Drug screening system, drug screening method based thereon, and drug screening platform

Abstract

The present invention discloses a drug screening system, a drug screening method and a drug screening platform based thereon, wherein the drug screening system comprises: a modified electrode loaded with beta amyloid protein (Aβ), and a Cu-2+-containing electrode selected from Cu-2+-containing electrodes bound to the Aβ. 2+ and Zn 2+ The metal ions bind to the Aβ by applying a solution containing the metal ions to the surface of the Aβ-loaded modified electrode, allowing the solution to stand for 0.5-2 hours, and then washing away excess metal ion solution, wherein the concentration of the metal ions in the solution is not less than 20 μM. This drug screening system can simultaneously screen for drugs that have the effect of disaggregating Aβ-metal ion complexes and determine whether the drugs also have the effect of reducing and / or eliminating oxidative stress, thereby achieving dual-effect drug screening.

Description

Technical Field

[0001] The present invention relates to the field of drug screening, and in particular to a drug screening system, a drug screening method based thereon, and a drug screening platform. Background Art

[0002] The pathogenic mechanism of Alzheimer's disease (AD) remains unclear. Among the many related pathological hypotheses, the classic protein cascade hypothesis believes that the deposition and aggregation of β-amyloid protein (Aβ) in the brain is the main cause of AD. Aβ is a 38-43 residue peptide produced by the action of two enzymes on amyloid precursor protein (APP), of which Aβ40 fragment is the most abundant, accounting for about 90%. Aβ deposition is often accompanied by metal ion imbalance. In the Aβ deposits in the brains of AD patients, Cu 2+ 、Zn 2+ Metal ion concentrations are significantly elevated. Metal ions not only accelerate Aβ aggregation but also exacerbate ROS production, leading to oxidative stress. Research into potential drugs targeting the metal ion-induced Aβ aggregation hypothesis continues to advance, but few have ultimately achieved international acceptance. Effective screening of existing potential drugs is more efficient and necessary than developing new drugs.

[0003] Currently, the effectiveness of potential drugs targeting Aβ is primarily assessed through functional assays that inhibit aggregation / disaggregation. Mainstream assays include chemical dye fluorescence spectroscopy, Western blotting, turbidimetric analysis, immunosensors, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Each of these methods has its own limitations, including the potential for false positives, lengthy assay times, complex pretreatment steps, high sample volume requirements, high cost, and low throughput, limiting their utility as convenient and universal approaches for drug screening. Electrochemical techniques offer high sensitivity, simplicity, and low cost. Numerous electrochemical sensors have been designed for the quantitative detection of various forms of Aβ complexes, where the signal primarily originates from the capture of Aβ in solution by antibodies or aptamers-modified electrodes. Furthermore, these studies typically focus solely on the aggregated form of Aβ, neglecting the generation of reactive oxygen species (ROS), which is closely associated with oxidative stress. Summary of the Invention

[0004] The object of the present invention is to provide a drug screening system, a drug screening method and a drug screening platform based thereon, for screening drugs having the effect of disaggregating Aβ-metal ion complexes and / or having the effect of reducing and / or eliminating oxidative stress.

[0005] To achieve the above objectives, the present invention provides a drug screening system in a first aspect, comprising:

[0006] Aβ-loaded modified electrode, and

[0007] The Aβ-binding 2+ and Zn 2+ of metal ions, among which

[0008] The metal ions bind to the Aβ by the following method:

[0009] The solution containing the metal ions is applied to the surface of the modified electrode loaded with Aβ, left to stand for 0.5-2 hours, and then the excess metal ion solution is washed away. The concentration of the metal ions in the solution is not less than 20 μM.

[0010] In some embodiments of the present invention, the metal ion binds to the Aβ by:

[0011] The solution containing the metal ions was applied to the surface of the modified electrode loaded with Aβ, and allowed to stand for 1 hour, and then the excess metal ion solution was washed away. The concentration of the metal ions in the solution was 50 μM.

[0012] The “excess metal ion solution” here can be understood as the portion of the metal ion solution that is not combined with the Aβ modified electrode.

[0013] In some embodiments of the present invention, the modified electrode loaded with Aβ comprises:

[0014] Conductive electrodes,

[0015] A polydiallyldimethylammonium chloride film covering the surface of the conductive electrode,

[0016] Gold nanoparticles fixed on the polydiallyldimethylammonium chloride film, wherein the gold nanoparticles are connected to glutathione;

[0017] Aβ is linked to the glutathione via an amidation reaction.

[0018] In some embodiments of the present invention, the conductive electrode is selected from a gold electrode, a glassy carbon electrode and an ITO electrode, preferably a gold electrode.

[0019] In some embodiments of the present invention, the polydiallyldimethylammonium chloride film is formed by the following method:

[0020] Coat the surface of the conductive electrode with a polydiallyldimethylammonium chloride solution, let it stand for 10-30 minutes, and then wash away excess polydiallyldimethylammonium chloride solution.

[0021] The “excess polydiallyldimethylammonium chloride solution” herein may be understood as the portion of the polydiallyldimethylammonium chloride solution that is not fixed to the conductive electrode.

