, wherein the functional organic molecule in the first sensing unit includes a first organic molecule, the end of the first organic molecule is a carboxyl group, and the carboxyl group is connected to the hydroxyl functional group of the boehmite functional layer through an ester bond.

[0009] Optionally, the aptamer molecule in the first sensing unit includes a first aptamer, which is connected to the first organic molecule.

[0010] Optionally, the plurality of sensing units includes a second sensing unit, wherein the functional organic molecule in the second sensing unit includes a second organic molecule, the end of which is an amino group, and the amino group is connected to the hydroxyl functional group of the boehmite functional layer through an amide bond.

[0011] Optionally, the aptamer molecule in the second sensing unit includes a second aptamer, which is connected to the second organic molecule.

[0012] Optionally, the first aptamer specifically binds to dopamine, and the second aptamer specifically binds to serotonin; The first ratio is defined as the proportion of the number of effective binding sites for dopamine in the first aptamer to the total number of binding sites in the first aptamer, and the second ratio is defined as the proportion of the number of effective binding sites for serotonin in the second aptamer to the total number of binding sites in the second aptamer, wherein the first ratio is greater than the second ratio.

[0013] Optionally, a central electrode is provided between the first electrode and the second electrode. One end of the central electrode is spaced apart from the first electrode to form a first sub-gap, and the other end is spaced apart from the second electrode to form a second sub-gap. The boehmite functional layer is disposed within the first sub-gap and the second sub-gap.

[0014] Optionally, the boehmite functional layer has a gradient thickness distribution perpendicular to the extension direction of the electrode pair; The thickness of the boehmite functional layer gradually decreases from the edge of the gap region toward the center of the gap region.

[0015] Optionally, an insulating barrier is provided between adjacent sensing units.

[0016] Optionally, the inner surface of the insulating barrier is provided with a plurality of grooves, which are arranged in an array and filled with a hydrophobic material.

[0017] Secondly, embodiments of this application provide a method for preparing a biosensor, comprising: Multiple sensing units are formed on a substrate. Each sensing unit includes an electrode pair, which includes a first electrode and a second electrode disposed opposite to each other to form a gap region. Selective deposition of boehmite functional layers in the gap region of each electrode pair; Functional organic molecules are covalently linked to the boehmite functional layer; An aptamer molecule is covalently linked to the functional organic molecule; Among them, the aptamer molecules of different sensing units have different spatial configurations.

[0018] Optionally, the step of forming a plurality of sensing units on a substrate, each sensing unit including an electrode pair, the electrode pair including a first electrode and a second electrode disposed opposite to each other to form a gap region, includes: Photoresist is spin-coated onto the substrate; The photoresist is patterned using a maskless laser direct writing device to define the electrode pattern; A photoresist template with electrode openings is formed by dissolving the photoresist in the exposed or unexposed areas with a developer solution. A metal layer is deposited on the surface of the photoresist template, so that the metal simultaneously covers the exposed area of ​​the substrate and the photoresist surface; Subsequently, through a stripping process, the photoresist that was not removed by development and the metal attached to its surface are dissolved and removed together, leaving only the metal pattern deposited on the substrate, thereby obtaining an electrode pair composed of the first electrode and the second electrode.

[0019] Thirdly, embodiments of this application provide a neurotransmitter detection system, comprising: Biosensors as described in the first aspect; A microfluidic chip, connected to the biosensor, is used to deliver biological samples; A signal processing unit, connected to the biosensor, is used to process the detection signal; The display unit, connected to the signal processing unit, is used to display the detection results of the neurotransmitter detection system.

[0020] In the biosensors described in the first aspect above, the following can be understood: The biosensor provided in this application effectively solves the core problems of traditional sensors in the detection of multiple neurotransmitters, namely, severe cross-interference caused by the similarity of target molecule structures and the inability to detect multiple targets simultaneously, by integrating multiple sensing units and giving different sensing units different spatial configurations of aptamer molecules. This significantly improves the detection specificity and multi-target analysis efficiency. In detail, the sensor has multiple independent sensing units on a substrate. Each unit contains a first electrode and a second electrode arranged opposite each other to form a gap region. A boehmite functional layer is provided in the gap region to electrically connect the electrodes. Functional organic molecules are covalently anchored to the hydroxyl functional groups of the boehmite functional layer. Aptamer molecules are then covalently linked to the functional organic molecules to form a recognition interface. The aptamer molecules of different sensing units have different spatial configurations, so that the molecular recognition interface of each unit has a unique three-dimensional orientation. Thus, when the sample flows through, the sensor can accurately distinguish structurally similar target molecules by the difference in geometric matching degree, reducing the cross-interference rate. At the same time, the parallel layout of multiple sensing units can simultaneously capture multiple target signals without additional operation, realizing high sensitivity and high throughput analysis of multiple neurotransmitters in a single detection, and completely overcoming the defects of false detection and inefficiency caused by traditional sensors that rely on a single aptamer configuration.

[0021] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, embodiments of this application are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of a biosensor provided in an embodiment of this application from a first-view perspective; Figure 2 This is a schematic diagram of the structure of a biosensor provided in an embodiment of this application from a second perspective; Figure 3 A schematic diagram of the structure of another biosensor provided in the embodiments of this application from a second perspective; Figure 4 This is a schematic diagram of the structure of a sensing unit in a biosensor provided in an embodiment of this application; Figure 5 This is a schematic diagram of the structure of another sensing unit in a biosensor provided in an embodiment of this application; Figure 6 A schematic diagram of another biosensor provided in this application embodiment; Figure 7 for Figure 6 Enlarged schematic diagram of the insulating retaining wall; Figure 8 A schematic flowchart illustrating a method for fabricating a biosensor provided in an embodiment of this application; Figure 9 This is a schematic diagram of a neurotransmitter detection system provided in an embodiment of this application.

[0024] Explanation of reference numerals in the attached figures: 10. Base; 20. Sensing unit; 201. First sensing unit; 202. Second sensing unit; 21. Electrode pair; 211. First electrode; 212. Second electrode; 213. Central electrode; 22. Boehmite functional layer; 23. Functional organic molecule; 231. First organic molecule; 232. Second organic molecule; 24. Aptamer molecule; 241. First aptamer; 242. Second aptamer; 30. Insulating barrier; 301. Groove; 302. Hydrophobic material. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0026] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0027] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0028] It should be noted that, where there is no conflict, the features in the embodiments of this application can be combined with each other.

