High-sensitivity biosensor for enhancing photoelectric response based on multi-exciton effect and preparation method of high-sensitivity biosensor

Abstract

The invention discloses a high-sensitivity biosensor for enhancing photoelectric response based on multi-exciton effect, which is of a layered stacking structure along the vertical direction and sequentially comprises an ITO (indium tin oxide) conductive glass substrate, an HQ-CdS hierarchical quantum structure homojunction photoelectrode and a 5-HT aptamer modification layer from bottom to top, wherein the HQ-CdS photoelectrode is composed of a B-CdS film and an in-situ grown C-modified CdS two-dimensional nanosheet and forms a homojunction structure, the preparation method of the sensor comprises the steps of preparing the B-CdS photoelectrode through chemical bath deposition and annealing treatment, constructing the HQ-CdS photoelectrode through in-situ photoelectrochemical treatment, and then completing sensor assembly through aptamer fixation and target molecule combination. According to the invention, the multi-exciton effect is used as a core signal amplification strategy, and the problem of insufficient resolution of the existing PEC aptamer sensor is solved.

Description

Technical Field

[0001] This invention relates to the field of photoelectrochemical biosensing technology, and in particular to a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response and its preparation method. Background Technology

[0002] Current research on PEC aptamer biosensors mainly focuses on the sensor's limit of detection (LOD), linear detection region, and specificity, neglecting research on sensor signal amplification strategies, i.e., resolution. Resolution is a crucial factor in bringing PEC sensors to practical applications. The shortcomings of existing photoelectrochemical biosensors in terms of sensitivity, signal stability, and practicality can be specifically divided into three points: Traditional photoelectrochemical biosensors have weak photoelectric response signals and insufficient sensitivity, making it difficult to accurately detect low concentrations of biological targets (such as trace amounts of nucleic acids and small molecule biomarkers). The photoelectric signals of existing sensors are easily affected by environmental interference (such as background current and non-specific adsorption), resulting in poor accuracy and stability of detection results. Some methods to improve sensitivity rely on complex structural designs or additional signal amplification steps, which increases both the manufacturing cost of the sensor and the operational complexity. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response and its fabrication method. By using the multiexciton effect as an efficient signal amplification strategy, it solves the problem of neglecting resolution in existing PEC aptamer biosensors.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response. The sensor has a layered stacked structure along the vertical direction, and from bottom to top, it includes a substrate, a photoelectrode layer, and an aptamer modification layer. The photoelectrode layer is an HQ-CdS hierarchical quantum structure homojunction photoelectrode, and the aptamer modification layer is a 5-HT aptamer molecular layer. The projection range of the aptamer modification layer completely covers the effective working area of ​​the photoelectrode layer, and the coverage area of ​​the photoelectrode layer completely overlaps with the conductive area of ​​the substrate.

[0005] Preferably, the substrate is ITO conductive glass, and the HQ-CdS photoelectrode is prepared by in-situ photoelectrochemical treatment of the surface of a B-CdS photoelectrode, and the surface is modified with C-doped CdS two-dimensional nanosheets.

[0006] This invention provides a method for fabricating a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response, specifically including the following steps: (1) Fabrication of B-CdS photoelectrode: a. Select ITO conductive glass as the substrate, clean it according to standard procedures, and then dry it with high-purity nitrogen. b. Preparation of CBD deposition solution: Disperse 1.5 mM CdSO4·8 / 3H2O and 75 mM CH4N2S solution evenly by ultrasonication, and slowly add NH3·H2O to adjust the pH value to 9; c. Vertically insert the clean ITO conductive substrate into the deposition solution and continuously stir the reaction. After the chemical bath deposition is completed, thoroughly rinse the photoelectrode surface with deionized water to remove any impurities and deposits that may remain during the deposition process. The deposition process can be repeated to ensure the uniformity and thickness of the film. d. Anneal the deposited photoelectrode film and allow it to cool naturally to obtain a B-CdS photoelectrode; (2) Fabrication of HQ-CdS photoelectrode: a. A three-electrode system was adopted, with B-CdS photoelectrode as the working electrode, a mixed solution of 1 M NaOH and 5 vol% methanol as the electrolyte, and a 300 W xenon lamp simulating sunlight through an AM 1.5 cutoff filter; b. Under alternating on / off light conditions, a bias voltage of -1.4 V ~ 0.4 V vs. Ag / AgCl is applied to synthesize C-modified CdS two-dimensional nanosheets on the surface of the B-CdS photoelectrode. This process is repeated twice to obtain the HQ-CdS photoelectrode. (3) Fabrication of HQ-CdS sensor: a. Take 10 μL of 1.8 μM 5-HT aptamer solution and coat it evenly on the surface of HQ-CdS photoelectrode. Incubate at 37℃ for 1 hour to fix the aptamer. Then rinse the photoelectrode surface thoroughly with ultrapure water to remove unbound aptamer molecules, thereby obtaining a series of DNA aptamer-modified photoelectrodes to prepare for subsequent experiments. b. Coat the modified photoelectrode surface with 5-HT solutions of different concentrations, incubate at a constant temperature of 37°C for 30 minutes. After incubation, thoroughly wash the photoelectrode surface with ultrapure water to remove unbound 5-HT molecules, and allow it to air dry at room temperature to obtain a series of sensors.

