Preparation method and application of photoelectrochemical aptamer sensor based on porphyrin cofs

Abstract

This invention relates to the field of photoelectrochemical aptamer sensor technology, specifically disclosing a method for preparing a rapid and sensitive photoelectrochemical aptamer sensor for detecting β-amyloid (Aβ) protein. A porphyrin-based covalent organic framework (COF) photoelectrochemical conversion layer is grown in situ on the electrode surface via a Schiff base reaction. After the reaction, the layer is placed on an activated Aβ protein. 42 In the aptamer solution, after covalent binding with amide, non-specific recognition sites were blocked by adding mercaptohexanol solution to the surface; finally, different concentrations of Aβ were incubated on the electrode surface. 42 That is, Aβ 42 Photoelectrochemical aptamer sensor. The Aβ sensor of this invention... 42 A photoelectrochemical aptamer sensor capable of realizing Aβ in the range of 1.0 pmol / L to 1.0 µmol / L. 42 Rapid and sensitive detection of content has advantages such as low background, high selectivity, low cost, simple instrument operation and portability. It is expected to play an important role in the field of biomedical analysis and detection, and provide important theoretical and experimental support for the diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of photoelectrochemical aptamer sensor technology, specifically to a method for preparing a Schiff base-based photoelectrochemical aptamer sensor based on covalent organic frameworks (COFs) and its application in subtype β-amyloid protein Aβ. 42 Applications in detection. Background Technology

[0002] With the continuous growth of my country's economy and significant changes in its social population structure, the age-related diseases brought about by an aging society have received increasing attention, especially neurodegenerative diseases such as Alzheimer's disease (AD), which have become a major challenge seriously affecting the health and quality of life of the elderly. Increasing evidence suggests that Aβ protein aggregation-induced neurotoxicity may be a major cause of AD. Aβ protein aggregation is a highly complex and reversible process. Due to the incorrect hydrolysis of amyloid precursor protein (APP) by β-secretase, Aβ monomers rapidly aggregate to form oligomers during the nucleation stage. Metastable oligomers further aggregate into fibrils, which undergo secondary nucleation to aggregate into fibers and plaques, accumulating in the interstitial space of brain cells, leading to a series of cranial nerve dysfunctions. The most common Aβ subtype in the human body is Aβ2. 40 and Aβ 42 Due to Aβ 42 With two more amino acids and its more compact secondary conformation, Aβ 42 It is more prone to misfolding and aggregation, more likely to form plaque-like deposits, and generally has stronger neurotoxicity. Studies have shown that plasma Aβ levels in patients with mild AD are higher. 42 The concentration increases significantly, but decreases as the condition worsens. Therefore, Aβ... 42 The test not only helps in early diagnosis, but also reflects the progression of the disease.

[0003] Covalent organic frameworks (COFs) are a class of crystalline organic porous polymers with permanent porosity, composed of small monomers linked by covalent bonds. They possess characteristics such as pre-designability, tunable and uniform pore size, and ease of functionalization. Due to the diversity of monomers, topologies, and bonds used in their construction, COFs provide a new platform for the structural design and functional development of organic polymers. Over the past two decades, COF materials have shifted their focus from structural design to functional design. In recent years, their optical and electrical properties have also been gradually explored. The tunable pore size of COFs and their ease of linking to functional groups targeting Aβ proteins demonstrate significant advantages for the specific recognition of Aβ proteins.

[0004] Photoelectrochemical (PEC) sensors are an analytical method that combines electrochemistry and photochemistry, possessing the advantages of both. They can detect substances with and without electrochemical activity and have attracted much attention due to their advantages of low background, high sensitivity, rapid and stable response, and no need for sample pretreatment. Because the energy forms of the excitation source (light) and the detection signal (electricity) are different, PEC sensing platforms have higher sensitivity and signal-to-noise ratios than traditional electrochemical sensing methods. Furthermore, the electronic readout system of PEC sensing platforms makes them simple and easy to miniaturize, while optical bioanalysis requires complex and expensive equipment. Highly sensitive PEC biosensors can achieve the detection of low concentrations of Aβ protein.