[0029] This application provides a technical solution including: a biosensor and its preparation method, and a neurotransmitter detection system. The technical solution provided in this application will be described below with reference to the accompanying drawings.

[0030] First, please see Figure 1 and Figure 2 This application introduces a biosensor provided in an embodiment, including a substrate 10 and a plurality of sensing units 20 disposed on the substrate 10. Each sensing unit 20 includes an electrode pair 21, a boehmite functional layer 22, a functional organic molecule 23, and an aptamer molecule 24.

[0031] The electrode pair 21 includes a first electrode 211 and a second electrode 212 disposed opposite to each other to form a gap region. A boehmite functional layer 22 is disposed within the gap region to connect the first electrode 211 and the second electrode 212. A functional organic molecule 23 is covalently bonded to the hydroxyl functional groups of the boehmite functional layer 22. An aptamer molecule 24 is covalently bonded to the functional organic molecule 23. The aptamer molecules 24 of different sensing units 20 have different spatial configurations.

[0032] The technical solution provided in this application effectively solves the core problems of severe cross-interference and inability to simultaneously detect multiple targets caused by the similarity of target molecule structures in traditional sensors when detecting multiple neurotransmitters by integrating multiple sensing units 20 and giving different aptamer molecules 24 to different sensing units 20, thereby significantly improving detection specificity and multi-target analysis efficiency. In detail, the sensor has multiple independent sensing units 20 on the substrate 10. Each unit includes a first electrode 211 and a second electrode 212 arranged opposite to each other and forming a gap region. A boehmite functional layer 22 is provided in the gap region to electrically connect the electrodes. Functional organic molecules 23 are covalently anchored to the hydroxyl functional groups of the boehmite functional layer 22. Aptamer molecules 24 are then covalently connected to the functional organic molecules 23 to form a recognition interface. The aptamer molecules 24 of different sensing units 20 have different spatial configurations, so that the molecular recognition interface of each unit has a unique three-dimensional orientation. Thus, when the sample flows through, the target molecules with similar structures can be accurately distinguished by the difference in geometric matching degree, reducing the cross-interference rate. At the same time, the parallel layout of multiple sensing units 20 can simultaneously capture multiple target signals without additional operation, realizing high sensitivity and high throughput analysis of multiple neurotransmitters in a single detection, and completely overcoming the defects of false detection and inefficiency caused by traditional sensors relying on a single aptamer configuration.

[0033] It should be noted that the biosensors provided in this application can be metal oxide semiconductor resistive sensors, conductive polymer sensors, carbon nanomaterial (such as graphene, carbon nanotube) based sensors, and memristor-type biosensors. These sensors all achieve detection by detecting changes in the interfacial electrical properties (such as resistance, conductance, or impedance) caused by the binding of target molecules to recognition elements (such as aptamers).

[0034] The memristor-type biosensor is a preferred embodiment of this invention. As the fourth basic passive circuit element after resistors, capacitors, and inductors, the core characteristic of a memristor is that its resistance state depends not only on the currently applied voltage or current but also on the historical electrical excitation process, and it can non-volatilely maintain this state after power is turned off. In electrical testing, memristors exhibit typical "pinched hysteresis I-V characteristics"—that is, under bipolar voltage scanning, the current-voltage curve forms a closed hysteresis loop near the origin, and as the scanning frequency increases, the hysteresis loop area gradually shrinks and tends to close.

[0035] In the memristor-type biosensor of this application, the boehmite functional layer serves as the switching medium, containing migratable oxygen vacancies or proton defects. When a sufficiently high electric field is applied between the first and second electrodes, these defects undergo directional migration, leading to the formation or breakage of local conductive filaments, thereby achieving a reversible switching between a high-resistivity state and a low-resistivity state. More importantly, when a target neurotransmitter (such as dopamine or serotonin) specifically binds to an aptamer immobilized on the boehmite surface, it introduces an additional dipole moment or alters the local dielectric environment at the sensing interface, thereby modulating the energy barrier required for oxygen vacancy migration. This effect is directly reflected as a positional shift, for example, from −0.6 V to −0.4 V. Since this shift is related to the target molecule concentration and the response process is reversible (threshold recovery after buffer rinsing), highly sensitive, reproducible, and label-free quantitative detection of neurotransmitters can be achieved.

[0036] The memristor-based biosensor of this application not only exhibits a stable on / off ratio in physiological buffer solution but also maintains resistance stability after multiple read / write cycles, fully demonstrating its unique advantages as an intelligent bioelectronic device integrating sensing, storage, and signal processing. Compared to traditional resistive sensors that only provide a single resistance reading, the memristor-based biosensor provides richer and more reliable information dimensions for the dynamic analysis of neurochemical signals through multi-dimensional electrical parameters such as threshold voltage shift, hysteresis loop change, and resistance retention time variation.

[0037] In some embodiments, the boehmite functional layer 22 is alumina hydrate with the chemical formula AlOOH, a nanomaterial with a high specific surface area and abundant hydroxyl functional groups, which can serve as active sites for covalently linking functional organic molecules 23. The first and second electrodes form a gap region of at least 5 μm in the horizontal direction, and the boehmite functional layer 22 fills this gap region. The thickness of the boehmite functional layer 22 ranges from 30 to 50 nm. It should be particularly noted that although the Debye length is only about 0.7–0.8 nm in typical physiological buffer solutions (such as phosphate buffer containing 150 mM NaCl, pH=7.4), significantly inhibiting long-range electrostatic interactions between charged biomolecules (such as dopamine and serotonin) and the sensing interface, the detection mechanism of this invention does not rely on such long-distance electric field coupling.

[0038] Specifically, after the target neurotransmitter diffuses to the sensing surface via Brownian motion or microfluidic-driven diffusion, it binds to its specific aptamer through short-range non-covalent interactions, typically less than 1 nm, achieving high affinity binding. This chemical recognition process occurs at the molecular-scale interface and is unaffected by the Debye shielding effect caused by ionic strength in solution. By combining specific aptamer recognition with a solid-state memristor switching mechanism, the limitations of Debye shielding on biosensing are successfully bypassed, enabling highly sensitive real-time detection of neurotransmitters in complex samples with high ionic strength, such as undiluted organoid culture media and cerebrospinal fluid.

