A bimodal sensor for detecting bace1 and preparation method and application thereof

Abstract

The present application belongs to the field of biosensors, and relates to a dual-mode sensor for detecting BACE1, in particular to a dual-mode sensor for detecting BACE1 and a preparation method and application thereof. The present application first uses silver nanoparticles co-modified by luminol and short peptides as an electrochemiluminescence and electrochemical dual-mode signal unit, uses a three-dimensional aggregate formed by CB[8] induced aggregation of silver nanoparticles co-modified by luminol and short peptides P2 as a signal probe, carries out signal labeling and signal amplification, and thus improves the sensitivity of BACE1 activity detection. It is worth noting that the dual-mode sensor provides diversified analysis data for the detection of BACE1 activity, and shows high selectivity and repeatability. Compared with a single-mode sensor, the dual-mode sensor can significantly improve the analysis efficiency and accuracy, and has important scientific significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of biosensors and relates to a dual-mode sensor for detecting BACE1, specifically a dual-mode sensor for detecting BACE1, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease is a neurodegenerative disease characterized by the loss of function of nerve cells in different areas of the brain. The gradual formation of extracellular aggregates composed of β-amyloid peptides is a pathological feature of Alzheimer's disease. β-secretase and γ-secretase sequentially cleave amyloid precursor proteins to generate β-amyloid peptides. β-amyloid precursor protein cleavage enzyme 1 (BACE1) is the rate-limiting step in the amyloidosis pathway. Compared with cognitively normal individuals, the concentration and activity of BACE1 protein are elevated in the brains of Alzheimer's patients. Therefore, BACE1 is considered a therapeutic target for early-stage Alzheimer's disease, and the inhibitory effects of different BACE1 inhibitors have been studied.

[0003] Traditional methods for detecting BACE1 activity include high-performance liquid chromatography (HPLC) and mass spectrometry (MS). While reliable, these methods are time-consuming and involve expensive equipment. In recent years, several new methods have been reported for detecting BACE1 activity, such as surface plasmon resonance (SPR), colorimetry, electrochemistry, and fluorescence. Electrochemiluminescence (ECL), a product of the combination of chemiluminescence and electrochemistry, offers advantages such as low background signal, wide dynamic range, high sensitivity, simple control, and simple equipment, making it a powerful tool for immunoassay and clinical diagnosis. However, traditional ECL detection only utilizes the light signal, neglecting the current signal. Electrochemical methods offer advantages such as low cost and miniaturized equipment, but suffer from poor reproducibility and weak resistance to interference. Patent CN113820298A discloses a self-assembled superquenched gold nanoparticle nanosensor. This sensor includes: a peptide probe, a peptide chain cleavable by BACE-1, labeled with a fluorophore; helper DNA labeled with a quencher; and gold nanoparticles. At least one peptide probe and at least one helper DNA are assembled on the surface of the gold nanoparticles. The connection end between the helper DNA and the gold nanoparticle is labeled with a fluorophore, and the fluorophore on the peptide probe and the quencher on the helper DNA are in close proximity. While calculating the concentration of BACE-1 by quenching the Cy5 fluorescence signal significantly improves the sensitivity of BACE-1, it does not achieve dual-mode cross-calibration detection.

[0004] Compared to using any single sensing mode, the combination of electrochemiluminescence and electrochemical techniques can improve the reliability of analysis, while providing more diverse information, lower detection limits, and higher accuracy, and correcting the limitations of any single technique used as a single mode (detection conditions, instrument bias, and signal capacity limitations). Compared to dual-mode sensors labeled with two different probes, sensors constructed with a single probe not only simplify the complex labeling process but also avoid potential interference between the two probes. However, most existing dual-mode sensors refer to the combination of electrochemistry and colorimetry or electrochemiluminescence and colorimetry; there is no dual-mode sensor that combines electrochemistry and electrochemiluminescence for mutual cross-referencing in the detection of BACE1 protein. To further explore the feasibility of a dual-mode sensor for the detection of BACE-1, our research group has conducted long-term research. Summary of the Invention

[0005] This invention proposes a dual-mode sensor for detecting BACE1, its preparation method, and its application. The electrochemiluminescence and electrochemical dual-mode sensor of this invention have good specificity, wide detection range, and high sensitivity, and can be used for screening statin inhibitors. The detection limit of electrochemiluminescence is 33.11 pM, and the detection limit of electrochemical is 53.19 pM.

[0006] The technical solution of this invention is implemented as follows:

[0007] A dual-mode sensor for detecting BACE1 includes a glassy carbon electrode modified with three-dimensional aggregates, the three-dimensional aggregates being modified onto the glassy carbon electrode by β-cyclodextrin, wherein the three-dimensional aggregates refer to aggregates of cucurbita[8]urea, peptide P1 and signal unit; the signal unit is silver nanoparticles co-modified with luminol and short peptide P2; the sequence of peptide P1 is shown in SEQ ID No.1, and its 5' end is modified with adamantane; the sequence of short peptide P2 is shown in SEQ ID No.2.