[0005] This invention, supported by the Natural Science Foundation of Hubei Province (2024AFB496), the Open Fund of Hubei Three Gorges Laboratory (SK240006), the Enshi Prefecture "Sailing Special Project" Science and Technology Program (Research on the Performance of Photoelectric Responsive Covalent Organic Framework Materials), and the Hubei Minzu University Internal Research Project (BS24057, XN24034), develops a method for real-time monitoring of Aβ. 42 A photoelectrochemical aptamer sensor has been developed. This sensor has a simple preparation method and excellent performance, and is expected to play an important role in the field of biomedical analysis and detection. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a photoelectrochemical aptamer sensor based on porphyrin COFs and its application in Aβ. 42 Applications in protein monomer identification and detection.

[0007] In a first aspect, the present invention provides a method for real-time monitoring of Aβ based on porphyrin COFs. 42 The method for preparing a protein monomer photoelectrochemical aptamer sensor includes the following steps:

[0008] S1. Using two small organic molecules with symmetrical structures, each containing two aldehyde groups and four amino groups respectively, as building monomers, COFs thin films are grown in situ on the surface of indium tin oxide (ITO) or fluorine-doped tin oxide (FTO) electrodes via Schiff base condensation reaction under the catalysis of Lewis acids, such as acetic acid, aspartic acid, hydroxyproline, and proline, to form a photoelectric conversion layer and obtain modified electrode COFs / M, where M is ITO or FTO.

[0009] S2. Activation of carboxyl-modified Aβ by a coupling agent EDC / NHS mixed solution. 42 The aptamer Apt solution was used to place the COFs / M modified electrode obtained in S1 into the activated Apt solution. The amino groups at the ends of COFs and the carboxyl groups modified by Apt were covalently bonded through amide bonds to obtain the Apt / COFs composite film modified electrode Apt / COFs / M.

[0010] S3. A blocking agent is added to the surface of the modified electrode Apt / COFs / M to block non-specific recognition sites. After blocking, the composite electrode MCH / Apt / COFs / M is obtained, which enables rapid and sensitive detection of Aβ. 42 Photoelectrochemical aptamer sensors.

[0011] Furthermore, before the in-situ synthesis of COFs thin films in step S1, the electrodes are pretreated, specifically by alternately washing the ultrasonic electrodes with anhydrous ethanol and ultrapure water.

[0012] Furthermore, the small organic molecules in step S1 are 2,5-dihydroxyterephthalaldehyde (DHTP) and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP), and even further, the molar ratio of DHTP to TAPP is 2:1. Preferably, the amino group of TAPP reacts with the aldehyde group of DHTP in a Schiff base reaction for 3 hours.

[0013] Furthermore, in step S1, a solvent is added during the Schiff base condensation reaction. The solvent is a mixed solvent of chloroform and dichloroform (preferably chloroform and dichloroform in a volume ratio of 3:1).

[0014] Furthermore, in the EDC / NHS mixed solution of the coupling agent activating Apt in step S2, the molar concentration ratio of EDC and NHS is 1:1, preferably both are 100 mmol / L.

[0015] Furthermore, in step S2, the concentration of Apt ranges from 0.5 to 3.0 μmol / L (optimal concentration 2 μmol / L), and the incubation time ranges from 0.5 to 4 h (optimal time 3 h).

[0016] Furthermore, in step S3, the blocking agent for non-specific adsorption is 6-mercaptohexanol (MCH) with a concentration of 2 mmol / L and a blocking time of 30 min.

[0017] Secondly, the present invention also provides a rapid and sensitive detection method for Aβ. 42 The photoelectrochemical aptamer sensor of the protein monomer was prepared using the above-described method.