[0039] It should be noted that the Debye length refers to the characteristic length in an electrolyte solution that describes the charge shielding effect, and its value is determined by the ionic strength of the solution.

[0040] In some embodiments, see Figure 2 The electrode pairs 21 of multiple sensing units 20 collectively form a comb-like layout. Within each sensing unit 20, multiple first electrodes 211 and multiple second electrodes 212 are arranged alternately in parallel strips along a first direction, forming an interlaced structure similar to comb teeth. Simultaneously, the multiple first electrodes 211 are spaced apart along a second direction perpendicular to the first direction, and the multiple second electrodes 212 are also spaced apart along the second direction. Here, "comb-like layout" refers to an electrode arrangement resembling the teeth of a comb. The first electrodes 211 and second electrodes 212 act as "comb teeth," closely interlaced in the first direction, while maintaining a regular interval in the second direction, thereby constructing a two-dimensional gridded sensing area on the sensor substrate 10.

[0041] Specifically, the alternating strip structure of the first electrode 211 and the second electrode 212 forms a continuous electric field interaction region in the first direction, while the spaced arrangement along the second direction effectively isolates the signal paths of adjacent sensing units 20 in the second direction. For example, in a physiological buffer environment, this layout makes the electric field distribution of each sensing unit 20 in the second direction independent, effectively reducing the problem of crosstalk among multiple sensing units 20. At the same time, the comb-like structure significantly increases the electrode-solution interface per unit area, which is beneficial to improving the effective sensing area and making it easier for neurotransmitter molecules to diffuse to the sensing interface, thereby improving detection efficiency.

[0042] In addition, the alternating arrangement of the first electrode 211 and the second electrode 212 in the first direction can be further defined such that the spacing between the first electrode 211 and the second electrode 212 in a sensing unit 20 is smaller than the spacing between adjacent first electrodes 211 and second electrodes 212 in two adjacent sensing units 20 in the first direction, thereby reducing the crosstalk problem between adjacent sensing units 20 in the first direction.

[0043] In some embodiments, see Figure 1 and Figure 2 Multiple sensing units 20 include a first sensing unit 201. The functional organic molecule 23 in the first sensing unit 201 includes a first organic molecule 231, the terminal of which is a carboxyl group. This carboxyl group is connected to the hydroxyl functional group of the boehmite functional layer 22 via an ester bond. It should be noted that the ester bond connection refers to a stable chemical bond formed through a dehydration condensation reaction, rather than physical adsorption or non-covalent bonding. Its formation mechanism depends on the activation of the hydroxyl groups in the boehmite functional layer 22 and the directional reaction of the carboxyl groups in the first organic molecule 231.

[0044] Exemplarily, during the fabrication of the biosensor, the boehmite functional layer 22 undergoes surface treatment to activate hydroxyl functional groups. Subsequently, a first organic molecule 231 (e.g., an aptamer molecule 24 with a carboxyl terminus or a small molecule ligand) is introduced into the interstitial region. This allows the carboxyl groups to undergo a specific esterification reaction with the activated hydroxyl groups, ensuring that the first organic molecule 231 is uniformly anchored on the boehmite surface in a monolayer form. The carboxyl groups are oriented towards the interstitial region, providing precise binding sites for subsequent target analytes, such as neurotransmitters. This embodiment achieves molecular-level stability through a chemical bonding mechanism: the bond energy of ester bonds is significantly higher than that of hydrogen bonds or van der Waals forces, allowing the first organic molecule 231 to maintain a rigid configuration in physiological buffer solution, avoiding signal drift caused by conformational fluctuations.

[0045] Further, please see Figures 1 to 5 The aptamer molecule 24 in the first sensing unit 201 includes a first aptamer 241, which is connected to a first organic molecule 231. Specifically, the first aptamer 241 in the first sensing unit 201 is stably connected to the carboxyl group of the first organic molecule 231 via a covalent bond. The first aptamer 241 is a single-stranded DNA molecule designed for the target neurotransmitter. After one end is modified with an amino group, it undergoes a directional amidation reaction with the carboxyl group at the end of the first organic molecule 231 under the action of a cross-linking agent, forming a strong chemical bond. This ensures that the first aptamer 241 maintains precise spatial orientation and structural integrity in the physiological buffer environment, effectively avoiding signal drift caused by molecule shedding or conformational changes, and significantly improving the recognition specificity and binding efficiency of the target neurotransmitter.

[0046] Furthermore, the multiple sensing units 20 include a second sensing unit 202. The functional organic molecule 23 in the second sensing unit 202 includes a second organic molecule 232, the second organic molecule 232 having an amino group at its terminal. This amino group is connected to the hydroxyl functional group of the boehmite functional layer 22 via an amide bond. Specifically, the amino group is covalently linked to the hydroxyl functional group (-OH) on the surface of the boehmite functional layer 22 via an amide bond (-CONH-). This connection process employs a standard cross-linking process. After the boehmite functional layer 22 is activated using an EDC / NHS system, a solution containing the amino-terminated second organic molecule 232 is introduced. The reaction is carried out at room temperature for 30 minutes, allowing the amino group and the activated hydroxyl group to form a stable amide bond. This ensures that the second organic molecule 232 maintains a rigid configuration in the physiological buffer environment, effectively preventing molecule shedding or conformational fluctuations. This provides a reliable basis for the subsequent directional anchoring of the second aptamer 242, enhancing the specificity of recognition for specific neurotransmitters.

[0047] Similarly, please see Figures 1 to 5 The aptamer molecule 24 in the second sensing unit 202 includes a second aptamer 242, which is connected to a second organic molecule 232. Specifically, after one end of the second aptamer 242 is modified with a carboxyl group, it reacts directionally with the amino group at the end of the second organic molecule 232 in a physiological buffer solution at room temperature for a certain period of time, such as 20 minutes, under the action of the EDC / NHS crosslinking system, to form a bond with stable amide bond. This ensures that the second aptamer 242 maintains precise spatial orientation and structural integrity in the physiological environment, effectively avoiding molecule shedding or conformational fluctuations, thereby improving the recognition specificity of specific neurotransmitters.