[0007] Preferably, in step (1)c, the deposition reaction temperature is 70°C and the reaction time is 1 hour.

[0008] Preferably, in step (1)d, the annealing process is carried out in an air atmosphere, the annealing temperature is 350°C, the holding time is 1 hour, and the heating rate is 5°C / min.

[0009] Preferably, in step (3)b, a coating with a concentration of 1×10⁻¹ is applied to the surface of the modified photoelectrode. 7 A 5-HT solution with g / mL ~ 1×10⁻¹¹ g / mL. Beneficial effects

[0010] Compared with existing technologies, it has the following advantages: This invention achieves efficient amplification of sensing signals by regulating the generation and suppression of multiexcitons, solving the problem of neglecting resolution in existing PEC aptamer biosensors. The biosensor of this invention consists of a vertical layered structure composed of an ITO substrate, an HQ-CdS photoelectrode, and a 5-HT aptamer modification layer. Each layer is tightly bound together through chemical deposition, in-situ growth, and non-covalent adsorption. Furthermore, the coverage areas of the aptamer modification layer and the photoelectrode layer are precisely aligned, ensuring both structural stability and photoelectric signal transmission efficiency and molecular recognition specificity. The HQ-CdS hierarchical quantum structure homojunction photoelectrode of this invention consists of a bottom B-CdS thin film and a top C-modified CdS two-dimensional nanosheet. The C-modified CdS two-dimensional nanosheet is grown in situ on the surface of the B-CdS thin film to form a homojunction structure. The structure optimizes the properties of the B-CdS substrate through chemical bath deposition and annealing, and then constructs the homojunction through in-situ photoelectrochemical treatment. This not only solves the problem of the sensitivity of the multiexciton effect to the surface state, but also modulates the quantum confinement effect through C modification, providing a structural basis for multiexciton generation and signal conversion. In the preparation process, the chemical bath precipitation of this invention uses a concentration ratio of 1.5 mM CdSO4・8 / 3H2O and 75 mM CH4N2S, and the pH value is adjusted to 9 to ensure that the crystallinity and defect level of the B-CdS film are suitable for subsequent multiexciton generation. During the aptamer modification and incubation process, the aptamer concentration is determined to be 1.8 μM, and the incubation temperature is 37℃. The aptamer is fixed for 1 hour and molecular binding is carried out for 30 minutes to ensure uniform adsorption of aptamers and maintain their biological activity.

[0011] Compared to traditional B-CdS sensors, the biosensor prepared in this invention exhibits improved signal resolution ΔI from 21.3 μA / cm² to 419.0 μA / cm², a sensitivity of 345 μA·cm⁻², and a detection limit as low as 3.33 × 10⁻¹. 7 g / mL, and within 1×10⁻¹ 7It maintains excellent linearity (R²≥0.990) over a wide concentration range of ~1×10⁻¹¹ g / mL, completely solving the core problem of weak photoelectric response and difficulty in detecting low concentrations of biological targets by traditional sensors. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the HQ-CdS sensor structure; Figure 2 The comparison chart shows the detection results of 5-HT in the comparative example and Example 1, compared with the standard curve. Figure 3 The curves showing the specificity and stability of 5-HT detection obtained in Example 1 are shown. Figure 4 The surface fluorescence intensity diagrams for the comparative example and Example 1 before and after aptamer adsorption are shown. Figure 5 The results and standard curve for the detection of 5-HT in artificial saliva in Example 1 are shown. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0014] like Figure 1 As shown, this embodiment provides a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response. The sensor has a layered stacked structure along the vertical direction, which includes a substrate, a photoelectrode layer, and an aptamer modification layer from bottom to top. The substrate is ITO conductive glass, the photoelectrode layer is an HQ-CdS hierarchical quantum structure homojunction photoelectrode, and the aptamer modification layer is a 5-HT aptamer molecular layer. The projection range of the aptamer modification layer completely covers the effective working area of ​​the photoelectrode layer, and the coverage area of ​​the photoelectrode layer completely overlaps with the conductive area of ​​the substrate. The HQ-CdS photoelectrode is prepared by in-situ photoelectrochemical treatment of the surface of a B-CdS photoelectrode, and the surface is modified with C element-doped CdS two-dimensional nanosheets.