[0018] Thirdly, the present invention also provides the above-mentioned photoelectrochemical aptamer sensor for detecting Aβ. 42 Applications of protein monomer concentrations, via Aβ 42 Quantitative analysis of the difference between photocurrent and dark current in protein-modified sensors (Aβ) 42 Protein monomer concentration, if the sample contains Aβ 42 Protein monomers will reduce the photocurrent response, thereby affecting Aβ. 42Qualitative analysis of protein monomers was performed; the magnitude of the decrease in photocurrent response was related to the Aβ content in the sample. 42 The concentration of protein monomers is directly proportional to the concentration of Aβ in the sample, thereby allowing for the determination of Aβ in the sample. 42 Quantitative analysis of protein monomers was performed.

[0019] Furthermore, the photoelectrochemical aptamer sensor detects Aβ. 42 The specific steps for determining protein monomer concentration are as follows:

[0020] (1) Different concentrations of Aβ were drop-coated onto the photoelectrochemical aptamer sensor respectively. 42 Protein monomer solution;

[0021] (2) Using a saturated silver chloride electrode as the reference electrode and a platinum wire electrode as the counter electrode, Aβ 42 A three-electrode system was constructed by modifying the MCH / Apt / COFs / ITO electrode as the working electrode.

[0022] (3) Place the three-electrode system in a phosphate buffer solution;

[0023] (4) Using 0V as the bias potential, without the need for additional electron donor / acceptor adjustment, current-time curves were measured under alternating light-on / off conditions, and the concentrations of Aβ at different concentrations were calculated. 42 The photocurrent response value of the protein monomer is lower than that without Aβ. 42 The difference between protein monomers is used to fit a linear curve;

[0024] (5) Take different concentrations of Aβ from (1) 42 Replace the protein monomer with the Aβ-containing monomer to be detected. 42 The protein monomer solution was subjected to the above (1)-(4) to calculate the photocurrent response value compared to that without Aβ. 42 After calculating the difference in protein monomers, the Aβ-containing protein to be detected is calculated based on the linear curve. 42 Aβ in protein monomer solution 42 Protein monomer concentration.

[0025] The photoelectrochemical aptamer sensor prepared above was applied to Aβ 42 The detection of protein monomers has a wide linear range of 1.0 pmol to 1.0 μmol / L. Within this concentration range, the sensor sensitivity is 0.6470 μA / (pmol / L) and the detection limit is 0.8131 pmol / L.

[0026] Compared with the prior art, the advantages and beneficial effects of the method of the present invention are as follows:

[0027] (1) The present invention provides a rapid and sensitive detection of Aβ 42The photoelectrochemical aptamer sensor based on protein monomers was developed by in-situ synthesis of a MCH / Apt / COFs / ITO composite film on the surface of an ITO electrode. This composite film is a novel sensing film, prepared for the first time, and is used for Aβ. 42 Protein monomer photoelectrochemical aptamer sensor;

[0028] (2) The rapid and sensitive detection of Aβ in this invention 42 A method for fabricating a protein monomer photoelectrochemical aptamer sensor was developed. A simple Schiff base condensation and amide covalent bonding method were used to prepare the MCH / Apt / COFs / ITO photoelectrochemical aptamer sensor. Scanning electron microscopy characterization showed that the MCH / Apt / COFs / ITO surface was uneven, resulting in a large specific surface area of ​​the electrodes and high photoelectric conversion efficiency, which is beneficial for improving the sensor's sensitivity. Experimental results showed that the prepared photoelectrochemical aptamer sensor has advantages such as a wide linear range, high sensitivity, low detection limit, short response time, high selectivity, good stability, and good reproducibility. Attached Figure Description

[0029] Figure 1 For the rapid and sensitive detection of Aβ of the present invention 42 Fabrication route of protein monomer photoelectrochemical aptamer sensors;

[0030] Figure 2 A roadmap for the synthesis of TAPP / DHTP-COFs;

[0031] Figure 3 Scanning electron microscope (SEM) characterization images of the COFs / ITO modified electrode surface;

[0032] Figure 4 The Fourier transform infrared (FT-IR) spectra of COFs are shown.