[0048] In some embodiments, the first aptamer 241 specifically binds to dopamine, and the second aptamer 242 specifically binds to serotonin. Of course, the binding of the first aptamer 241 to dopamine and the binding of the second aptamer 242 to serotonin are merely examples of this embodiment. In other embodiments, the first aptamer 241 and the second aptamer 242 may also bind to other neurotransmitters, as long as the neurotransmitters bound to the first aptamer 241 and the second aptamer 242 are different. The ratio of the number of effective binding sites for dopamine in the first aptamer 241 to the total number of binding sites in the first aptamer 241 is a first ratio, and the ratio of the number of effective binding sites for serotonin in the second aptamer 242 to the total number of binding sites in the second aptamer 242 is a second ratio, wherein the first ratio is greater than the second ratio.

[0049] For example, the first aptamer 241 specifically binds to dopamine molecules, and the second aptamer 242 specifically binds to serotonin molecules. Because dopamine molecules have a rigid benzene ring structure, they can achieve efficient directional matching with the binding site of the first aptamer 241. However, serotonin molecules, due to their flexible indole ring, require greater conformational adjustments, resulting in some binding sites failing to effectively participate in recognition. Therefore, this embodiment improves the response sensitivity and specificity of dopamine detection by optimizing the utilization rate of the binding sites of the first aptamer 241. Specifically, the proportion of effective binding sites in the first aptamer 241 to the total number of binding sites is 85%, and the proportion of effective binding sites in the second aptamer 242 to the total number of binding sites is 70%. The first proportion is higher than the second proportion, making the detection limit of dopamine much lower than that of serotonin, thereby significantly reducing the cross-interference rate between dopamine and serotonin, effectively avoiding signal crosstalk between neurotransmitters in complex biological samples, and meeting the reliability requirements of simultaneous multi-target detection in clinical settings.

[0050] It should be noted that the technical solution of reducing crosstalk in multi-neurotransmitter detection by setting the ratio of effective binding sites in the first aptamer 241 and the second aptamer 242 is applicable not only to dopamine and serotonin but also to the simultaneous detection of other neurotransmitters. Of course, the relationship between the first and second ratios is mainly determined by the structure and characteristics of the neurotransmitters being detected. In this embodiment, because dopamine and serotonin are being detected, the first ratio needs to be greater than the second ratio. In other embodiments, the first ratio can also be set to be greater than the second ratio as needed, without limitation.

[0051] In some embodiments, see Figure 5 A central electrode 213 is provided between the first electrode 211 and the second electrode 212. One end of the central electrode 213 is spaced from the first electrode 211 to form a first sub-gap, and the other end is spaced from the second electrode 212 to form a second sub-gap. The boehmite functional layer 22 is disposed within the first and second sub-gap. In this embodiment, by introducing the central electrode 213, a single sensing unit 20 is divided into two independent memristor channels, ensuring that the boehmite functional layer 22 forms a Debye shielding effect suppression region in the physiological buffer solution, thereby effectively suppressing the interference of ion shielding on charge transport. At the same time, the central electrode 213 serves as a differential reference point, which can cancel environmental noise and baseline drift in real time, improving the signal-to-noise ratio and detection stability of the sensor.

[0052] Further, please see Figure 5The boehmite functional layer 22 deposited in the first sub-gap formed between the first electrode 211 and the central electrode 213 is modified with a first aptamer 241 to specifically bind dopamine molecules, while the boehmite functional layer 22 deposited in the second sub-gap formed between the central electrode 213 and the second electrode 212 is modified with a second aptamer 242 to specifically bind serotonin molecules. Based on the inherent characteristics of the boehmite functional layer 22, the two sensing channels are ensured to be electrically independent, while sharing the central electrode 213 as a differential reference point. By synchronously acquiring the resistive switching signals of the first and second sub-gap, environmental noise and baseline drift are effectively canceled, and signal crosstalk caused by electrode sharing is avoided. Thus, synchronous and high-precision detection of two neurotransmitters is achieved within a single sensing unit 20.

[0053] Please see Figure 3 In some embodiments, a sensing unit 20 may include multiple electrode pairs arranged in an array, and a central electrode 213 having multiple first ends and multiple second ends. Each first end is spaced apart from a first electrode 211 to form a first sub-gap, and each second end is spaced apart from a second electrode 212 to form a second sub-gap. Each first sub-gap and each second sub-gap contains a boehmite functional layer 22. Compared with the previous embodiment where each electrode pair has one central electrode 213, this embodiment has a simpler structure. In a sensing unit 20, only one central electrode 213 is needed to connect multiple electrode pairs, and each electrode pair can form two sub-gap, effectively increasing the number of boehmite functional layers 22 that can be provided.

[0054] In some embodiments, the boehmite functional layer 22 exhibits a gradient thickness distribution along the direction perpendicular to the extension of the electrode pair 21; the thickness of the boehmite functional layer 22 gradually decreases from the edge of the gap region toward the center of the gap region. Based on the principles of electric field modulation and ion transport optimization, the thicker boehmite functional layer 22 at the edge provides structural stability and effectively suppresses edge leakage current and non-specific adsorption, while the thinner boehmite functional layer 22 at the center enhances the local electric field strength due to the capacitance effect when a working bias voltage is applied, thereby improving the binding dynamic efficiency of neurotransmitter molecules and functionalized aptamers.

[0055] In some embodiments, see Figure 6An insulating barrier 30 is provided between adjacent sensing units 20. This insulating barrier 30 can be made of silicon dioxide and is formed on the surface of the substrate 10 using photolithography and dry etching processes. Its height can be controlled and selected according to actual conditions to ensure complete coverage of the boehmite functional layer 22 region and extension to the edge of the sensing unit 20. In this embodiment, based on the principles of electrochemical isolation and physical barrier, the diffusion path of neurotransmitter molecules and ion migration channels between adjacent sensing units 20 are effectively blocked, avoiding signal crosstalk caused by electric field coupling or cross-contamination.