[0015] This embodiment also provides a method for fabricating a high-sensitivity biosensor based on the multiexciton effect to enhance photoelectric response, specifically including the following steps: (1) Fabrication of B-CdS photoelectrode: a. Select ITO conductive glass as the substrate, clean it according to standard procedures, and then dry it with high-purity nitrogen. b. Preparation of CBD deposition solution: Disperse 1.5 mM CdSO4·8 / 3H2O and 75 mM CH4N2S solution evenly by ultrasonication, and slowly add NH3·H2O to adjust the pH value to 9; c. Vertically insert the clean ITO conductive substrate into the deposition solution and continuously stir the reaction. The deposition reaction temperature is 70℃ and the reaction time is 1 hour. After the chemical bath deposition is completed, rinse the photoelectrode surface thoroughly with deionized water to remove any impurities and deposits that may remain during the deposition process. The deposition process can be repeated to ensure the uniformity and thickness of the film. d. The deposited photoelectrode film was annealed in an air atmosphere at a temperature of 350°C for 1 hour at a heating rate of 5°C / min. After natural cooling, the B-CdS photoelectrode was obtained. (2) Fabrication of HQ-CdS photoelectrode: a. A three-electrode system was adopted, with B-CdS photoelectrode as the working electrode, a mixed solution of 1 M NaOH and 5 vol% methanol as the electrolyte, and a 300 W xenon lamp simulating sunlight through an AM 1.5 cutoff filter; b. Under alternating on / off light conditions, a bias voltage of -1.4 V ~ 0.4 V vs. Ag / AgCl was applied to synthesize C-modified CdS two-dimensional nanosheets on the surface of the B-CdS photoelectrode. This process was repeated twice to obtain HQ-CdS photoelectrodes, which were labeled as MEG-CdS-1C (one surface treatment) and MEG-CdS-2C (two surface treatments), respectively. (3) Fabrication of HQ-CdS sensor: a. Take 10 μL of 1.8 μM 5-HT aptamer solution and coat it evenly on the surface of HQ-CdS photoelectrode. Incubate at 37℃ for 1 hour to fix the aptamer. Then rinse the photoelectrode surface thoroughly with ultrapure water to remove unbound aptamer molecules, thereby obtaining a series of DNA aptamer-modified photoelectrodes to prepare for subsequent experiments. b. Different concentrations of 5-HT solutions (1×10-17 g / mL, 1×10-16 g / mL, 1×10-15 g / mL, 1×10-14 g / mL, 1×10-13 g / mL, 1×10-12 g / mL, 1×10-11 g / mL) were coated onto the prepared DNA aptamer photoelectrodes and incubated at a constant temperature of 37℃ for 30 minutes. After incubation, the photoelectrode surface was thoroughly washed with ultrapure water to remove unbound 5-HT molecules and then allowed to air dry at room temperature to obtain a series of sensors.