[0008] In the above-described method for preparing a dual-mode sensor for detecting BACE1, an adamantane (ADA)-functionalized peptide P1 (FEADLNVESIEETK-ADA) is used as a substrate for BACE1 and is linked to a β-cyclodextrin-modified glassy carbon electrode via a host-guest interaction between β-cyclodextrin and adamantane. This modified electrode is then incubated in a supramolecular cucurbit[8]urea solution, where cucurbit[8]urea is assembled at the nitrogen terminus of the substrate peptide via a host-guest interaction between cucurbit[8]urea and the substrate peptide. Silver nanoparticles co-modified with luminol and short peptide P2 (FGDDPPPPC) exhibit both electrochemical and electrochemiluminescent activities and are used as a dual-mode signal unit. This signal unit is induced to aggregate into a three-dimensional aggregate by supramolecular cucurbit[8]urea. The three-dimensional aggregate is used as a signal probe for signal amplification, and the signal probe is labeled at the nitrogen terminus of the substrate peptide via a host-guest interaction between cucurbit[8]urea and short peptide P2 (FGDDPPPPC). In the presence of BACE1, the substrate peptide is cleaved, causing the short peptide (FEAD) to detach from the glassy carbon electrode surface. The signal probe labeled on the substrate peptide is also reduced, resulting in a decrease in electrochemiluminescence and electrochemical signal. The activity of BACE1 is detected by using the relationship between the decrease in signal and BACE1 activity.

[0009] Specifically, the preparation method of the dual-mode sensor for detecting BACE1 described above includes the following steps:

[0010] (1) Synthesis of silver nanoparticle aggregates co-modified with luminol and short peptide P2 induced by cucurbit[8]urea

[0011] First, 3 mL of 5 mM silver nitrate was added to a solution containing 5 mL of ultrapure water and 9 mL of anhydrous ethanol, and the mixture was stirred vigorously. Then, 0.5 mL of a 0.01 M luminol sodium hydroxide solution (0.1 M) was added to the above solution, resulting in a yellow solution. All these steps were performed at room temperature with continuous stirring for 2 h. The color changed from light yellow to dark yellow, indicating the successful formation of luminol-modified silver nanoparticles. Finally, the mixture was centrifuged at 10,000 rpm for 10 minutes to obtain a soft precipitate.

[0012] A certain volume of the P2 peptide stock solution (1 μM) was diluted to 10 nM with 0.1 M phosphate buffer (pH = 7.0) containing 1% tris(dicarboxyethyl)phosphonic acid hydrochloride and incubated at room temperature for 1 hour to activate the thiol groups in the peptide. Subsequently, the prepared luminol-modified silver nanoparticles were incubated overnight at 4°C with a P2 (FGDDPPPPC) stock solution containing 1 mg / mL bovine serum albumin. Bovine serum albumin was used to stabilize the luminol-modified silver nanoparticles and minimize nonspecific adsorption. The mixture was then centrifuged at 12000 rpm for 20 minutes at 4°C to remove excess peptides. Finally, the precipitate was resuspended in 200 μL of ultrapure water to obtain a suspension of luminol and short peptide P2 co-modified silver nanoparticles. After adding 10 μL of cucurbit[8]urea solution to the suspension for 1.5 hours (final concentration of 47.62 μM), a three-dimensional aggregate of silver nanoparticles co-modified with luminol and short peptide P2 was obtained. After centrifugation, the aggregate was redispersed in 100 μL of ultrapure water to obtain a three-dimensional aggregate suspension.

[0013] (2) Assembly of the dual-mode sensor for detecting BACE1

[0014] ① Glassy carbon electrodes with a diameter of 2.5-3.5 mm were successively treated with 1.0, 0.3, and 0.05 μm aluminum oxide polishing powder to polish the electrode surface into a mirror finish. The electrodes were then washed in ethanol and pure water and dried under an infrared lamp.

[0015] ② The glassy carbon electrode treated in step ① was immersed in a 0.05 M PBS solution (pH = 5.0) containing 6 mM β-cyclodextrin as the electrolyte, and β-cyclodextrin was electropolymerized onto the surface of the glassy carbon electrode by cyclic voltammetry. After electropolymerization, the adsorbed substances on the electrode surface were carefully washed with deionized water.

[0016] ③ Immerse the glassy carbon electrode treated in step ② in a 5 μM solution of substrate peptide P1 and incubate overnight at 4°C;

[0017] ④ Remove the electrode treated in step ③, wash it with 0.1 M pH 7.4 PBS solution, and then soak it in 1.0 mg∙mL⁻¹ water. -1 After incubation in bovine serum albumin for 1 h, the sample was washed with 0.1 M pH 7.4 PBS solution and air-dried.