[0022] In some embodiments of the present invention, the gold nanoparticles are fixed on the polydiallyldimethylammonium chloride film by the following method:

[0023] A dispersion of gold nanoparticles linked to glutathione is coated on a polydiallyldimethylammonium chloride film, allowed to stand for 20-60 minutes, and then excess dispersion is washed off.

[0024] The “excess dispersion” here can be understood as the dispersion of the gold nanoparticles that is not fixed on the polydiallyldimethylammonium chloride film.

[0025] In some embodiments of the present invention, the gold nanoparticles linked to glutathione are prepared by the following method:

[0026] Add chloroauric acid trihydrate and glutathione to a mixed solution of methanol and glacial acetic acid. After stirring until transparent, add sodium borohydride solution. After the reaction solution turns brown, continue stirring for 1-4 hours.

[0027] In some embodiments of the present invention, the Aβ is linked to the glutathione by the following method:

[0028] Applying an Aβ buffer solution with a concentration of 20-80 μM, preferably 50 μM, to the conductive electrode immobilized with the gold nanoparticles, and letting it stand for 1-3 hours, preferably 2 hours, to allow the amino group of the Aβ to undergo an amidation reaction with the carboxyl group of the glutathione;

[0029] Preferably, before applying the Aβ buffer, the carboxyl group of the glutathione is activated;

[0030] More preferably, after the amidation reaction is completed, the unreacted carboxyl groups of the glutathione are capped.

[0031] The second aspect of the present invention also provides a method for screening drugs capable of disaggregating Aβ-metal ion complexes, wherein the metal ions are selected from Cu 2+ and Zn 2+ , wherein, using the drug screening system provided in the first aspect, the method comprises:

[0032] The drug screening system was placed in [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy was performed in the solution to determine the first charge transfer resistance R CT 0 ;

[0033] allowing the drug screening system and a solution of a first drug to be screened to react;

[0034] The drug screening system after the reaction is placed in the [Fe(CN)6] 3- / 4-Electrochemical impedance spectroscopy was performed in the solution to determine the second charge transfer resistance R CT ';

[0035] Determine the second charge transfer resistance R CT 'With the first charge transfer resistor R CT 0 The charge transfer resistance change rate R' between the two is used to determine whether the first drug to be screened has the effect of disaggregating the Aβ-metal ion complex.

[0036] Specifically, when R' is less than a first threshold, it is determined that the first drug to be screened has the effect of disaggregating the Aβ-metal ion complex.

[0037] In a specific implementation process, the static reaction between the drug screening system and the solution of the first drug to be screened can be performed by inserting the drug screening system into the solution of the first drug to be screened and allowing it to stand for a period of time for reaction.

[0038] [Fe(CN)6] 3- / 4- The solution is a commonly used probe solution in the art, preferably at a concentration of 5 mM, wherein 5 mM [Fe(CN)6] 3- / 4- The composition is 5mM[Fe(CN)6] 3- 、5mM[Fe(CN)6] 4- and 0.1 M NaCl in HEPES buffer.

[0039] In the specific implementation process, the charge transfer resistance change rate R' can be (R CT '-R CT 0 ) / R CT 0 .

[0040] The third aspect of the present invention further provides a method for screening a drug capable of reducing and / or eliminating oxidative stress, wherein the drug screening system provided in the first aspect is used, and the method comprises:

[0041] After the modified electrode loaded with Aβ is immersed in a reducing agent solution for 10-60 minutes, the reducing agent solution is characterized by ultraviolet spectroscopy to determine the first absorbance value A 265 0 , wherein the reducing agent solution contains a second drug to be screened;

[0042] After the drug screening system is immersed in the reducing agent solution for 10-60 minutes, the reducing agent solution is characterized by ultraviolet spectroscopy to determine the second absorbance value A 265 ';

[0043] Determine the first absorbance value A 2650 With the second absorbance value A 265 'The difference ΔA 265 , used to determine whether the second drug to be screened has the effect of reducing and / or eliminating oxidative stress.

[0044] Specifically, when ΔA 265 When the oxidative stress is less than the second threshold, it is determined that the second drug to be screened has the effect of reducing and / or eliminating oxidative stress.

[0045] In some embodiments of the invention, ΔA 265 =A 265 0 -A 265 '.