[0056] Further, please see Figure 7 The inner surface of the insulating barrier 30 is provided with multiple grooves 301, which are arranged in an array. Each groove 301 is filled with a hydrophobic material 302. For example, the depth of the grooves 301 is controlled within the range of 50 to 100 nm, and they are filled with plasma-treated silane-based hydrophobic material 302. The structure of the grooves 301 filled with hydrophobic material 302 is based on the principle of surface energy modulation. The array of grooves 301 significantly expands the contact area between the hydrophobic interface and the biological sample. The low surface energy of the hydrophobic material 302 inhibits the spreading behavior of water molecules on the barrier surface, thereby blocking the diffusion channels of neurotransmitter molecules along the inner surface of the barrier. Furthermore, by fully filling the grooves 301 with hydrophobic material 302, the integrity of the barrier structure is not affected. This effectively improves the signal independence and detection reliability of the high-density integrated sensor in long-term continuous monitoring, providing physical isolation for simultaneous analysis of multiple neurotransmitters.

[0057] Please see Figure 8 The embodiments of this application provide a method for fabricating a biosensor, comprising: S1. Multiple sensing units are formed on a substrate. Each sensing unit includes an electrode pair, which includes a first electrode and a second electrode disposed opposite to each other to form a gap region.

[0058] In some embodiments, step S1 may include the following technical solutions: In the step of forming multiple sensing units 20 on the substrate 10, a silicon wafer is used as the substrate 10 material, and the electrode pair 21 structure is constructed by photolithography. For example, positive photoresist AR5350 is used as the mask material. After spin-coating at 4000 rpm for 40 seconds, it is soft-baked at 105°C for 4 minutes to remove solvent and enhance adhesion. Subsequently, ultraviolet exposure is performed using a high-resolution hard mask, with the exposure dose controlled at 60 mJ / cm² to ensure clear definition of the electrode pattern. After exposure, a developer solution prepared with AR300-26 and deionized water at a ratio of 7:1 was used for development to remove unexposed areas. Finally, a gold layer was deposited by magnetron sputtering at room temperature with a sputtering rate of 0.04 Å / s and an argon flow rate maintained at 44 SCCM to form an electrode pad with a size of 100 µm and a thickness of 30 nm. The active area was precisely set to 1 µm², and the electrode gap was controlled at 1 µm. This process is based on the principle of micro-nano fabrication. By optimizing the exposure dose and development parameters, it ensures that the electrode pair 21 forms a stable micron-level spacing in the gap region, providing a precise physical framework for subsequent functional layer deposition, while avoiding signal distortion caused by gap size deviation.

[0059] In another embodiment, step S1 includes steps S11 to S15, as follows: S11. Spin-coat photoresist onto the substrate.

[0060] Spin-coat a layer of positive photoresist (such as AR5350) onto the substrate. The process parameters can be as follows: spin-coat at 3000 rpm for 5 seconds, then spin-coat at 4000 rpm for 40 seconds to form a uniform film; then soft bake at 105°C for 240 seconds to fully evaporate the solvent and cure the photoresist layer.

[0061] S12. Use a maskless laser direct writing device to pattern the photoresist to define the electrode pattern.

[0062] A maskless laser direct writing device (such as the MicroWriter system) is used to pattern the photoresist. This device uses a 365 nm wavelength ultraviolet light source, with a resolution of 0.4 μm and an exposure dose of 65 mJ / cm². The desired electrode pattern is written directly onto the photoresist under computer control, without the need for a physical mask.

[0063] S13. The photoresist in the exposed or unexposed areas is dissolved by the developer to form a photoresist template with electrode openings.

[0064] After exposure, the substrate is immersed in a developer (such as ARP 300-26) for 60 seconds to selectively dissolve the photoresist in the exposed or unexposed areas. The choice between exposed and unexposed areas depends mainly on the properties of the photoresist, thereby forming a photoresist template with precise electrode openings on the substrate, exposing the areas that will be used to form the first and second electrodes.

[0065] S14. Deposit a metal layer on the surface of the photoresist template so that the metal simultaneously covers the exposed area of ​​the substrate and the photoresist surface.

[0066] A metal layer was deposited on the entire sample surface using electron beam evaporation. Specific conditions were: gold (Au) as the electrode material, a deposition rate controlled at 0.5 Å / s, and a final thickness of 50 nm. During this process, the metal not only covered the exposed areas of the substrate (i.e., the electrode openings) but also simultaneously covered the top surface of the remaining photoresist.

[0067] S15. Subsequently, through a stripping process, the photoresist that was not removed by development and the metal attached to its surface are dissolved and removed together, leaving only the metal pattern deposited on the substrate, thereby obtaining an electrode pair composed of the first electrode and the second electrode.

[0068] The lift-off process involves immersing the sample in acetone for 2 minutes, followed by ultrasonic treatment at 40 W for 20 seconds. This process completely dissolves and removes the photoresist (i.e., the remaining unexposed or exposed areas) and the attached metal layer from the substrate. After this step, only the metal pattern directly deposited on the substrate remains, clearly forming an electrode pair consisting of a first electrode and a second electrode positioned opposite each other, with a micron-sized gap between them. This provides the structural basis for the subsequent deposition of the boehmite functional layer.

[0069] The beneficial effects of steps S11 to S15 are as follows: First, the introduction of maskless laser direct writing technology completely eliminates the physical mask fabrication step required by traditional photolithography, saving not only the high cost of masks (typically several thousand yuan per mask) but also significantly shortening the process cycle. More importantly, this technology has sub-micron resolution (up to 0.4 μm), sufficient to accurately construct the micron-level gap region between the first and second electrodes, providing geometric constraints for the subsequent localized deposition of boehmite functional layers and ensuring the repeatability of memristor switching behavior.

[0070] Secondly, by employing positive photoresist in conjunction with a lift-off process, high-fidelity pattern transfer of metal electrodes was achieved. After metal deposition, only the metal in the exposed area of ​​the substrate is retained to form electrodes, while excess metal covering the photoresist surface is dissolved and removed along with the photoresist by acetone, avoiding metal damage or edge roughness problems that may occur with dry etching.