[0018] ⑤ Remove the electrode treated in step ④, immerse it in the test solution, incubate at 37°C for 60 minutes, then remove it, wash it with 0.1 M pH=7.4 PBS solution and air dry it;

[0019] ⑥ Soak in the three-dimensional aggregate suspension of step (1) and incubate for 2 h. After washing and drying with 0.1 M pH=7.4 PBS solution, an electrochemiluminescence and electrochemical dual-mode sensor for signal amplification detection of BACE1 based on supramolecular cucurbit[8]urea-induced aggregation is obtained;

[0020] The above-described method for using the dual-mode sensor to detect BACE1 involves the following steps:

[0021] a. Immerse the modified glassy carbon electrode in the test solution, incubate at 37°C for 60 minutes, then remove, wash with PBS solution and air dry;

[0022] b. The modified glassy carbon electrode treated in step a was immersed in a 47.62 μM cucurbita[8]urea solution and a 2 mg / mL three-dimensional aggregate suspension in sequence. After incubation, cleaning and drying, the dual-mode sensor electrode was obtained.

[0023] c. Using the dual-mode sensor electrode as the working electrode, the Ag / AgCl (3M KCl) electrode as the reference electrode, and the platinum wire electrode as the counter electrode, electrochemical detection and electrochemiluminescence detection were performed.

[0024] d. The decrease in electrochemical response signal ΔI compared to the blank signal LSV and the decrease in electrochemiluminescence response signal ΔL ECL Substituting these values ​​into the electrochemical linear equation and the electrochemiluminescence linear equation respectively yields the concentration of BACE1.

[0025] The above electrochemical linear equation is ΔI LSV =-2.5948 + 1.5584 log C,R 2 = 0.9980; the linear equation for electrochemiluminescence is ΔL ECL = -1130 + 821 log C,R 2 = 0.9982.

[0026] The application of the aforementioned dual-mode sensor for detecting BACE1 in screening inhibitors of BACE1 is characterized by the following steps: BACE1 at a final concentration of 10 nM is mixed with inhibitors of different gradient concentrations in a solution for 30 min; then the dual-mode sensor is immersed in the mixed solution and incubated at 37 °C for 60 min. After incubation, the sensor is removed, washed with 0.1 M pH=7.4 PBS solution, and dried. The relationship between the intensity of the obtained electrochemical signal or electrochemiluminescence signal and the inhibitor concentration is recorded by detection, and a working curve is plotted to evaluate the inhibitory effect.

[0027] The present invention has the following beneficial effects:

[0028] (1) The electrochemiluminescence and electrochemical dual-mode sensor for detecting BACE1 described in this invention has good specificity, wide detection range, high sensitivity, and fast detection speed. In the presence of BACE1, the substrate peptide is cleaved, causing the short peptide (FEAD) to detach from the glassy carbon electrode surface. The signal probe labeled on the substrate peptide is also reduced, resulting in a decrease in electrochemiluminescence and electrochemical signals. The activity of BACE1 is detected by utilizing the relationship between the decrease in signal and BACE1 activity. The electrochemiluminescence and electrochemical dual-mode sensor described in this invention has good specificity, wide detection range, and high sensitivity, and can be used for screening statin inhibitors. The detection limit of electrochemiluminescence is 33.11 pM, and the detection limit of electrochemical is 53.19 pM.

[0029] (2) This invention is the first to use silver nanoparticles co-modified with luminol and short peptides as electrochemiluminescence and electrochemical dual-mode signal units. The three-dimensional aggregates formed by the aggregation of silver nanoparticles co-modified with luminol and short peptide P2 induced by CB[8] are used as signal probes for signal labeling and signal amplification, thereby improving the sensitivity of BACE1 activity detection. It is worth noting that this dual-mode sensor provides diverse analytical data for the detection of BACE1 activity and shows high selectivity and repeatability. Compared with single-mode sensors, this dual-mode sensor can significantly improve analytical efficiency and accuracy, and has important scientific significance and application value. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The image shows the morphology of the signal probe in Example 1. A is a TEM image of luminol-modified silver nanoparticles, B is an HRTEM image of luminol-modified silver nanoparticles, and C is a TEM image of a three-dimensional aggregate formed by the aggregation of silver nanoparticles modified with luminol and P2 induced by cucurbit[8]urea.

[0032] Figure 2 The fabrication and working principle diagram of an electrochemiluminescence and electrochemical dual-mode sensor for detecting BACE1.

[0033] Figure 3 The electrochemical impedance spectroscopy diagrams for glassy carbon electrodes at different modification stages are shown.

[0034] Figure 4 This is a graph showing the electrochemiluminescence intensity of the dual-mode sensor at different concentrations of substrate peptides.

[0035] Figure 5 The image shows the electrochemiluminescence intensity of the dual-mode sensor at different concentrations of cucurbita[8]urea.