[0046] The fourth aspect of the present invention further provides an intelligent logic drug screening platform, which includes: a digital comparator, the digital comparator including:

[0047] a first input terminal E, for inputting an EIS experimental result R' of whether the third drug to be screened has an effect of disaggregating the Aβ-metal ion complex; when R' is less than or equal to a first threshold, a high-level signal is input to the first input terminal E; and when R' is greater than the first threshold, a low-level signal is input to the first input terminal E, wherein the EIS experimental result R' is determined according to the method of the second aspect;

[0048] The second input terminal U is used to input the UV test result ΔA of whether the third drug to be screened has the effect of reducing and / or eliminating oxidative stress 265 , in ΔA 265 When the second input terminal U inputs a high level signal, the second input terminal U inputs a high level signal, and ... 265 When the UV test result ΔA is greater than the second threshold, the second input terminal U inputs a low level signal, wherein the UV test result ΔA is determined according to the method described in the third aspect. 265 ;

[0049] a first output terminal D, when a high-level signal is input to the first input terminal E and a low-level signal is input to the second input terminal U, the first output terminal D outputs a high-level signal, which indicates that the third drug to be screened has an effect of disaggregating the Aβ-metal ion complex but does not have an effect of reducing and / or eliminating oxidative stress;

[0050] a second output terminal R, when a low-level signal is input to the first input terminal E and a high-level signal is input to the second input terminal U, the second output terminal R outputs a high-level signal, which indicates that the third drug to be screened has an effect of reducing and / or eliminating oxidative stress, but does not have an effect of disaggregating the Aβ-metal ion complex;

[0051] The third output terminal B, when the first input terminal E inputs a high level signal and the second input terminal U inputs a high level signal, the third output terminal B outputs a high level signal, which indicates that the third drug to be screened has the effect of disaggregating the Aβ-metal ion complex and has the effect of reducing and / or eliminating oxidative stress.

[0052] In a specific implementation process, determining the EIS experimental result R' according to the method of the second aspect includes: placing the drug screening system in [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy was performed in the solution to determine the first charge transfer resistance R CT 0 ;

[0053] allowing the drug screening system and a solution of a third drug to be screened to react;

[0054] The drug screening system after the reaction is placed in the [Fe(CN)6] 3- / 4- Electrochemical impedance spectroscopy was performed in the solution to determine the second charge transfer resistance R CT ';

[0055] Determine the second charge transfer resistance R CT 'With the first charge transfer resistor R CT 0 The charge transfer resistance change rate R' between them.

[0056] In a specific implementation process, determining the UV test result according to the method described in the third aspect includes:

[0057] After the modified electrode loaded with Aβ is immersed in a reducing agent solution for 10-60 minutes, the reducing agent solution is characterized by ultraviolet spectroscopy to determine the first absorbance value A 265 0 , wherein the reducing agent solution contains a third drug to be screened;

[0058] After the drug screening system is immersed in the reducing agent solution for 10-60 minutes, the reducing agent solution is characterized by ultraviolet spectroscopy to determine the second absorbance value A 265 ';

[0059] Determine the first absorbance value A 265 0 With the second absorbance value A 265 'The difference ΔA 265 .

[0060] It can be understood that in the present invention, the first drug to be screened, the second drug to be screened, and the third drug to be screened may be the same or different.

[0061] Beneficial effects

[0062] The present invention provides a drug screening system having at least one of the following advantages.

[0063] First, it can effectively screen out drugs that have the effect of disaggregating Aβ-metal ion complexes, thereby providing a guarantee for the rapid development of anti-AD drugs.

[0064] Furthermore, the drug screening system provided by the present invention can also screen out drugs that have the effect of reducing and / or eliminating oxidative stress, or while screening drugs that have the effect of disaggregating Aβ-metal ion complexes, determine whether the drug also has the effect of reducing and / or eliminating oxidative stress, thereby achieving dual-effect screening of drugs.

[0065] In addition, the drug screening system provided by the present invention has the advantages of short screening time, reusability, and saving Aβ dosage costs.

[0066] The drug screening method and drug screening platform based on the drug screening system also have the above advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The electrochemical impedance spectroscopy characterization results of the modified electrode loaded with Aβ prepared in Example 1 are shown, wherein: Figure 1 (A) shows 5mM [Fe(CN)6] 3- / 4- Nyquist curves of the probe response to (a) bare gold electrode, (b) PDDA film, (c) PDDA / AuNPs film, (d) PDDA / AuNPs / Aβ film, and (e) PDDA / AuNPs / Aβ / EA film electrodes; Figure 1 (B) shows the R at different modification steps. CT Value (the Nyquist curve of the electrode with PDDA layer is approximately a straight line, and the fitted R CT The value is a thick line close to zero), and the illustration in (B) is the Randles equivalent circuit model.

[0068] Figure 2A 、 Figure 2B 、 Figure 2C The AFM (atomic force microscope) characterization results of the modified electrode loaded with Aβ prepared in Example 1 during different modification processes are shown respectively.

[0069] Figure 3 The electrochemical impedance spectroscopy characterization results of the drug screening system corresponding to different preparation conditions when the modified electrode loaded with Aβ prepared in Example 1 is used to prepare the drug screening system are shown, wherein: Figure 3(A) is the Nyquist curve of the modified electrode loaded with Aβ immersed in 50μM Cu(II) buffer solution for different time periods, and the inset shows: R CT The trend of the R value with immersion time in buffer solution containing and not containing 50 μM Cu(II); (B) is the Nyquist curve of the modified electrode loaded with Aβ after immersion in different concentrations of Cu(II) for 2 h, and the inset shows: R CT The value changes with Cu(II) concentration.