[0071] Third, this process is based entirely on standard microfabrication equipment (such as spin coaters, laser direct writing systems, and electron beam evaporation stages), eliminating the need for highly corrosive piranha solutions or expensive silicon-on-insulator (SOI) wafers, significantly reducing material and safety costs. Furthermore, the entire process requires only 3–4 core steps, significantly improving manufacturing efficiency and yield compared to the 5–7 steps of electron beam lithography and multiple alignment processes required for traditional silicon nanowire memristors. Crucially, this method supports wafer-level parallel manufacturing. Maskless laser direct writing allows for rapid pattern switching, easily integrating different sensing units (such as functionalized units for dopamine and serotonin) on the same chip, providing a scalable manufacturing foundation for simultaneous detection of multiple neurotransmitters.

[0072] S2. Selectively deposit boehmite functional layers in the gap region of each electrode pair.

[0073] In the step of selectively depositing boehmite functional layers 22 in the interstitial regions of each electrode pair 21, atomic layer deposition (ALD) technology is used to achieve regioselectivity. Specifically, the substrate 10 is placed in the deposition chamber, and trimethylaluminum and water vapor are used as precursors for cyclic deposition at 150°C. By adjusting the number of deposition cycles, the boehmite layer is nucleated and grown only in the interstitial regions, while the electrode surface forms a self-limiting barrier due to surface energy differences. Ultimately, the thickness of the functional layer in the central interstitial region is controlled within the range of 30-50 nm. This process is mainly based on the principle of interfacial chemical regulation, utilizing the electric field concentration effect in the interstitial regions to promote precursor adsorption while avoiding covering the electrode edges. This ensures that the functional layer forms a continuous thin film only within the interstitial regions, effectively preventing electrode short circuits and optimizing memristor switching characteristics, laying the foundation for subsequent biomolecular modification.

[0074] S3. Functional organic molecules are covalently linked on the boehmite functional layer.

[0075] In the step of covalently linking functional organic molecules 23 to the boehmite functional layer 22, a silane coupling agent is used for surface activation treatment. Specifically, the substrate 10 with the boehmite functional layer 22 deposited is immersed in a 5% (v / v) solution of 3-aminopropyltriethoxysilane (APTES) in ethanol and reacted at 60°C for 2 hours. Afterward, it is thoroughly rinsed with anhydrous ethanol and dried under nitrogen. This process is based on the principle of surface chemical bonding. The ethoxy groups in the silane molecules undergo hydrolysis and condensation with the hydroxyl groups on the boehmite surface to form stable Si-O-Si covalent bonds, while simultaneously exposing amino active groups. This provides high-density reaction sites for the directional linking of aptamer molecules 24, which is beneficial for improving the binding efficiency and stability of the subsequent biorecognition layer.

[0076] S4. Adaptor molecules are covalently linked to functional organic molecules, and the aptor molecules of different sensing units have different spatial configurations.

[0077] In the step of covalently linking aptamer molecule 24 to functional organic molecule 23, specific binding is achieved through a carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinking method. Specifically, the amino-modified functional layer is activated for 15 minutes by immersing it in a phosphate buffer containing 1 mM EDC and 5 mM NHS, followed by reaction with a 0.1 mM aptamer molecule 24 solution at 4°C for 12 hours. After the reaction, unbound molecules are removed by washing with PBS buffer. This process is based on the principle of bioconjugation chemistry. The EDC / NHS system activates amino groups to form active esters, which then form efficient amide bonds with the carboxyl groups of aptamer molecule 24. This ensures that aptamer molecule 24 is fixed in a directional manner on the surface of the functional layer, avoiding the loss of active sites due to random adsorption, and providing a molecular recognition basis for highly sensitive neurotransmitter detection.

[0078] It should be noted that the aptamer molecules 24 of different sensing units 20 have different spatial configurations, achieving multi-target detection capability through partitioned modification. In specific operation, the chip can be divided into 4-8 independent sub-arrays, each corresponding to a different sensing unit 20, which is modified with dopamine aptamers (with a rigid benzene ring configuration) and serotonin aptamers (with a flexible indole ring configuration), respectively. The modification process is carried out sequentially under the control of microfluidic channels to ensure the spatial orientation difference of the aptamer molecules 24 in each unit. This process is mainly based on the principle of molecular recognition specificity. Different spatial configurations enable the aptamer molecules 24 to form selective binding pockets for target neurotransmitters. The rigid configuration improves the detection sensitivity of dopamine to 0.05 nM, while the flexible configuration optimizes the detection specificity of serotonin to 0.3 nM, thereby achieving simultaneous and high-precision detection of multiple neurotransmitters within a single chip, meeting the reliable analysis needs in complex clinical sample environments.

[0079] For example, after the functional organic molecule modification is completed, the aptamer is directionally immobilized. Taking the dopamine detection unit as an example: 5 μL of a 1 μM aqueous solution of hydroxyl-modified dopamine aptamer (sequence: 5'-OH-(CH2)6-AGG GCA GGC TTAGGG CAC GTG GG-3') is dropped onto the surface of the corresponding sensing unit and left to stand overnight (approximately 12 hours) at 4°C. This allows the hydroxyl groups of the aptamer and the terminal carboxyl groups of the MUA to react with the terminal carboxyl groups of 1-,4-phthalic acid (also known as terephthalic acid) in a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) activation system, forming stable ester bonds. For the serotonin detection unit, the carboxyl-modified serotonin aptamer is covalently linked to the surface of 4-aminobenzoic acid to achieve covalent cross-linking. After fixation, the aptamers were gently rinsed five times with phosphate-buffered saline (PBS, pH 7.4) to remove unbound aptamers, and non-specific adsorption sites were blocked with 1% bovine serum albumin (BSA) solution for 30 minutes. This process ensures that the aptamers are fixed to the sensing interface in a high-density, highly oriented manner, maintaining their three-dimensional conformation and thus preserving high affinity and specific recognition of the target neurotransmitter.

[0080] In some embodiments, the step of covalently linking the functional organic molecule 23 to the boehmite functional layer 22 includes: A solution of functional organic molecules 23 with carboxyl groups is applied to a portion of the boehmite functional layer 22 of the sensing unit 20, and a solution of functional organic molecules 23 with amino groups is applied to another portion of the boehmite functional layer 22 of the sensing unit 20.