[0036] Figure 6 This is a graph showing the electrochemiluminescence intensity of the dual-mode sensor when the signal probe connection time is different.

[0037] Figure 7 This is a graph showing the electrochemiluminescence intensity of the dual-mode sensor at different pH values.

[0038] Figure 8 The image shows the electrochemiluminescence intensity of the dual-mode sensor at different concentrations of the co-reacting reagent H2O2.

[0039] Figure 9 The images show the electrochemical and electrochemiluminescence signals of the dual-mode sensor when detecting BACE1.

[0040] Figure 10 Standard curves for the decrease in electrochemical signal and the concentration of BACE1 when detecting BACE1 using a dual-mode sensor, and standard curves for the decrease in electrochemiluminescence signal and the concentration of BACE1.

[0041] Figure 11 Electrochemical intensity and electrochemiluminescence intensity signals were obtained when five modified electrodes detected the same concentration of BACE1.

[0042] Figure 12 This is a selective test diagram for a dual-mode sensor.

[0043] Figure 13 This is a graph showing the relationship between inhibitor concentration and inhibitory effect. Detailed Implementation

[0044] 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 some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1

[0046] (1) Synthesis of silver nanoparticle aggregates co-modified with luminol and short peptide P2 induced by cucurbit[8]urea

[0047] First, silver nitrate was added to a mixed solution containing ultrapure water and anhydrous ethanol and stirred vigorously. Then, a sodium hydroxide solution containing luminol was added to the above solution. All these steps were carried out at room temperature with continuous stirring for 2 hours. The color changed from light yellow to dark yellow, indicating the successful formation of luminol-modified silver nanoparticles. Finally, the mixture was centrifuged at 10,000 rpm for 10 minutes to obtain a soft precipitate.

[0048] A certain volume of the P2 peptide stock solution (1 μM) was diluted to 10 nM with 0.1 M phosphate buffer (pH = 7.0) containing 1% tris(dicarboxyethyl)phosphonic acid hydrochloride and incubated at room temperature for 1 hour to activate the thiol groups in the peptide. Subsequently, the prepared luminol-modified silver nanoparticles were incubated overnight at 4°C with P2 stock solution containing 1 mg / mL bovine serum albumin. Bovine serum albumin was used to stabilize the luminol-modified silver nanoparticles and minimize nonspecific adsorption. Then, the mixture was centrifuged at 12000 rpm for 20 minutes at 4°C to remove excess peptide. Finally, the precipitate was resuspended in 200 μL of ultrapure water to obtain a suspension of luminol and short peptide P2 co-modified silver nanoparticles. After adding 10 μL of cucurbit[8]urea solution to the suspension for 1.5 hours (final concentration of 47.62 μM), aggregates of silver nanoparticles co-modified with luminol and short peptide P2 were obtained. After centrifugation, the aggregates were redispersed in 100 μL of ultrapure water for subsequent measurements.

[0049] The morphology of the nanocomposite material synthesized in this invention is as follows: Figure 1 As shown. Figure 1 A shows that the luminol-modified silver nanoparticles are uniform spheres with a particle size of approximately 30 nm; Figure 1 B shows that the luminol-modified silver nanoparticles have high-resolution lattice fringes with a crystal plane spacing of 0.227 nm, which is determined by the (111) spacing of Ag. Figure 1 C shows that cucurbita[8]urea induces the aggregation of silver nanoparticles modified with luminol and short peptide P2 to form a three-dimensional aggregate. The noble metal silver nanoparticles have electrochemical activity, luminol has electrochemiluminescence activity, and the silver nanoparticles have a strong catalytic effect on the electrochemiluminescence of luminol. Therefore, the three-dimensional aggregate can be used as a dual-signal probe for electrochemiluminescence and electrochemical signals, realizing the signal amplification function of the sensor.

[0050] (2) Construction process of electrochemiluminescence and electrochemical dual-mode sensor for signal amplification detection of BACE1 based on supramolecular cucurbit[8]urea-induced aggregation.

[0051] a. Glassy carbon electrodes with a diameter of 2.5-3.5 mm were successively treated with 1.0, 0.3, and 0.05 μm aluminum oxide polishing powder to polish the electrode surface into a mirror finish. The electrodes were then washed in ethanol and pure water and dried under an infrared lamp.

[0052] b. Immerse the glassy carbon electrode treated in step a into a 0.05 M PBS solution (pH = 5.0) containing 6 mM β-cyclodextrin as the electrolyte, and electropolymerize the β-cyclodextrin onto the surface of the glassy carbon electrode using cyclic voltammetry. After electropolymerization, carefully wash the adsorbed material on the electrode surface with deionized water;

[0053] c. Immerse the glassy carbon electrode treated in step b in a 5 μM substrate peptide P1 solution and incubate overnight at 4°C;

[0054] d. Remove the electrode treated in step c, wash it with 0.1 M PBS solution (pH 7.4), and then immerse it in 1.0 mg·mL⁻¹ water. -1 After incubation in bovine serum albumin for 1 h, the sample was washed with 0.1 M pH 7.4 PBS solution and air-dried.