[0070] Figure 4 Shown are the Nyquist plots (A) and R' changes (B) of the Aβ-Cu(II) modified electrode when it was alternately treated with 50 μM CQ solution and 50 μM Cu(II) solution.

[0071] Figure 5 (A) shows the UV absorption spectra of AA solution in contact with Aβ-modified electrode and Aβ-Cu(II)-modified electrode in the absence of TMPD; Figure 5 (B) shows the UV absorption spectra of AA solution in contact with Aβ-modified electrode and Aβ-Cu(II)-modified electrode in the presence of TMPD; Figure 5 Middle (C) is the difference in absorbance changes under different conditions; Figure 5 Middle (D) is the ΔA when the Aβ-Cu(II) modified electrode is alternately exposed to 50μM MTPPD and 50μM Cu(II). 265 .

[0072] Figure 6 Shown are the logic circuit (A) and truth table (B) of a single-digit binary comparator.

[0073] Figure 7 A shows the R' of different potential drugs, Figure 7 Panel B shows the ΔA of different potential drugs 265 , where the dotted lines represent the corresponding thresholds, and the error bars represent the standard deviation of the experimental results (n=3); Figure 7 C represents the logic circuit of the comparator logic system and the meaning of each output; Figure 7 Middle (D) shows the logic circuit truth table and classification of four representative drugs; Figure 7 The meanings of the abbreviations are as follows: Buffer represents buffer control, α-KG represents α-ketoglutaric acid, TEMPO represents tetramethylpiperidine, EPI represents epinephrine, DA represents dopamine, VD represents vitamin D, EDTA represents ethylenediaminetetraacetic acid, TMPD represents N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride, Cys represents L-cysteine, and CQ represents chloroiodohydroxyquinoline.

[0074] Figure 8 Indicates that the selection is different from Figure 7 After the threshold is reached, the drug screening results are formed, where A shows the R' of different potential drugs and B shows the ΔA of different potential drugs. 265 , dotted lines indicate the corresponding thresholds, and error bars indicate the standard deviation of the experimental results (n=3); C is the logic circuit truth table and classification of four representative drugs. DETAILED DESCRIPTION

[0075] In order to have a clearer understanding of the technical solution, purpose and effect of the present invention, the specific implementation of the present invention is now described with reference to the accompanying drawings.

[0076] It should be noted that, in the following examples, if specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used without manufacturer specified are all commercially available conventional products.

[0077] First, the preparation process of various raw materials used in the present invention will be described below.

[0078] 1. Preparation of polydiallyldimethylammonium chloride (PDDA) solution:

[0079] Weigh 4 mg of PDDA (MW 20000-35000, 20%) and 58.4 mg of NaCl, add them into 2 mL of deionized water, dissolve and mix thoroughly to obtain 2 mL of 2 mg / mL PDDA solution containing 0.5 M NaCl, store at room temperature, and use 10 μL each time to modify the electrode.

[0080] 2. Preparation of Glutathione-Linked Gold Nanoparticles

[0081] Gold nanoparticles (AuNPs) were prepared by sodium borohydride reduction method.

[0082] To a mixture of 3.0 mL of methanol and 0.5 mL of glacial acetic acid, 19.7 mg of chloroauric acid trihydrate and 7.7 mg of glutathione were added and stirred to dissolve, yielding a bright yellow solution. After rapid stirring for 5 minutes, 1.3 mL of a 0.6 M sodium borohydride solution in ice water was added dropwise, gradually turning brown during the addition. After continued high-speed stirring for 2 hours, the solution was centrifuged at 4000 rpm for 10 minutes in a 30 kD centrifuge tube. The solvent was removed, and the resulting gold nanoparticles were redispersed in 20 mM HEPES buffer, pH 8.0. Washing was repeated several times until the filtrate became colorless, thereby removing undersized particles and obtaining uniformly sized AuNPs. Finally, the prepared AuNPs were diluted and dispersed in 12 mL of 20 mM HEPES buffer and stored at 4°C.

[0083] 3. Preparation of a mixed solution of 400 mM EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 100 mM NHS (N-hydroxysuccinimide)

[0084] Dissolve 38.3 mg of EDC and 5.8 mg of NHS in 0.5 mL of deionized water and mix thoroughly to obtain a fresh mixture of EDC and NHS. Prepare the mixture immediately before each use.

[0085] 4. Preparation of Aβ Buffer

[0086] Aβ was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a concentration of 1 mg / mL and allowed to stand overnight at 4°C to dissolve the prepolymer formed in advance. The solution was aliquoted into 100 μL / tube, i.e., each tube contained 0.1 mg Aβ. The solvent HFIP was blown dry under a nitrogen atmosphere to obtain an Aβ monomer film, which was stored at -18°C. Each time it was used, 460 μL of 20 mM HEPES buffer was added to a single tube and sonicated to dissolve it to obtain a 50 μM Aβ solution.

[0087] 5. Preparation of 0.5 M ethanolamine solution

[0088] Add 2 mL of deionized water to 61.1 mg of ethanolamine liquid to obtain a 0.5 M ethanolamine solution.