[0016] A method for fabricating a B-CdS sensor specifically includes the following steps: Step 1: Fabrication of B-CdS photoelectrode: ITO conductive glass was selected as the photoelectrode substrate and thoroughly cleaned according to the standard cleaning procedure to remove surface oil, impurities and oxide layer. After cleaning, the surface was dried with high-purity nitrogen to obtain a clean, dry and pollution-free conductive substrate. To prepare a chemical bath deposition (CBD) solution, a 1.5 mM CdSO4·8 / 3H2O solution was mixed with a 75 mM CH4N2S solution. The solution was then dispersed by ultrasonication or thoroughly stirred to ensure homogeneity. Subsequently, the solution was continuously stirred with a magnetic stirrer, and an appropriate amount of NH3·H2O was slowly added dropwise while the pH was monitored in real time with a pH meter and adjusted to 9. The pretreated ITO conductive substrate was vertically inserted into the prepared deposition solution and stirred continuously at 70°C for 1 hour. After the reaction, the photoelectrode surface was thoroughly rinsed with deionized water to remove any residual impurities and unreacted substances from the deposition process. To ensure the uniformity of the film and the target thickness, the deposition process was repeated three times. Annealing: The deposited photoelectrode film was placed in an air atmosphere for annealing. The annealing temperature was set at 350℃, the holding time was 1 hour, and the heating rate was precisely controlled at 5℃ / min. After annealing, the film was allowed to cool naturally to room temperature to obtain the B-CdS photoelectrode. Step 2: Fabrication of the B-CdS sensor: Take 10 μL of 1.8 μM 5-HT aptamer solution and uniformly coat it onto the surface of the prepared B-CdS photoelectrode (the effective area of ​​the photoelectrode is 1×0.5 cm²). Place the coated photoelectrode in an environment of 37℃ for 1 hour to allow the aptamer molecules to be stably bound to the surface of the photoelectrode. After incubation, thoroughly rinse the surface of the photoelectrode with ultrapure water to remove unbound free aptamer molecules. Different concentrations of 5-HT solution (concentration range 1×10⁻¹) were coated onto the aptamer-modified photoelectrode surface. 7 (g / mL ~ 1×10⁻¹¹ g / mL); incubate at a constant temperature of 37℃ for 30 minutes to allow 5-HT molecules to specifically bind to the aptamers on the photoelectrode surface; after incubation, wash the photoelectrode surface with ultrapure water to remove non-specifically bound 5-HT molecules, and allow it to air dry at room temperature to obtain a series of B-CdS sensors. See Figure 2It can be seen that although the comparative B-CdS photoelectrode is a photocurrent-enhanced sensor, it is limited by its relatively small photocurrent density, which is within 1×10⁻¹. 6 Within the linear detection range of g / mL to 1×10⁻¹¹ g / mL, the error bar is large and the linearity is poor, resulting in insufficient reliability of the sensing results. However, Example 1 of this invention effectively utilizes the multiexciton generation effect to enable the HQ-CdS photoelectrode to form a photocurrent suppression sensing mechanism. Its sensing signal resolution ΔI reaches 419.0 μA / cm², which is nearly 20 times higher than the 21.3 μA / cm² of the B-CdS photoelectrode. This significantly reduces the detection error, greatly enhances the stability and reliability of the sensing results, breaks through the performance bottleneck of the prior art, and demonstrates excellent PEC sensing performance, providing an efficient solution for PEC sensing of ultra-low concentration biomolecules.

[0017] Figure 3 The specificity and stability test curves for 5-HT detection obtained in Example 1 are shown. A commercially available 5-HT aptamer was used as a specific probe during the preparation of this sensor. The HQ-CdS sensor exhibits a specific response to 5-HT molecules. BSA, histamine, dopamine, and Aβ42 were selected as interference sources, and all detectable concentrations were kept equal at 1×10⁻¹¹ g / mL. Specificity tests were conducted under identical conditions. After 7 days of storage, the photocurrent density of the HQ-CdS sensor showed virtually no decrease from its initial value, indicating good stability and application potential.

[0018] Figure 4 To compare the surface fluorescence intensity before and after aptamer adsorption with that of Example 1, A and B represent the B-CdS photoelectrode and the B-CdS photoelectrode after aptamer adsorption, while 4C and D represent the HQ-CdS photoelectrode and the HQ-CdS photoelectrode after aptamer adsorption. Before aptamer incubation, no obvious fluorescence signal was observed on the surface of the B-CdS photoelectrode, but after incubation, a green fluorescence signal was observed, indicating that the aptamer had been successfully adsorbed onto the B-CdS photoelectrode surface. Similarly, no fluorescence signal was generated on the surface of the HQ-CdS photoelectrode before aptamer modification, but a significant green fluorescence signal appeared after modification. The uniform distribution of the green fluorescence signal indicates that the aptamer can be stably and uniformly adsorbed on the HQ-CdS photoelectrode surface. This phenomenon reflects the excellent surface characteristics of the HQ-CdS photoelectrode, such as the uniformity of the active sites on the material surface and the stability of the surface chemical state, which can effectively support the adsorption of aptamer molecules on its surface. In addition, the uniformity of fluorescence intensity also indicates that the HQ-CdS photoelectrode has good controllability in the adsorption process of aptamers and is not prone to local adsorption or enrichment, which is crucial for maintaining the high sensitivity and high specificity of the PEC sensor.