[0055] e. Remove the electrode treated in step d, immerse it in the test solution, incubate at 37°C for 60 minutes, and then remove BACE1;

[0056] f. The glassy carbon electrode treated in step e was washed with 0.1 M pH=7.4 PBS solution and then incubated for 1 h and 2 h in 5 μM cucurbit[8]urea solution and cucurbit[8]urea-induced aggregate suspension, respectively. After washing with 0.1 M pH=7.4 PBS solution and drying, an electrochemiluminescence and electrochemical dual-mode sensor for signal amplification detection of BACE1 based on supramolecular cucurbit[8]urea-induced aggregation was obtained.

[0057] In step (1), 3 mL of 5 mM silver nitrate was added to a solution containing 5 mL of ultrapure water and 9 mL of anhydrous ethanol, and the mixture was stirred vigorously. Then, 0.5 mL of 0.01 M luminol sodium hydroxide solution (0.1 M) was added to the above solution to obtain a yellow solution.

[0058] The sequence of the short peptide P2 in step (1) is FGDDPPPPC.

[0059] The substrate peptide P1 sequence in step (2) is an adamantane (ADA) labeled sequence, denoted as: FEADLNVESIEETK-ADA.

[0060] The construction process of electrochemiluminescence and electrochemical dual-mode sensors is as follows: Figure 2As shown, β-cyclodextrin was modified onto the surface of a treated glassy carbon electrode by electropolymerization. An adamantane (ADA)-functionalized peptide (FEADLNVESIEETK-ADA) was used as a substrate for BACE1 and linked to the β-cyclodextrin-modified glassy carbon electrode via a host-guest interaction between β-cyclodextrin and adamantane. After blocking the active sites on the modified electrode surface with bovine serum albumin, BACE1 cleaved the substrate peptide. The modified electrode was then incubated in a supramolecular cucurbit[8]urea solution, and cucurbit[8]urea was assembled to the N-terminus of the substrate peptide via a host-guest interaction between cucurbit[8]urea and the uncleaved substrate peptide. A signal probe was labeled to the N-terminus of the uncleaved substrate peptide via a host-guest interaction between cucurbit[8]urea and the short peptide (FGDDPPPPC). The activity of BACE1 was detected based on the relationship between electrochemiluminescence and electrochemical signal intensity and BACE1 activity.

[0061] (3) Electrochemiluminescence and electrochemical dual-mode sensors were used for BACE1 activity detection and evaluation of inhibitory effects. The steps are as follows:

[0062] a. A three-electrode system was constructed using an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared working electrode. This system was connected to an electrochemical device. Electrochemical detection was performed by adding 1 M KCl solution to the electrolytic cell (using LSV to record the electrochemical signal, with a potential range of −0.1 V to 0.2 V, a pulse amplitude of 50 mV, a pulse width of 50 ms, and a scan rate of 100 mV s). −1 Based on the relationship between the obtained electrochemical signal quenching values ​​and the concentration of BACE1 buffer, a working curve was plotted;

[0063] b. A three-electrode system was formed using an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared working electrode, and connected to an electrochemiluminescence device: 0.1 M PBS (pH=10.0) containing 0.2 mM H2O2 solution was added to the electrolytic cell for electrochemiluminescence detection. A voltage was applied to the assembled working electrode using cyclic voltammetry (the voltage range of the cyclic voltammetry scan was -0.6~0.6 V, the scan rate was 100 mV / s, and the photomultiplication voltage was 800 V). The working curve was plotted based on the relationship between the obtained electrochemiluminescence signal quenching value and the BACE1 buffer concentration.

[0064] c. BACE1 was mixed with an inhibitor at a specific concentration gradient for 30 min, with the final concentration of BACE1 maintained at 10 nM. The mixture was then drop-coated onto a modified electrode and incubated at 37 °C for 1 h. Subsequent steps were similar to those for BACE1 detection. The feasibility of the prepared electrochemiluminescence and electrochemical dual-mode sensor was tested using the half-inhibition concentration (WIC) of the obtained inhibitor.