[0033] Figure 5 Photocurrent response curves for different modified electrodes;

[0034] Figure 6 The photoelectric response current curves for different concentrations of Apt are shown.

[0035] Figure 7 The photoelectric response current curves of Apt at different incubation times;

[0036] Figure 8 The prepared MCH / Apt / COFs / ITO modified electrode was tested for different concentrations of Aβ. 42 Time-current curve (linear graph);

[0037] Figure 9 For Aβ 42Stability curves of protein monomer photoelectrochemical aptamer sensors;

[0038] Figure 10 For Aβ 42 Selectivity histogram of protein monomer photoelectrochemical aptamer sensor;

[0039] Figure 11 For Aβ 42 Bar chart showing the reproducibility of protein monomer photoelectrochemical aptamer sensors. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the implementation methods of the present invention, and not all of the implementation methods. Based on the implementation methods of the present invention, all other implementation methods obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] In the following examples, the indium tin oxide (ITO) conductive glass electrode was purchased from South China Xiangcheng Technology Co., Ltd. (size: 40mm × 10mm × 1mm). Apt was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0042] Example 1

[0043] A rapid and sensitive detection method for Aβ 42 Methods for preparing photoelectrochemical aptamer sensors based on protein monomers, such as... Figure 1 As shown, it includes the following steps:

[0044] S1. At room temperature (25℃), COFs-modified ITO electrodes (COFs / ITO) were synthesized in situ on an ITO electrode via Schiff base condensation reaction using 2,5-dihydroxyterephthalaldehyde (DHTP) and the photosensitizer 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP) as monomers, catalyzed by acetic acid (HAc). The reaction route is as follows: Figure 2 As shown;

[0045] S2. The COFs / ITO modified electrode surface obtained in S1 was then drop-coated with activated 2.0 μmol / L Aβ. 42 An Apt / COFs composite film modified ITO electrode (Apt / COFs / ITO) is prepared by covalently bonding the amino group at the end of COFs with the carboxyl group modified by Apt through an amide bond in an aptamer Apt solution.

[0046] S3. MCH was added to the surface of the modified electrode Apt / COFs / ITO obtained in S2 to block the non-specific recognition sites. After blocking, the composite electrode MCH / Apt / COFs / ITO was obtained to detect different concentrations of Aβ.42 Protein monomers, i.e., rapid and sensitive detection of Aβ 42 A photoelectrochemical aptamer sensor for protein monomers; wherein the MCH concentration is 2 mmol / L, the addition volume is 20 μL / ITO, and the blocking time after addition is 30 min.

[0047] S4. Drop-coat 20 μL of Aβ onto the surface of the prepared modified electrode MCH / Apt / COFs / ITO. 42 Proteins were kept moist to incubate different concentrations of Aβ. 42 Protein monomers were used to obtain different concentrations of Aβ 42 The MCH / Apt / COFs / ITO was placed in PBS solution and subjected to current-time curve testing under LED white light (40mW) to obtain the photoelectric response curve.

[0048] It should be noted that S1 in Example 1 specifically includes: placing ITO in a 7mL centrifuge tube, adding 2,5-dihydroxyterephthalaldehyde (DHTP), photosensitizer 5,10,15,20-tetratetra(4-aminophenyl)porphyrin (TAPP), solvent, and acetic acid. The masses of TAPP and DHTP used for in-situ growth on the ITO electrode surface are 0.84mg and 0.40mg, respectively. The solvent is a mixed solution of chloroform and dichloroform (volume ratio 3:1), with a volume of 1400μL. The amino group of TAPP reacts with the aldehyde group of DHTP in a Schiff base reaction for 3 hours. The TAPP and DHTP used are purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the chloroform and dichloroform are from China National Pharmaceutical Group Co., Ltd. The ITO electrode of the in-situ grown COFs film has an indium tin oxide conductive film on one side, with a range of 10mm × 10mm and an area of ​​1cm². 2 The concentration of acetic acid catalyst was 10 mol / L, and the volume added to each centrifuge tube was 10 μL. The in-situ grown DHTP / TAPP-COFs film is more uniform, thus exhibiting better photoelectric response performance and improving the sensor's detection sensitivity.