[0089] Example 1 Preparation of Aβ-loaded modified electrode

[0090] In this example, a gold electrode was used as a conductive electrode to prepare a modified electrode. First, the gold electrode was polished on deerskin with aluminum oxide powder of particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm in sequence to activate the electrode and improve its electrochemical performance.

[0091] At room temperature, 10 μL of polydiallyldimethylammonium chloride (PDDA) solution (2 mg / mL) was applied to the polished gold electrode surface and allowed to stand for 20 min. The excess solution was washed off, and then 10 μL of AuNPs dispersion was applied and allowed to stand for 40 min. After washing off the excess dispersion, 10 μL of a mixed solution of 400 mM EDC and 100 mM NHS was applied to the electrode surface and allowed to stand for 15 min to activate the carboxyl groups of glutathione on AuNPs. Then, 10 μL of 50 μM Aβ buffer (20 mM HEPES buffer, pH 7.4) was applied and allowed to stand for 2 h. Finally, 0.5 M ethanolamine solution was used to block the excess active sites on the AuNPs to obtain an Aβ-loaded modified electrode, hereinafter also referred to as Au / PDDA / AuNPs / Aβ / EA modified electrode.

[0092] Example 2 Preparation of modified electrode loaded with Aβ-Cu(II)

[0093] The modified electrodes loaded with Aβ prepared in Example 1 were immersed in CuCl2 buffer solutions of different concentrations (0, 25, 50, and 100 μM) formed by dissolving CuCl2 in 20 mM HEPES buffer.

[0094] After a period of time (0.5, 1.0, 15, 2.0 and 3.0 h) in the Cu(II) buffer solution (hereinafter referred to as Cu(II) solution), the electrode was taken out and the excess Cu was washed off. 2+ The solution was obtained by 2+ The modified electrode loaded with Aβ-Cu(II) complex, referred to as Aβ-Cu(II) modified electrode, can be used in the drug screening system provided by the present invention.

[0095] Characterization and testing

[0096] 1. Electrochemical impedance spectroscopy characterization of Au / PDDA / AuNPs / Aβ / EA modified electrode:

[0097] Electrochemical tests were performed using a CHI 660E electrochemical workstation (CH Instruments, Shanghai) with a three-electrode configuration using an Au / PDDA / AuNPs / Aβ / EA modified electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum electrode as the counter electrode. Electrochemical impedance spectroscopy (EIS) measurements were performed using 5 mM [Fe(CN)6] 3- / 4- (5mM[Fe(CN)6] 3- 、5mM[Fe(CN)6] 4- and 0.1M NaCl HEPES buffer), frequency 0.1~10 5 Hz, amplitude 5 mV, voltage 0.17 V. The obtained EIS data were fitted using ZsimpWin software.

[0098] Using the electroactive probe [Fe(CN)6] 3- / 4- The Nyquist curve response of the counter electrode indicates the successful immobilization of the PDDA / AuNPs / Aβ / EA film on the gold electrode surface. In pH 7.0 buffer, the bare gold electrode exhibits a small impedance ( Figure 1 Middle (A), curve a), after the bare gold electrode was modified with positively charged PDDA, the negatively charged probe [Fe(CN)6] 3- / 4- Easier to reach the electrode surface, lower impedance ( Figure 1 (A), curve b). The AuNPs obtained by glutathione reduction are negatively charged in a pH 7.4 buffer solution but have good conductivity. After further modification onto the electrode, the impedance is slightly increased ( Figure 1(A), curve c). However, after the carboxyl groups on the AuNPs surface were activated by EDC / NHS and Aβ was further covalently attached to the electrode surface, the Nyquist semicircle increased significantly ( Figure 1 (A), curve d). This is because Aβ, as a poorly conductive peptide, forms a large steric hindrance on the electrode surface, hindering the probe from diffusing to the electrode surface and conducting electron transfer signals. Finally, EA, which is used to block the excess active sites on the AuNPs surface, enhances electron transfer ( Figure 1 (A), curve e). During the modification process, the R CT The value was also estimated by software fitting, verifying the electrode modification process ( Figure 1 In (B), cyclic voltammetry and atomic force microscopy were also used to verify the successful preparation of Aβ-modified electrodes.

[0099] 2. AFM characterization of Au / PDDA / AuNPs / Aβ / EA modified electrode:

[0100] Considering the relatively simple electrochemical measurements, the height profile and root mean square roughness (Rq) of the modified films were measured and evaluated by atomic force microscopy (AFM) images. The surface coated with PDDA is relatively smooth ( Figure 2A ), compared with the surface roughness significantly increased from 1.7 nm to 7.0 nm after AuNPs modification ( Figure 2B ), which may be due to the different degrees of aggregation of AuNPs, resulting in large fluctuations in the microscopic surface. However, when the flexible long-chain Aβ is further connected to the surface of AuNPs, the grooves are filled and the surface roughness is reduced from 7.0nm to 2.8nm ( Figure 2C ).