[0065] Example 2

[0066] (1) Construction process of electrochemical impedance spectroscopy sensor

[0067] This experiment used electrochemical impedance spectroscopy to characterize the sensor construction process. The electrode interfaces at different stages of the construction process were measured at 5 mM [Fe(CN)6]. 3- / 4- Tested in solution. For example... Figure 3 As shown, after electropolymerization of β-cyclodextrin on a glassy carbon electrode, the charge transfer resistance (Rct) increased from 41.28 ohms to 62.88 ohms. This is due to the decrease in the electronic conductivity of the polyβ-cyclodextrin film. Figure 3 (Curve b in the figure). After assembling the negatively charged peptide fragment P1 onto a poly-β-cyclodextrin-modified glassy carbon electrode, Rct further increased to 215.52 ohms ( Figure 3 Curve c). Subsequently, bovine serum albumin was used to block the remaining active sites on the electrode surface to prevent non-specific adsorption of the electrode, and Rct continued to increase to 267.37 ohms ( Figure 3 Curve d in the figure). When the electrode is incubated with BACE1, Rct decreases to 238.77 ohms ( Figure 3 The curve e in the figure indicates that the presence of BACE1 leads to the cleavage of the substrate peptide. Then cucurbituril [8] is assembled into the N-terminus of the uncleaved substrate peptide P1 via host-guest recognition, and Rct is significantly increased ( Figure 3 The curve f in the figure has an Rct value of 417.06 ohms. Finally, the signal probe was labeled by the host-guest recognition interaction between cucurbita[8]urea and the short peptide P2 on the signal probe, and the Rct value rose to the maximum value ( Figure 3 The curve g in the figure has an Rct value of 492.91 ohms. In summary, each step in the sensor construction process causes a significant change in the electrode surface impedance, indicating that the sensor construction was successful.

[0068] (2) Optimization of detection conditions

[0069] a. Effect of substrate peptide P1 concentration on electrochemiluminescence intensity

[0070] Figure 4 The effect of substrate peptide concentration on electrochemiluminescence intensity was demonstrated. From Figure 4It can be seen that when the substrate peptide concentration is increased to 5 μM, the electrochemiluminescence intensity remains at a relatively stable value, indicating that the amount of substrate peptide modification on the electrode has reached saturation.

[0071] b. Effect of cucurbit[8]urea concentration on electrochemiluminescence intensity

[0072] Figure 5 The effect of cucurbituril concentration on electrochemiluminescence intensity was demonstrated. From [8] Figure 5 It can be seen that when the concentration of cucurbit[8]urea increases to 5 μM, the electrochemiluminescence intensity remains at a relatively stable value.

[0073] c. Effect of incubation time of signal probe on electrochemiluminescence signal

[0074] Figure 6 The effect of incubation time of the signal probe on the electrochemiluminescence signal was demonstrated. The signal probe was modified at the optimal concentrations of substrate peptide and cucurbit[8]urea. After the incubation time of the signal probe was increased to 120 min, the electrochemiluminescence intensity signal value did not change much. Finally, 120 min was selected as the optimal incubation time of the signal probe.

[0075] d. Electrochemiluminescence supports the optimization of electrolyte pH.

[0076] Figure 7 This study demonstrates the effect of electrolyte pH on electrochemiluminescence. For most luminol electrochemiluminescence processes, the optimal reaction conditions are alkaline. In this invention, the pH of the solution was adjusted from 7 to 12, with the highest electrochemiluminescence intensity obtained at pH 10.

[0077] e. Optimization of the concentration of co-reactant H2O2

[0078] Figure 8 The effect of the concentration of the co-reactant H2O2 on the electrochemiluminescence intensity was demonstrated. Under optimal conditions, the concentration of the co-reactant H2O2 was optimized, and the electrochemiluminescence intensity tended to stabilize when the H2O2 concentration was increased to 0.2 mM.

[0079] (3) Drawing the standard curve

[0080] The electrochemical and electrochemiluminescence signals of the dual-mode sensor of the present invention were detected at different concentrations of BACE1. Standard curves were plotted between the quenched electrochemical signal intensity and the BACE1 concentration, as well as between the quenched electrochemiluminescence intensity and the BACE1 concentration. The specific detection steps are as follows:

[0081] a. The glassy carbon electrode modified sequentially with BACE1-cyclodextrin and substrate peptide P1 was immersed in a series of active BACE1 buffer solutions with concentrations of 50 pM, 100 pM, 200 pM, 600 pM, 1 nM, 5 nM, 10 nM, 50 nM and 100 nM. After incubation at 37°C for 60 minutes, the electrode was removed, washed with 0.1 M pH=7.4 phosphate buffer solution and dried. Then, it was incubated sequentially with 5 μM cucurbita[8]urea and signal probe for 1 h and 2 h respectively, washed with 0.1 M pH=7.4 PBS solution and dried.

[0082] b. Using the sensor electrode processed in step a as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode, a three-electrode system is formed and connected to an electrochemical device: 1 M KCl solution is added to the electrolytic cell for electrochemical detection (using LSV to record the electrochemical signal, with a potential range of −0.1 V ~ 0.2 V, a pulse amplitude of 50 mV, a pulse width of 50 ms, and a scan rate of 100 mV s). −1 ).

[0083] c. Using an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the working electrode prepared in step a, a three-electrode system is formed and connected to an electrochemical device: 0.1 M PBS (pH=10.0) containing 0.2 mM H2O2 solution is added to the electrolytic cell for electrochemiluminescence detection (the voltage range of the cyclic voltammetry scan is -0.6~0.6 V, the scan rate is 100 mV / s, and the photomultiplication voltage is 800 V).