[0049] In Example 1, S2 specifically includes: In Example 1, the coupling agent for activating Apt is an EDC / NHS mixed solution, wherein the concentration of EDC is 100 mmol / L and the concentration of NHS is 100 mmol / L. 20 μL of the EDC / NHS mixed solution is used to activate 20 μL of 2 μmol / L Apt for 30 min. The concentration of Apt is 2 μmol / L, and the incubation time is 3 h, thus obtaining the Apt / COFs composite film modified ITO electrode (Apt / COFs / ITO).

[0050] In Example 1, S3 specifically involves: drop-coating 20 μL of 2 mmol / L mercaptohexanol (MCH) onto the surface of the Apt / COFs / ITO obtained in S2 to seal non-specific recognition sites, thus preparing the composite electrode MCH / Apt / COFs / ITO, i.e., a photoelectrochemical aptamer sensor. The π-conjugated structure and excellent stability of COFs give the COFs / ITO electrode good photoelectric conversion efficiency, thereby shortening the Aβ... 42 The response time of a protein monomer photoelectrochemical aptamer sensor. In the construction of the photoelectrochemical aptamer sensor, based on Aβ... 42 Apt, customized with amino acid sequence, is used as an electrode modifier to improve sensor selectivity.

[0051] In Example 1, S4 specifically includes: a PBS solution with a concentration of 0.1 μmol / L, a pH of 7.40, and a volume of 20 μL. The electrolytic cell used in the test has a volume of 5 mL and a light window of 1 cm. 3 The light used in the test was white LED light with a wavelength range of 400–700 nm, and the on / off time was alternated between 30 s and 20 s.

[0052] It should be noted that in Example 1, before the in-situ synthesis of DHTP / TAPP-COFs film on the surface of ITO electrode in S1, the following steps are also included: pre-treatment of ITO electrode, specifically: cleaning ITO electrode in an ultrasonic cleaner by alternating use of anhydrous ethanol and ultrapure water, and then drying the electrode at room temperature for later use.

[0053] Example 1: A DHTP / TAPP-COFs film was generated on an ITO electrode using an in-situ synthesis method. Then, the amino groups at the ends of the COFs were covalently bonded to the carboxyl groups modified by Apt using an amide method. Finally, MCH was added to block the non-specific recognition sites of the modified electrode Apt / COFs / ITO, thus preparing a novel Aβ-coated COFs film. 42 Protein monomer photoelectrochemical aptamer sensor, this Aβ 42 Protein monomer photoelectrochemical aptamer sensors possess excellent properties such as wide linear range, high sensitivity, low detection limit, short response time, high selectivity, good stability, and good reproducibility.

[0054] Performance testing

[0055] 1. SEM characterization of COFs-modified electrode surface

[0056] The surface morphology of the COFs-modified ITO electrode in Example 1 was characterized using SEM technology, and the results are as follows: Figure 3 As shown.

[0057] from Figure 3As can be seen, a highly uniform, autumn-leaf-shaped thin film structure is clearly visible on the surface of the COFs-modified ITO electrode. The entire surface is uneven and possesses a large specific surface area. Therefore, the uneven surface structure of the COFs-modified ITO electrode endows it with higher photoelectric conversion efficiency and better enrichment ability, thereby increasing the number of exposed amino groups on the surface, enabling it to connect with more aptamers, and further enhancing its affinity for Aβ. 42 Sensing sensitivity of protein monomers.