[0101] 3. Response of Au / PDDA / AuNPs / Aβ / EA Modified Electrode to Aβ-Cu(II) Aggregation

[0102] The binding of multivalent metal ions, particularly copper, zinc, and iron, to proteins is crucial for maintaining normal cellular function. Disturbances in metal ion homeostasis can affect protein structure and induce oxidative stress, leading to severe neurodegenerative diseases. Using the modified electrode described above as a detection platform and Cu(II) as a representative metal ion, the effects of Aβ on aggregation and reactive oxygen species (ROS) generation at the electrochemical interface were investigated.

[0103] As a sensitive monitoring method related to electron transfer rate, EIS can reflect the changes in the morphology of Aβ on the electrode surface. When Aβ aggregates and hinders the electron transfer on the electrode surface, R CTWhen the Aβ modified electrode was immersed in Cu(II) solution (see Example 2), the electrode impedance gradually increased with the extension of immersion time. After 1 hour, R CT The value reaches the maximum value and remains basically unchanged ( Figure 3 A), 2 hours at R CT The decrease in the value is due to experimental error. CT The value remains basically unchanged. The change trend in Cu(II) solution is obviously different from the control experiment of Aβ electrode in buffer solution ( Figure 3 (A), inset). This indicates that although the slight self-aggregation of Aβ during the immersion time cannot be completely avoided, under this condition, R CT The change in the Aβ concentration is primarily due to Cu(II)-induced Aβ aggregation, forming Aβ-Cu(II) complexes, rather than Aβ self-aggregation. Furthermore, for the Au / PDDA / AuNPs / Aβ / EA-modified electrode, Cu(II)-induced Aβ aggregation can be detected within 1 hour, much faster than the 24 hours required by other common detection methods.

[0104] 4. Response of Au / PDDA / AuNPs / Aβ / EA Modified Electrode to Aβ-Cu(Ⅱ) Depolymerization

[0105] The Au / PDDA / AuNPs / Aβ / EA modified electrode was immersed in Cu(II) solution for 1 hour to obtain an Aβ-Cu(II) modified electrode, which was used to further study the drug depolymerization effect. Chloroquinoline (CQ), which has been reported to regulate metal ion-induced Aβ aggregation, was used as the detection object to verify the depolymerization effect of CQ on Aβ-Cu(II) from the perspective of EIS. CT The rate of change of the value R' (defined as (R CT '-R CT 0 ) / R CT 0 ) for evaluation, where R CT 0 represents the charge transfer resistance value of the Aβ-Cu(II) modified electrode before static reaction with 50 μM CQ in HEPES buffer; R CT ' represents the charge transfer resistance value of the Aβ-Cu(II) modified electrode after 2h of static reaction with CQ solution. The surface impedance of the Aβ-Cu(II) modified electrode was measured again after CQ treatment. CT The value of R decreases significantly, indicating that CQ promotes the disaggregation of Aβ-Cu(II) aggregated on the electrode surface. The electrode is then treated with Cu(II) solution, and Aβ aggregates again under the induction of Cu(II). The above process is repeated three times. According to the Nyquist plot, it can be seen that the R value of the electrode surface under different treatments is CTTo achieve repeated increase and decrease ( Figure 4 (A)), the corresponding R' switches between greater than 0 and less than 0 ( Figure 4 (B)), demonstrating that the modified electrode can be reused as a drug screening system.

[0106] 5. Response of the Au / PDDA / AuNPs / Aβ / EA-modified electrode to ROS generation catalyzed by Aβ-Cu(II) The generation of ROS is a major factor in oxidative stress and is associated with neurodegenerative diseases, including AD. Aβ itself only catalyzes the production of small amounts of ROS, while the Aβ-Cu(II) complex, with the help of common reducing agents in the human body, such as ascorbic acid (AA) and vitamin E, catalyzes the incomplete reduction of O2 to form large amounts of ROS, such as superoxide anions and hydrogen peroxide. Removing Cu(II) from the Aβ-Cu(II) complex can prevent ROS generation and reduce oxidative stress. Therefore, metal chelation therapy is the most commonly used method to reduce ROS.

[0107] The Aβ modified electrode prepared in Example 2 can also be used as a screening system for potential drugs against oxidative stress caused by Aβ-Cu(II). AA is a common antioxidant in the human body, and its ultraviolet absorption peak at 265nm can be used to characterize the generation and consumption of ROS in the system. In the absence of effective drugs, the Aβ-Cu(II) modified electrode was inserted into a 100μM AA HEPES buffer for a contact reaction for 30 minutes, and then the absorbance of the AA solution at 265nm (A) was measured after the Aβ-Cu(II) modified electrode was removed from the AA solution. 265 ), and it was found that it was significantly lower than the absorbance of AA solution in contact with Aβ-modified electrode (not connected to Cu(II)) ( Figure 5 (A)). This indicates that the Aβ-Cu(II) complex catalyzes the reduction of dissolved oxygen to produce ROS, which consumes part of the AA. In the presence of effective drugs such as 50μM TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) (AA solution contains 50μM TMPD), the AA solution A after contacting with the Aβ-Cu(II) modified electrode or the Aβ modified electrode 265 The values ​​are almost the same ( Figure 5 (B)) This demonstrates that TMPD effectively scavenged ROS and protected AA from being consumed. The difference in AA absorbance (ΔA) in the absence and presence of Cu(II) was used to determine the effect of TMPD on the ROS. 265 ) reflects the drug's clearance of ROS induced by metal ions. ΔA 265 If it approaches 0, it means the drug is effective. Otherwise, ΔA 265A larger value indicates that AA is consumed by ROS and the drug fails to work. The electrode was treated alternately with Cu(II) and TMPD to verify the repeatability of the modified electrode in the UV experiment. The experimental data showed that in the measurement of multiple changes in conditions, ΔA 265 There are also significant changes, reflecting the interaction between metal ions and drugs ( Figure 5 The UV experiments demonstrate the role of drugs in eliminating the adverse effects of ROS caused by Cu(II) and the reusability of the modified electrodes as a screening system for compounds that can reduce and / or eliminate oxidative stress.