[0019] Figure 5 The results and standard curve for 5-HT detection in artificial saliva in Example 1 are shown. Even in artificial saliva containing various interfering substances, the HQ-CdS sensor still exhibits excellent 5-HT detection performance. The standard curve obtained through transient photocurrent response testing shows that the sensor performs well at 1×10⁻⁶ ppm. -17 ~ 1×10 -11 It exhibits excellent linear response over a wide concentration range of g / mL, with a sensitivity as high as 345 μA·cm⁻¹ within this range. -2 The detection limit is as low as 3.33×10 -17 The g / mL value indicates that the sensor has strong anti-interference ability in complex matrices.

[0020] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0021] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-sensitivity biosensor based on multi-exciton effect enhanced photoelectric response, the sensor is in a layered stack structure along the vertical direction, characterized in that: From bottom to top, it comprises a substrate, a photoelectrode layer and an aptamer modification layer in sequence, wherein the photoelectrode layer is a HQ-CdS hierarchical quantum structure homojunction photoelectrode, the aptamer modification layer is a 5-HT aptamer molecule layer, and the projection range of the aptamer modification layer completely covers the effective working area of the photoelectrode layer, and the coverage range of the photoelectrode layer completely coincides with the conductive area of the substrate.

2. The high-sensitivity biosensor based on the enhancement of photoelectric response by multi-exciton effect according to claim 1, characterized in that: The substrate is ITO conductive glass, the HQ-CdS photoelectrode is prepared by in-situ photoelectrochemical treatment on the surface of a B-CdS photoelectrode, and the surface is modified with C element doped CdS two-dimensional nanosheets.

3. The method of claim 1, wherein the method is characterized by: Specifically comprising the following steps: (1) Preparation of B-CdS photoelectrode: a. Select ITO conductive glass as the substrate, clean it according to the standard procedure, and then dry it with high-purity nitrogen; b. Prepare the CBD deposition solution: uniformly ultrasonically disperse 1.5 mM CdSO4·8 / 3H2O and 75 mM CH4N2S solution, and slowly add NH3·H2O to adjust the pH value to 9; c. Insert the clean ITO conductive substrate vertically into the deposition solution, continuously stir the reaction, and after the chemical bath deposition is completed, rinse the surface of the photoelectrode with deionized water to remove impurities and attachments that may be left over during the deposition process, and the deposition process can be repeated to ensure the uniformity and thickness of the film; d. Anneal the deposited photoelectrode film, and after natural cooling, obtain the B-CdS photoelectrode; (2) Preparation of HQ-CdS photoelectrode: a. Use a three-electrode system, with the B-CdS photoelectrode as the working electrode, the electrolyte being a mixture of 1 M NaOH and 5 vol% methanol, and the light source being a 300 W xenon lamp with an AM 1.5 cutoff filter simulating sunlight; b. Under the condition of intermittent light, apply a bias of -1.4 V ~ 0.4 V vs. Ag / AgCl to the surface of the B-CdS photoelectrode to synthesize C-modified CdS two-dimensional nanosheets, and repeat the process twice to obtain the HQ-CdS photoelectrode; (3) Preparation of HQ-CdS sensor: a. Take 10 μL of 1.8 μM 5-HT aptamer solution and evenly coat it on the surface of the HQ-CdS photoelectrode, incubate at 37℃ for 1 hour to fix the aptamer, and then thoroughly rinse the surface of the photoelectrode with ultrapure water to remove unbound aptamer molecules, thereby obtaining a series of DNA aptamer-modified photoelectrodes for subsequent experiments; b. Coat different concentrations of 5-HT solution on the surface of the modified photoelectrode, incubate at 37℃ for 30 minutes, and after the incubation is completed, thoroughly wash the surface of the photoelectrode with ultrapure water to remove unbound 5-HT molecules, and naturally dry at room temperature to obtain a series of sensors.

4. The method according to claim 3, wherein the method is characterized by: In step (1)c, the deposition reaction temperature is 70℃, and the reaction time is 1 hour.

5. The method of claim 3, wherein the method is characterized by: In step (1)d, the annealing treatment is performed in an air atmosphere, the annealing temperature is 350℃, the holding time is 1 hour, and the heating rate is 5℃ / min. In step (1)d, the annealing treatment is performed in an air atmosphere, the annealing temperature is 350℃, the holding time is 1 hour, and the heating rate is 5℃ / min.

6. The method of claim 3, wherein the method is characterized by: The step (3) b coats the modified photoelectrode surface with a 5-HT solution with a concentration of 1×10⁻¹ 7 g / mL ~ 1×10⁻¹¹ g / mL.

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