[0058] 2. FT-IR characterization of COFs materials

[0059] The chemical structure of COFs grown in situ in centrifuge tubes according to the S1 synthesis method in Example 1 was characterized by Fourier transform infrared spectroscopy to assess whether imine condensation between amino and aldehyde groups occurred. The results are as follows: Figure 4 As shown.

[0060] from Figure 4 As can be seen from the data, the COFs material grown in situ in Example 1 was grown at a height of 1619 cm⁻¹. -1 The presence of a distinct -C=N- bond characteristic peak indicates that a Schiff base condensation reaction successfully occurred between the amino group of TAPP and the aldehyde group of DHTP.

[0061] 3. Photoelectrochemical response on different modified electrodes

[0062] The COFs / ITO modified electrode prepared in Example 1S1, the Apt / COFs / ITO modified electrode prepared in Example 1S2, and the Aβ electrode prepared in Example 1S4 were tested respectively. 42 Aβ at a concentration of 1 nmol / L 42 The photoelectrochemical response on the / MCH / Apt / COFs / ITO modified electrode is as follows: Figure 5 As shown.

[0063] The specific testing method is as follows: using a saturated silver chloride electrode as the reference electrode and a platinum wire electrode as the counter electrode, Aβ... 42 A three-electrode system was constructed using either Apt / COFs / ITO or Apt / COFs / ITO modified electrodes as the working electrodes. Using 0.1 mol / L phosphate buffer solution (pH 7.40) as the substrate, and with a bias voltage of 0 V, the current-time curves were measured to evaluate the COFs / ITO modified electrode, the Apt / COFs / ITO modified electrode, and the Aβ-modified electrode. 42 Photoelectrochemical response on / MCH / Apt / COFs / ITO modified electrode. Figure 5The results showed that the COFs / ITO modified electrode exhibited excellent photoelectrochemical response, with a photocurrent response of approximately 11.11 μA. On the Apt / COFs / ITO modified electrode, due to the non-conductivity of Apt, electron transport was slowed, resulting in a photocurrent response of approximately 7.01 μA. The reaction between Apt and 100 pmol / L Aβ... 42 Following covalent linking of protein monomers, electron transport in the protein was further slowed down, and the photocurrent response subsequently decreased to approximately 5.23 μA. Different concentrations of Aβ... 42 The degree to which protein monomers delay electron transport varies; therefore, the sensor can detect Aβ in the sample. 42 Detection of protein monomers.

[0064] 4. Effects of Apt concentration and incubation time on photoelectric response

[0065] Following the method in Example 1S2, the concentrations of the Apt solution were changed to 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L, and 3 μmol / L. Then, the composite electrode Apt / COFs / ITO was prepared using the same method as in Example 1. The photoelectric response of the photoelectrochemical aptamer sensors prepared with different concentrations of Apt was tested, and the results are as follows: Figure 6 As shown. From Figure 6 As can be seen from this, when the concentration of Apt is 2 μmol / L, the photocurrent of the sensor decreases and gradually reaches a stable state. Therefore, the optimal concentration of Apt is 2 μmol / L.

[0066] Following the method in Example 1S2, the incubation time was changed to 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, and 4h. Then, the composite electrode Apt / COFs / ITO was prepared using the same method as in Example 1. The photoelectric response of the photoelectrochemical aptamer sensor prepared with different Apt incubation times was tested, and the results are as follows: Figure 7 As shown. From Figure 7 As can be seen from this, when the incubation time of Apt is 3 hours, the decreasing trend of sensor photocurrent decreases and gradually reaches stability. Therefore, the optimal incubation time of Apt is 3 hours.