[0081] For example, the chip is divided into 4 to 8 independent sensing unit regions. A microfluidic channel system is used to sequentially apply a solution of functional organic molecules 23 with carboxyl groups (e.g., a 5% (v / v) solution of 3-carboxypropyltriethoxysilane ethanol) to the surface of the boehmite functional layer 22 of a designated sensing unit 20. The reaction is carried out at 60°C for 2 hours to form a carboxylated modification layer. Subsequently, the channel is changed to apply a solution of functional organic molecules 23 with amino groups (e.g., a 5% (v / v) solution of 3-aminopropyltriethoxysilane ethanol) to other sensing units 20, and the amination modification is completed under the same conditions. This process is based on the principle of surface chemical partitioning. Through the directional introduction of different functional groups, complementary reaction sites are provided for the specific connection of subsequent aptamer molecules 24. The carboxylated regions can efficiently bind to the amination aptamer molecules 24, and the amination regions are adapted to the carboxylated aptamer molecules 24, ensuring high selectivity and low cross-interference in simultaneous detection of the multi-unit array, thereby meeting the reliability requirements of multi-neurotransmitter analysis in complex biological samples.

[0082] For example, to achieve the orientation and stable fixation of aptamers on the boehmite surface, functional organic molecules are introduced as molecular bridges onto the boehmite functional layer. The specific steps are as follows: First, the chip with completed boehmite deposition is placed in an oxygen plasma cleaner and treated for 60 seconds at 50 W power and 0.2 mbar oxygen atmosphere to significantly increase the density of hydroxyl groups (-OH) on the boehmite surface. Subsequently, for different sensing units, solutions of functional organic molecules containing specific end groups are added dropwise: for example, for the dopamine detection unit, a 1 mM 11-hydroxyundecanoic acid (MUA) ethanol solution is used; for the serotonin detection unit, a 1 mM 3-aminopropyltriethoxysilane (APTES) ethanol solution is used. The chip is incubated at room temperature in the dark for 2 hours, allowing carboxyl groups (-COOH) or amino groups (-NH2) to form covalent bonds with the hydroxyl groups on the boehmite surface through esterification or condensation reactions, respectively. After the reaction, the chip is rinsed three times with ethanol to remove unbound molecules and dried under nitrogen. Fourier transform infrared spectroscopy (FTIR) detection at 1730 cm⁻¹ -1 (C=O stretching) and 1560 cm -1 The presence of a characteristic peak at the (NH bend) point confirms the successful grafting of the functional organic molecule.

[0083] In some embodiments, prior to step S1, the following may also be included: The substrate was ultrasonically cleaned sequentially with acetone, methanol, and deionized water, and then dried with high-purity nitrogen. Details are as follows: To ensure good adhesion between the subsequent metal electrode and the substrate and reduce interfacial leakage, the silicon / silica substrate underwent a rigorous surface cleaning process. The specific procedure was as follows: the substrate was sequentially ultrasonically cleaned in analytical grade acetone for 5 minutes to remove grease and organic contaminants; then transferred to methanol and ultrasonically cleaned for 3 minutes to further dehydrate and remove residual solvent; finally, it was rinsed twice with deionized water for 1 minute each time to remove ionic impurities. After cleaning, the substrate surface was immediately dried with high-purity nitrogen (≥99.999%) at an angle to avoid water residue. The substrate surface after this three-solvent cleaning process had a contact angle of less than 10°, and X-ray photoelectron spectroscopy (XPS) showed a reduction of over 80% in the intensity of the carbon contamination peak, laying a clean interface foundation for subsequent high-fidelity patterning and stable memristor behavior.

[0084] In some embodiments, step S2 includes: adding boehmite nanosheet dispersion to the interstitial region and allowing it to dry at room temperature, so that the boehmite nanosheets self-assemble and fill the interstitial region.

[0085] Specifically, after the electrode pair is prepared, memristor active materials need to be selectively introduced into the gap region between the first and second electrodes. In this embodiment, boehmite (AlOOH) nanosheets are used as the switching dielectric layer, and the preparation method is as follows: Boehmite nanosheets synthesized by hydrothermal method are dispersed in anhydrous ethanol to prepare a uniform suspension with a concentration of 0.1 mg / mL; then, 5 μL of the dispersion is precisely added to the gap region of the sensing unit using a micropipette; the solution is left to stand for 2 hours in a clean environment at room temperature (25℃) and relative humidity <40% to allow the solvent to evaporate naturally. The boehmite nanosheets self-assemble under capillary action and bridge between the two electrodes to form a continuous dielectric functional layer. Scanning electron microscopy (SEM) observation shows that this drop casting process can achieve complete filling of the gap region, and the thickness of the boehmite layer can be controlled within the range of 30–50 nm. This method requires no complex equipment, avoids high-cost processes such as atomic layer deposition (ALD) or sputtering, and retains the abundant hydroxyl functional groups on the surface of boehmite, providing chemical anchoring points for subsequent biofunctionalization.

[0086] Please see Figure 9 Embodiments of this application also provide a neurotransmitter detection system, comprising: The biosensor as described in any of the foregoing embodiments; A microfluidic chip, connected to the biosensor, is used to deliver biological samples; A signal processing unit, connected to the biosensor, is used to process the detection signal; The display unit, connected to the signal processing unit, is used to display the detection results.

[0087] For example, the microfluidic chip achieves a hermetically tight connection with the sensing area of ​​the biosensor through a microchannel structure. The microchannel width is controlled within the range of 100 to 200 μm, and the flow rate is set to 0.5 to 2 μL / min. Based on the principle of fluid dynamics regulation, by controlling the channel size and flow rate parameters, diffusion or mixing of biological samples during delivery is effectively avoided, ensuring that neurotransmitter molecules reach each sensing unit 20 at a stable concentration. In response to the delivered biological sample, the aptamer molecules 24 in the biosensor's functional layer specifically bind to the target neurotransmitter, triggering a change in the resistivity of the boehmite functional layer 22. This process is based on the principle of molecular recognition and resistivity coupling. Aptamer molecules 24 with different spatial configurations selectively capture neurotransmitters such as dopamine or serotonin, resulting in a quantifiable change in the resistance value between electrodes. The insulating barrier structure 30 effectively blocks the ion migration path between adjacent units, ensuring rapid and reliable molecular recognition in complex biological environments.