[0084] Under optimal conditions, a series of BACE1 concentrations were detected, and the detected signals were significantly lower than the blank signal. Based on this, the electrochemical response signal reduction value ΔI was established. LSV The relationship curve between the concentration of β-amyloid precursor protein lyase 1 and the decrease in electrochemiluminescence response signal ΔL ECL The relationship curve between the concentration of β-amyloid precursor protein lyase 1 and the concentration of β-amyloid precursor protein lyase 1. Figure 9 A represents the concentration of β-amyloid precursor protein lyase 1 and its corresponding electrochemical response signal. Figure 9 B represents the BACE1 concentration and its corresponding electrochemiluminescence response signal. Figure 10 A is the relationship between BACE1 concentration and ΔI. LSV The linear correlation diagram between them shows that the linear regression equation is ΔI. LSV =-2.5948 + 1.5584 log C(pM, R 2 = 0.9980), detection limit 53.19 pM (3δ), Figure 10 B represents the relationship between BACE1 concentration and ΔL.ECL The linear correlation graph between them shows that the linear regression equation is ΔL. ECL = -1130 + 821 log C (pM, R 2 =0.9982), detection limit 33.11 pM (3δ)

[0085] (4) Sensor repeatability test

[0086] Figure 11 Five sensors constructed using the same process were used to detect 1 nM BACE1. The electrochemical results showed a relative standard deviation of 3.8% among the five sensors, and the electrochemiluminescence results showed a relative standard deviation of 3.6% among the five sensors, indicating that the prepared sensors have good reproducibility.

[0087] (5) Sensor selectivity test

[0088] To test the selectivity of the sensor, several different proteases (glucose oxidase, trypsin, horseradish catalase, and acid phosphatase) were selected for interference testing experiments. Figure 12 As shown, 1 nM BACE1 has a certain quenching effect on electrochemical and electrochemiluminescence signals, while 10 nM glucose oxidase, trypsin, horseradish catalase, and acid phosphatase have no significant quenching effect on electrochemical and electrochemiluminescence signals. The quenching effect of the five substances mixed together on electrochemical and electrochemiluminescence signals is almost the same as that of BACE1. Therefore, the dual-mode sensor prepared in this invention has good selectivity.

[0089] Application examples

[0090] To further demonstrate the application of this biosensor in the screening of BACE1 inhibitors, we evaluated the effect of a statin-based peptide mimicry inhibitor, KTEEISEVN-Sta-VAEF, on BACE1 activity. Figure 13 As shown, under constant BACE1 concentration, both electrochemical and electrochemiluminescence signals gradually increased with increasing KTEEISEVN-Sta-VAEF concentration, indicating that BACE1 activity was inhibited by KTEEISEVN-Sta-VAEF.

[0091] Based on the relationship between inhibitor concentration and inhibitor rate, the electrochemical half-inhibition concentration (IC50) is... 50 The estimated half-maximum concentration (IC50) for electrochemiluminescence is 41.83 nM. 50The estimated value is 32.40 nM. This value is consistent with values ​​obtained by other methods, indicating that peptide substrate cleavage depends on BACE1 activity. Our method provides a new technique for screening BACE1 inhibitors.

[0092] 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 dual-mode sensor for detecting BACE1, characterized in that: The invention comprises a glassy carbon electrode with adamantane-functionalized peptide P1, a cucurbita[8]urea solution, and a signal probe solution, wherein the adamantane-functionalized peptide P1 is modified on the glassy carbon electrode by β-cyclodextrin; the signal probe is a three-dimensional aggregate formed by the aggregation of silver nanoparticles co-modified with luminol and short peptide P2 induced by cucurbita[8]urea; the sequence of peptide P1 is shown in SEQ ID No.1, and its 5' end is modified with adamantane; the sequence of short peptide P2 is shown in SEQ ID No.

2.

2. The method for preparing the dual-mode sensor for detecting BACE1 according to claim 1, characterized in that: Using adamantane-functionalized peptide P1 as the detection substrate, it is linked to a β-cyclodextrin-modified glassy carbon electrode through the host-guest interaction between β-cyclodextrin and adamantane. The modified glassy carbon electrode is then incubated in a supramolecular cucurbit[8]urea solution. Cucurbit[8]urea is assembled to the nitrogen terminus of peptide P1 through the host-guest interaction between cucurbit[8]urea and peptide P1. Silver nanoparticles co-modified with luminol and short peptide P2 are used as signal units. These signal units are induced to aggregate into three-dimensional aggregates by cucurbit[8]urea. The three-dimensional aggregates are used as signal probes. The signal probes are labeled to the nitrogen terminus of peptide P1 through the host-guest interaction between cucurbit[8]urea and short peptide P2, thus obtaining a dual-mode sensor for detecting BACE1.