[0067] 5. Aβ 42 Linear range, sensitivity, and detection limit of protein monomer photoelectrochemical aptamer sensors

[0068] Current-time curves were tested under alternating light and dark conditions to evaluate the effect of the MCH / Apt / COFs / ITO modified electrode prepared in Example 1S3 on Aβ. 42The quantitative analysis capability of protein monomers, specifically the MCH / Apt / COFs / ITO modified electrode prepared in Example 1, for different concentrations of Aβ. 42 The decrease in photoelectric response of protein monomers, the results are as follows: Figure 8 As shown.

[0069] The specific experimental method is as follows: according to S4 in Example 1, the prepared MCH / Apt / COFs / ITO modified electrode surface is further incubated with different concentrations of Aβ. 42 The protein monomer was placed in 5 mL of 0.1 mol / L phosphate buffer solution (pH 7.40) at a bias voltage of 0 V. Simultaneously, light (LED white light, wavelength range 400–700 nm) and darkness were alternately provided to achieve the amperometric response. Specifically, 30 s (light) and 20 s (dark) cycles were alternated to perform current-time curve measurements. The difference between the current under light and dark conditions was calculated to evaluate its photoelectric response. The photoelectric response was also calculated for different concentrations of Aβ. 42 The photocurrent response value of the protein monomer is lower than that without Aβ. 42 The difference between protein monomers is fitted to a linear curve, such as... Figure 8 As shown. The data indicates that, with Aβ 42 As the concentration of the protein monomer increases, the difference in decrease gradually widens, starting from 0.57 μA (corresponding to 1.0 pmol / L of Aβ). 42 The protein monomer concentration increased to 4.40 μA (corresponding to 1.0 μmol / L Aβ). 42 (Protein monomers). Figure 8 It can be seen that, within the concentration range of 1.0 pmol / L to 1.0 μmol / L, the difference in photocurrent response is related to Aβ. 42 The logarithm of the protein monomer concentration showed a good linear relationship, i.e., ΔI(μA) = 0.6470logc(pmol / L) + 0.5311, R 2 =0.9991, within this concentration range, the sensor's sensitivity is 0.6470 μA / (pmol / L) (slope of the linear equation), and the detection limit is 0.8131 pmol / L. The detection limit is calculated using the formula D = 3N / S, where D is the detection limit, N is the noise signal (6.98 μA), and S is the sensitivity. The results indicate that the prepared Aβ... 42 Protein monomer photoelectrochemical aptamer sensors have advantages such as wide linear range, high sensitivity, and low detection limit.

[0070] 6. Aβ 42 Stability, selectivity and reproducibility of protein monomer photoelectrochemical aptamer sensors

[0071] Stability is a crucial evaluation metric for biosensors. The photoelectrochemical aptamer sensor prepared in this invention exhibits stability at 10 pmol / L Aβ. 42 After incubation in the protein monomer, tests were conducted under five conditions, and the photocurrent response was recorded under 14 on / off irradiation cycles. The results are as follows: Figure 9 As shown, the sensor maintains a stable photocurrent response after multiple cycles (680s) with a relative standard deviation of 7.7%, demonstrating that the sensor has good stability.

[0072] There are many factors in living organisms that affect the detection of Aβ. 42 Since protein monomers are biological substances, based on the detection in Example 5, the photoelectrochemical aptamer sensor in Example 1 was investigated for its effect on Aβ by adding various interfering agents. 42 Selectivity of protein monomers. Specifically, the Aβ prepared in Example 1 was studied using current-time curves. 42 The selectivity of the MCH / Apt / COFs / ITO modified electrode was assessed. The MCH / Apt / COFs / ITO modified electrode prepared in Example 1 was further incubated with 1.0 nmol / L of glucose, dopamine, insulin, and Aβ, respectively. 42 Fibrous tissue replaces 100 pmol / L of Aβ 42 Protein monomers, with a bias voltage of 0V, were placed in 5 mL of 0.1 mol / L phosphate buffer solution at pH 7.40. Simultaneously, alternating light and dark environments (30 s light and 20 s dark) were provided to achieve the amperometric response, and current-time curves were performed. The difference between the current under light and dark conditions was calculated to evaluate its photoelectric response. Figure 10 As shown, common interfering factors in biological samples such as dopamine, glucose, and insulin affect Aβ. 42 The photocurrent effects of the protein monomers were 5.4%, 1.5%, and 11%, respectively. These experimental results demonstrate that the Aβ prepared in Example 1... 42 Protein monomer photoelectrochemical aptamer sensors exhibit good selectivity for proteins composed of Aβ. 42 Aβ, a protein monomer polymer 42 The fiber also exhibits a certain degree of selectivity (photocurrent affects 54%).