[0088] The signal processing unit establishes an electrical connection with the electrode pair 21 of the biosensor through a shielded cable. It uses a low-noise preamplifier to amplify the original signal by 100 times and uses a digital filter to filter out 50 Hz power frequency interference and high-frequency noise. Based on the principle of signal integrity optimization, the signal-to-noise ratio is significantly improved to over 40 dB by matching the sensor output impedance and the amplifier input impedance. At the same time, an adaptive algorithm is used to correct the temperature drift effect, providing a high-fidelity data foundation for subsequent result analysis.

[0089] The display unit is connected to the signal processing unit via a standard interface and uses an LCD screen to display neurotransmitter concentration values ​​and detection status information in real time. Based on the principle of human-computer interaction optimization, the interface design includes concentration trend curves and threshold alarm functions. When the detection result exceeds the preset range, an audio-visual prompt is automatically triggered, thereby intuitively and promptly providing feedback to the user on the detection results, meeting the requirements of real-time information presentation and readability for rapid clinical diagnosis.

[0090] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A biosensor, characterized in that, It includes a substrate and a plurality of sensing units disposed on the substrate, each of the sensing units comprising: An electrode pair, comprising a first electrode and a second electrode disposed opposite to each other to form a gap region; A boehmite functional layer is disposed in the gap region to connect the first electrode and the second electrode; Functional organic molecules are covalently linked to the hydroxyl functional groups of the boehmite functional layer; The aptamer molecule is covalently linked to the aforementioned functional organic molecule; The aptamer molecules of different sensing units have different spatial configurations.

2. The biosensor according to claim 1, characterized in that, The electrode pairs of the plurality of sensing units together form a comb-like layout, and in the plurality of sensing units, the plurality of first electrodes and the plurality of second electrodes are arranged alternately in parallel strips along a first direction, the plurality of first electrodes are arranged at intervals along a second direction, the plurality of second electrodes are arranged at intervals along the second direction, and the first direction and the second direction are perpendicular.

3. The biosensor according to claim 1, characterized in that, The plurality of sensing units include a first sensing unit, wherein the functional organic molecule in the first sensing unit includes a first organic molecule, the end of which is a carboxyl group, and the carboxyl group is connected to the hydroxyl functional group of the boehmite functional layer through an ester bond.

4. The biosensor according to claim 3, characterized in that, The aptamer molecule in the first sensing unit includes a first aptamer, which is connected to the first organic molecule.

5. The biosensor according to claim 4, characterized in that, The plurality of sensing units include a second sensing unit, wherein the functional organic molecule in the second sensing unit includes a second organic molecule, the end of which is an amino group, and the amino group is connected to the hydroxyl functional group of the boehmite functional layer through an amide bond.

6. The biosensor according to claim 5, characterized in that, The aptamer molecule in the second sensing unit includes a second aptamer, which is connected to the second organic molecule.

7. The biosensor according to claim 6, characterized in that, The first aptamer specifically binds to dopamine, and the second aptamer specifically binds to serotonin; The first ratio is defined as the proportion of the number of effective binding sites for dopamine in the first aptamer to the total number of binding sites in the first aptamer, and the second ratio is defined as the proportion of the number of effective binding sites for serotonin in the second aptamer to the total number of binding sites in the second aptamer, wherein the first ratio is greater than the second ratio.

8. The biosensor according to claim 1, characterized in that, A central electrode is provided between the first electrode and the second electrode. One end of the central electrode is spaced apart from the first electrode to form a first sub-gap, and the other end is spaced apart from the second electrode to form a second sub-gap. The boehmite functional layer is disposed within the first sub-gap and the second sub-gap.

9. The biosensor according to claim 8, characterized in that, The central electrode has multiple first ends and multiple second ends. Each first end is spaced apart from a first electrode to form a first sub-gap. Each second end is spaced apart from a second electrode to form a second sub-gap. Each first sub-gap and each second sub-gap is provided with a boehmite functional layer.

10. The biosensor according to claim 1, characterized in that, The boehmite functional layer has a gradient thickness distribution perpendicular to the extension direction of the electrode pair; The thickness of the boehmite functional layer gradually decreases from the edge of the gap region toward the center of the gap region.

11. The biosensor according to claim 1, characterized in that, An insulating barrier is provided between adjacent sensing units.

12. The biosensor according to claim 11, characterized in that, The inner surface of the insulating barrier is provided with multiple grooves, which are arranged in an array and filled with a hydrophobic material.

13. A method for preparing a biosensor, characterized in that, include: Multiple sensing units are formed on a substrate. Each sensing unit includes an electrode pair, which includes a first electrode and a second electrode disposed opposite to each other to form a gap region. Selective deposition of boehmite functional layers in the gap region of each electrode pair; Functional organic molecules are covalently linked to the boehmite functional layer; An aptamer molecule is covalently linked to the functional organic molecule; Among them, the aptamer molecules of different sensing units have different spatial configurations.

14. The preparation method according to claim 13, characterized in that, The step of forming a plurality of sensing units on a substrate, each sensing unit including an electrode pair, the electrode pair including a first electrode and a second electrode disposed opposite to each other to form a gap region, includes: Photoresist is spin-coated onto the substrate; The photoresist is patterned using a maskless laser direct writing device to define the electrode pattern; A photoresist template with electrode openings is formed by dissolving the photoresist in the exposed or unexposed areas with a developer solution. A metal layer is deposited on the surface of the photoresist template, so that the metal simultaneously covers the exposed area of ​​the substrate and the photoresist surface; Subsequently, through a stripping process, the photoresist that was not removed by development and the metal attached to its surface are dissolved and removed together, leaving only the metal pattern deposited on the substrate, thereby obtaining an electrode pair composed of the first electrode and the second electrode.

15. A neurotransmitter detection system, characterized in that, include: The biosensor as described in any one of claims 1 to 12; A microfluidic chip, connected to the biosensor, is used to deliver biological samples; A signal processing unit, connected to the biosensor, is used to process the detection signal; The display unit, connected to the signal processing unit, is used to display the detection results.

Images