[0108] 5. Construction of an intelligent logic drug screening platform

[0109] A digital comparator is a commonly used device in digital circuits that can compare two binary numbers with the same bit and determine the relationship between their values. The simplest single-digit comparator has three outputs (such as Figure 6 A in the figure indicates that the numerical relationship between the two inputs is "greater than", "less than" and "equal to". The specific meaning of the comparison result output is shown in Figure 6 Middle B.

[0110] Based on the experimental results and the concept of digital comparator, the inventors tried to use R' and ΔA 265 As input, the drug effect is output, and the three specific outputs are defined as "disaggregation", "anti-ROS" and "dual effect", to construct a drug efficacy judgment device. In order to better combine the logic circuit with the experimental results, some adjustments were made to the digital comparator so that when the two inputs are equal, it can be specifically distinguished whether they are both "0" or both "1". The device uses Aβ modified electrodes as the working platform. Define R' as Input (input) E, R'≤-0.15 (-0.15 is the threshold) is "1" state (high level), otherwise it is "0" state (low level). Define ΔA 265 is Input U, ΔA 265 ≤0.1 (0.1 is the threshold) is the "1" state, otherwise it is the "0" state. The output value of "1" or "0" indicates that the drug works or does not work in the corresponding aspect. Drugs with different therapeutic effects are recorded as different output results, among which drugs that only have a disaggregation effect on the Aβ-Cu(II) complex correspond to Output (output) D = 1, drugs that only have a ROS inhibitory effect correspond to Output R = 1, and drugs that have both disaggregation and ROS scavenging functions correspond to Output B = 1. For completely ineffective drugs, all three output values ​​are "0". R' and ΔA caused by 9 different potential drugs and buffer control group 265 According to the above threshold definition and calculation, construct the truth table and comparator logic gate network, as shown in Figure 7 shown.

[0111] In this article, the threshold value of R' being in the "1" state is determined based on the specific effects of the various small molecules measured on the signal. It can be understood that the greater the degree of signal reduction, the more effective the disaggregation of the measured molecules. Therefore, in this article, the above threshold value of R' is roughly set at half of the difference between the experimental group and the control group.

[0112] In this paper, ΔA 265 The threshold value of the "1" state is determined based on the specific effects of various small molecules on the signal. It can be understood that protecting the AA signal as unchanged as possible indicates that the drug has a stronger ability to resist oxidative stress. Therefore, in this paper, ΔA 265 The above threshold is set roughly at halfway between the experimental group and the control group where there is a significant difference.

[0113] Of course, in practical applications, those skilled in the art can determine R' and ΔA according to actual needs. 265 The threshold for the "1" state.

[0114] When the input combination is (00), R' and ΔA 265 There is no significant difference with the control group, and all outputs are in the "0" state, which means that the drug has no therapeutic effect. α-KG belongs to this type. When the input combination is (11), it means R CT The value dropped significantly, and A 265 No significant changes occur, so Output B is in the "1" state, and all other outputs are "0". This type of drug includes CQ, TMPD, VD, etc. When the input combination is (01), although R' is close to zero and no depolymerization is shown, ΔA 265 This indicates that AA is protected and no ROS is generated. Therefore, the drug only exhibits the effect of inhibiting ROS, with Output R in the "1" state and other outputs in the "0" state. For example, the drug TEMPO corresponds to output (010). When the input combination is (01), the situation is similar, only the depolymerization effect is reflected, and only Output D is in the "1" state. Dopamine (DA) and epinephrine (EPI) are representative drugs with output (100).