[0073] Reproducibility is also an important reference performance of the sensor. Five MCH / Apt / COFs / ITO modified electrodes prepared in Example 1 were incubated with 1.0 pmol / L Aβ solution using the same method. 42 The relative standard deviation of the protein monomer detection results, when performed in parallel under the same conditions, was 9.1%. Figure 11 The results show that, in analytical applications, this photoelectrochemical aptamer sensor is effective for Aβ. 42The determination of protein monomers has good reproducibility.

[0074] In this embodiment, Aβ was prepared using in-situ condensation, covalent linkage, and aptamer-specific recognition methods. 42 / MCH / Apt / COFs / ITO photoelectrochemical aptamer sensor. Scanning electron microscopy characterization revealed that the COFs surface exhibited an uneven, leaf-like structure, resulting in a large specific surface area and high photoelectric conversion efficiency. This application optimized the detection conditions of this photoelectrochemical aptamer sensor, such as the concentration of Apt and the incubation time. Results showed that this Aβ... 42 Protein monomer photoelectrochemical aptamer sensors possess properties such as wide linear range, high sensitivity, low detection limit, high selectivity, and good stability. They exhibit good selectivity for common interfering biomolecules such as glucose, dopamine, and insulin, and are effective against Aβ. 42 Fibrous tissues also have a certain degree of recognition ability.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for real-time monitoring of Aβ based on porphyrin COFs 42 The method for preparing a protein monomer photoelectrochemical aptamer sensor includes the following steps: S1. Using 2,5-dihydroxyterephthalaldehyde (DHTP) and 5,10,15,20-tetra(4-aminophenyl)porphyrin (TAPP) as monomers with symmetrical structures containing two aldehyde groups and four amino groups respectively, COFs thin films are grown in situ on the surface of indium tin oxide (ITO) or fluorine-doped tin oxide (FTO) electrodes via Schiff base condensation reaction under Lewis acid catalysis to form a photoelectric conversion layer, thus obtaining modified electrode COFs / M, where M is ITO or FTO. S2. Activation of carboxyl-modified Aβ by a coupling agent EDC / NHS mixed solution. 42 The aptamer Apt solution was used to place the COFs / M modified electrode obtained in S1 into the activated Apt solution. The amino groups at the ends of COFs and the carboxyl groups modified by Apt were covalently bonded through amide bonds to obtain the Apt / COFs composite film modified electrode Apt / COFs / M. In the EDC / NHS coupling agent mixed solution, the molar concentration ratio of EDC to NHS is 1:1; the concentration range of Apt is 0.5–3.0 μmol / L, and the incubation time ranges from 0.5 to 4 h. S3. A blocking agent is added to the surface of the modified electrode Apt / COFs / M to block non-specific recognition sites. After blocking, the composite electrode MCH / Apt / COFs / M is obtained, which enables rapid and sensitive detection of Aβ. 42 The photoelectrochemical aptamer sensor; the blocking agent is 6-mercaptohexanol (MCH) at a concentration of 2 mmol / L, and the blocking time is 30 min.

2. The preparation method according to claim 1, characterized in that, The Lewis acid is any one of acetic acid, aspartic acid, hydroxyproline, and proline.

3. The preparation method according to claim 1, characterized in that, In step S1, a solvent is added during the Schiff base condensation reaction. The solvent is a mixture of chloroform and dichloroform.

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