3. The method for preparing a dual-mode sensor for detecting BACE1 according to claim 2, characterized in that, The steps are as follows: (1) Add silver nitrate to an alcoholic aqueous solution and stir vigorously. Then add sodium hydroxide solution containing luminol and continue stirring until the color changes from light yellow to dark yellow. Finally, centrifuge to separate the precipitate. The soft precipitate obtained is luminol-modified silver nanoparticles. (2) Prepare a short peptide P2 stock solution, incubate at room temperature, and then incubate the luminol-modified silver nanoparticles prepared in step (1) with the short peptide P2 stock solution containing bovine serum albumin at 4°C overnight. Then centrifuge to remove excess peptides, and resuspend the precipitate in ultrapure water to obtain a suspension of silver nanoparticles co-modified with luminol and short peptide P2. (3) Add the cucurbit[8]urea solution to the suspension in step (2). After the reaction is complete, centrifuge and redisperse it into ultrapure water to obtain a three-dimensional aggregate suspension. (4) The polished and cleaned glassy carbon electrode was immersed in a PBS solution containing β-cyclodextrin. After electropolymerization, the glassy carbon electrode was cleaned with deionized water and then immersed in peptide P1 solution. It was incubated overnight at 4°C. The resulting glassy carbon electrode was cleaned with PBS solution and then immersed in bovine serum albumin solution. After incubation, it was cleaned with PBS solution to obtain the modified glassy carbon electrode. (5) The modified glassy carbon electrode, the three-dimensional aggregate suspension and the cucurbit[8]urea solution form a dual-mode sensor system.

4. The method for preparing a dual-mode sensor for detecting BACE1 according to claim 3, characterized in that: In step (1), the molar ratio of silver nitrate to luminol is 3:

1.

5. The method for preparing a dual-mode sensor for detecting BACE1 according to claim 4, characterized in that: In step (2), the short peptide P2 stock solution is a phosphate buffer solution with a concentration of 10 nM short peptide P2, wherein the phosphate buffer solution is a 0.1 M phosphate buffer solution containing 1% tris(dicarboxyethyl)phosphine hydrochloride; the concentration of bovine serum albumin in the short peptide P2 stock solution is 1 mg / mL; and the concentration of silver nanoparticles co-modified with luminol and short peptide P2 in the suspension is 1 mg / mL.

6. The method for preparing a dual-mode sensor for detecting BACE1 according to claim 5, characterized in that: In step (3), the final concentration of cucurbita[8]urea was 47.62 μM; in step (4), the concentration of β-cyclodextrin in the PBS solution was 6 mM, the concentration of the PBS solution was 0.05 M and the pH was 5.0; the concentration of peptide P1 solution was 5 μM; and the concentration of PBS solution used to clean the electrode was 0.1 M and the pH was 7.

4.

7. The method for preparing a dual-mode sensor for detecting BACE1 according to claim 3, characterized in that: The concentration of the three-dimensional aggregate suspension in step (5) is 2 mg / mL.

8. The method of using the dual-mode sensor for detecting BACE1 as described in claim 1, characterized in that, The steps are as follows: a. Immerse the modified glassy carbon electrode in the test solution, incubate at 37°C for 60 minutes, then remove, wash with PBS solution and air dry; b. The modified glassy carbon electrode treated in step a was immersed in a 47.62 μM cucurbita[8]urea solution and a 2 mg / mL three-dimensional aggregate suspension in sequence. After incubation, cleaning and drying, the dual-mode sensor electrode was obtained. c. Using a dual-mode sensor electrode as the working electrode, an Ag / AgCl 3M KCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode, electrochemical detection and electrochemiluminescence detection were performed. d. The decrease in electrochemical response signal ΔI compared to the blank signal LSV and the decrease in electrochemiluminescence response signal ΔL ECL Substituting these values ​​into the electrochemical linear equation and the electrochemiluminescence linear equation respectively yields the concentration of BACE1.

9. The method of using the dual-mode sensor for detecting BACE1 according to claim 8, characterized in that: The electrochemical linear equation is ΔI LSV =-2.5948 + 1.5584 log C,R 2 = 0.9980; the linear equation for electrochemiluminescence is ΔL ECL = -1130 + 821 log C,R 2 = 0.9982.

10. The application of the dual-mode sensor for detecting BACE1 as described in claim 1 in screening inhibitors of BACE1, characterized in that, The steps are as follows: BACE1 at a final concentration of 10 nM is mixed with inhibitors of different gradient concentrations in a solution for 30 min. Then, the dual-mode sensor is immersed in the mixed solution and incubated at 37 °C for 60 min. After incubation, the sensor is removed, washed with 0.1 M pH=7.4 PBS solution, and dried. The relationship between the intensity of the obtained electrochemical signal or electrochemiluminescence signal and the inhibitor concentration is recorded by detection, and a working curve is plotted to evaluate the inhibitory effect.

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