[0115] In addition, different thresholds can be set as needed. For example, when the threshold of Input E is set to a lower value of -0.35, the requirement for the drug's disaggregation effect is more stringent, and when the threshold of Input U is set to -0.18, a lower standard is set for the drug to inhibit ROS ( Figure 8 ). In this case, α-KG is still determined to be a drug that does not show any benefit, and the output is (000). However, due to the reset of the threshold, the input E corresponding to EPI and DA is "0", and the input U is "1". The combination of inputs (01) indicates that R CTThe value did not decrease significantly but AA was protected, so the corresponding output R was in the "1" state and the other outputs were "0". Therefore, EPI and DA were redefined as representatives of this type of drug that only has an inhibitory effect on the production of ROS. In this case, there is no longer a representative drug with an output (100) that only reflects the depolymerization effect among the several small molecules tested. Other drugs, such as VD, TMPD and EDTA, which were once classified as dual-function drugs, are now reclassified as ROS inhibitors after adjusting the threshold setting. There are also some effective drugs that are not affected by the change in threshold setting, such as Cys and CQ.

[0116] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An intelligent logic drug screening platform, characterized in that: include: A digital comparator, the digital comparator comprising: The first input terminal E is used to input the EIS experimental result R' of whether the drug to be screened has the effect of disaggregating the Aβ-metal ion complex, wherein the high level signal and the low level signal of the first input terminal E correspond to R' being less than or equal to the first threshold and R' being greater than the first threshold, respectively, wherein the metal ion is selected from Cu 2+ or Zn 2+ , the R' is determined by the following method: The drug screening system is placed in [Fe(CN)6] 3- / 4- solution, determine the first charge transfer resistance R CT 0 and the second charge transfer resistor R CT ', wherein the drug screening system comprises: Aβ-loaded modified electrode, and The metal ion that binds to the Aβ, wherein The metal ions bind to the Aβ by the following method: Applying a solution of the metal ions with a concentration of not less than 20 μM to the surface of the modified electrode loaded with Aβ and allowing it to stand for 0.5-2 hours; Determine R CT 'with R CT 0 The charge transfer resistance change rate R' between The second input terminal U is used to input the UV experimental result ΔA of whether the drug to be screened has the effect of reducing and / or eliminating oxidative stress 265 , wherein the high level signal and the low level signal of the second input terminal U correspond to ΔA respectively 265 Less than or equal to the second threshold and ΔA 265 is greater than a second threshold, wherein the ΔA 265 Determined by: Determine the first absorbance value A of the modified electrode loaded with Aβ and the reducing agent solution immersed in the drug screening system for 10-60 minutes respectively 265 0 and the second absorbance value A 265 ', wherein the reducing agent solution contains the drug to be screened; Determine A 265 0 With A 265 'The difference ΔA 265 ; A first output terminal D is configured to output a high-level signal when a high-level signal is input to the first input terminal E and a low-level signal is input to the second input terminal U, indicating that the drug to be screened only has the effect of disaggregating the Aβ-metal ion complex; The second output terminal R is configured to output a high-level signal when a low-level signal is input to the first input terminal E and a high-level signal is input to the second input terminal U, indicating that the drug to be screened only has the effect of reducing and / or eliminating oxidative stress; The third output terminal B is used to output a high-level signal when a high-level signal is input to the first input terminal E and a high-level signal is input to the second input terminal U, which indicates that the drug to be screened has the effect of disaggregating the Aβ-metal ion complex and has the effect of reducing and / or eliminating oxidative stress.

2. The intelligent logic drug screening platform according to claim 1, characterized in that: The metal ions bind to the Aβ by the following method: The solution containing the metal ions was applied to the surface of the modified electrode loaded with Aβ, and allowed to stand for 1 hour, and then the excess metal ion solution was washed away. The concentration of the metal ions in the solution was 50 μM.

3. The intelligent logic drug screening platform according to claim 1, characterized in that: The modified electrode loaded with Aβ comprises: Conductive electrodes, A polydiallyldimethylammonium chloride film covering the surface of the conductive electrode, Gold nanoparticles fixed on the polydiallyldimethylammonium chloride film, wherein the gold nanoparticles are connected to glutathione; Aβ is linked to the glutathione via an amidation reaction.

4. The intelligent logic drug screening platform according to claim 3, characterized in that: The conductive electrode is a gold electrode.

5. The intelligent logic drug screening platform according to claim 3, characterized in that: The polydiallyldimethylammonium chloride film is formed by the following method: Coat the surface of the conductive electrode with a polydiallyldimethylammonium chloride solution, let it stand for 10-30 minutes, and then wash away excess polydiallyldimethylammonium chloride solution.

6. The intelligent logic drug screening platform according to claim 3, characterized in that: The gold nanoparticles are fixed on the polydiallyldimethylammonium chloride film by the following method: A dispersion of gold nanoparticles linked to glutathione is coated on a polydiallyldimethylammonium chloride film, allowed to stand for 20-60 minutes, and then excess dispersion is washed off.

7. The intelligent logic drug screening platform according to claim 3, characterized in that: The Aβ is linked to the glutathione by the following method: Coating an Aβ buffer solution with a concentration of 50 μM onto the conductive electrode immobilized with the gold nanoparticles and allowing it to stand for 2 hours to allow the amino group of the Aβ to undergo an amidation reaction with the carboxyl group of the glutathione; Wherein, before applying the Aβ buffer, the carboxyl group of the glutathione is activated; After the amidation reaction is completed, the unreacted carboxyl groups of the glutathione are capped.

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