Preparation method of porous membrane electrochemical biosensor for detecting A beta oligomer
By modifying gold nanoflowers and hydrophobically modified SiO2 nanoparticles on the conical nanopore membrane and utilizing the specific binding of Aβ oligomers to sialic acid to change the pore size of the nanopore, the false positive problem of detecting Aβ oligomers in the existing technology is solved, and high-sensitivity and low-cost Alzheimer's disease diagnosis is achieved.
Patent Information
- Application Number
- CN202511226577.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-10-17
AI Technical Summary
Existing Alzheimer's disease marker detection methods such as enzyme-linked immunosorbent assay and fluorescence analysis are costly or susceptible to background effects, and it is difficult to accurately detect Aβ oligomers in the blood, resulting in false positive results.
Gold nanoflowers and hydrophobically modified SiO2 nanoparticles were modified with conical nanoporous membranes, and thiolated sialic acid was connected through gold-sulfur bonds. The specific binding of Aβ oligomers to sialic acid changed the effective pore size of the nanopores, and the content of Aβ oligomers was detected.
It achieves high-sensitivity, low-cost, and accurate detection of Aβ oligomers, avoids false positive results caused by Aβ protein fibrillation, and improves the accuracy of detection.
Smart Images
Figure CN120801449A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of ion channel blocking effect sensor, and relates to a preparation method of a porous membrane electrochemical biosensor for detecting Aβ oligomers. BACKGROUND
[0002] Aβ protein is formed by a series of Aβ monomers, soluble Aβ oligomers and fibrils from amyloid precursor protein by the cleavage of β-secretase and γ-secretase. The soluble Aβ oligomers and fibrils are more toxic than the insoluble filaments and plaques, causing oxidative damage, inflammatory response, interference with the function and survival of neurons and various adverse behaviors. In summary, the Aβ oligomers are the most recognized theory in the pathogenesis of Alzheimer's disease. Therefore, detecting the content of Aβ oligomers in blood has important significance for the diagnosis of Alzheimer's disease. During the deposition of Aβ protein, the sialic acid residues located at the end of the cell membrane can serve as a bridge to combine with Aβ oligomers, promote the conformational transition of Aβ oligomers on the cell membrane surface, and further form fibrils with neurotoxicity.
[0003] The deposition of Aβ aggregation is easily affected by the chemical composition of the cell membrane surface. Among them, the sialic acid located at the end of the cell membrane specifically adsorbs Aβ oligomers and promotes the aggregation of Aβ oligomers. Therefore, the present application uses sialic acid as a modifier on the small pore end of the conical nanopore membrane gold nanoflower, and the specific binding of Aβ oligomers and sialic acid produces a difference in the blank nanopore membrane electrical signal.
[0004] Detecting the content of Aβ oligomers in blood has important significance for the prevention and early diagnosis of Alzheimer's disease. At present, there are many detection methods for Alzheimer's disease markers. Among them, there are many methods for detecting Aβ oligomers in blood. The enzyme-linked immunosorbent assay is simple to operate but high in cost, and false positive errors may occur; the fluorescence analysis method is low in cost and simple in imaging, but is easily affected by the background and prone to fluorescence quenching. At present, electrochemical aptamer sensors have been widely used in the detection of disease markers, and have the advantages of low price, easy operation, high sensitivity and strong selectivity.
[0005] The PET nuclear pore membrane of the conical nanopore is formed by using different etching liquids on both sides to form nanopore channels with different pore sizes at both ends of the nuclear pore membrane. The etched conical nanopore is modified by hydrophobic modified SiO2 nanoparticles to prevent Aβ protein fibrillation. Gold nanoflowers are formed at the small end of the conical nuclear pore membrane by one-step reduction method as a substrate for linking thiolated sialic acid. The sialic acid linked by gold sulfide bond with the gold nanoflower forms a switchable nanochannel. When the sialic acid contacts the Aβ oligomers, the two will specifically bind to the Aβ oligomers due to the action of hydrogen bond, resulting in a decrease in the effective pore size of the nanopore channel and a decrease in the ion flow to produce a specific electrical signal. Based on the above purpose, the present application adopts the following technical scheme. SUMMARY
[0006] The present application aims to provide an electrochemical sensor for detecting Aβ oligomers, and to provide a simple and effective diagnosis of Alzheimer's disease. The specific structure is shown in Figure 1 The ring-shaped gold nanoflower is modified on the small pore end of the conical nuclear pore membrane, and the thiolated sialic acid is connected. The specific binding of Aβ oligomers to sialic acid reduces the effective pore size of the nuclear pore membrane, thereby changing the ion flow. The content of Aβ oligomers in the blood to be tested is detected to determine whether the sample provider has Alzheimer's disease. The working principle of the sensor is shown in Figure 2 As shown in Figure 2 As shown in, a is the pore size of the sialic acid on the nuclear pore membrane gold nanoflower when it is not combined with Aβ oligomers, and b is the sialic acid on the nuclear pore membrane gold nanoflower, which is specifically combined with Aβ oligomers through hydrogen bonds, resulting in a reduction in the effective pore size of the nuclear pore membrane, and thus reducing the ion flow in the pore to change the electrical signal.
[0007] Aβ protein itself has amphiphilic properties, so both extremely hydrophobic materials and extremely hydrophilic materials can effectively prevent the fibrous aggregation of Aβ. Therefore, hydrophobic copolymer nanoparticles, i.e. hydrophobically modified SiO2 nanoparticles, are modified on the surface of the nuclear pore membrane to avoid false results caused by the aggregation of Aβ protein to form protein fibers.
[0008] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:
[0009] In the first aspect, the conical nuclear pore membrane is cut to the appropriate size and fixed in the electrolytic cell. Preferably, Na3Au(SO3)2 solution is added to the large pore end and allowed to stand for 1-10 min, and then NaBH4 solution is added to the small pore end. The solutions on both sides of the nuclear pore membrane are oxidized and reduced by diffusion to form gold nanoparticles, forming a gold nanoflower modified nuclear pore membrane.
[0010] Further, the concentration of Na3Au(SO3)2 solution is 1 mmol / L-0.05 mol / L, and the concentration of NaBH4 solution is 1 mmol / L-0.05 mol / L.
[0011] Further, the reaction time is 5-120 min.
[0012] In the second aspect, the interaction of Aβ protein with hydrophobic groups can inhibit the fibration of Aβ protein, prevent its fibration in the pore channel of the nuclear pore membrane due to the space squeezing effect, and thus affect the experimental results. Therefore, the hydrophobically modified SiO2 nanoparticles on the surface of the nuclear pore membrane can inhibit the fibration of Aβ protein to form polymers, and the aggregation of Aβ protein is inhibited by the hydrophobic groups.
[0013] Further, tetraethyl orthosilicate, hexamethyl disilazane and ethanol are mixed in a molar ratio of 1:2.5:60, and then condensed under stirring at 40°C in a water bath for 25 min to obtain hydrophobic modified SiO2 nanoparticles.
[0014] Further, the hydrophobic modified SiO2 is attached to the surface of the nuclear pore membrane, and γ-aminopropyl triethoxysilane is used as a coupling agent to mix with the hydrophobic modified SiO2 nanoparticles to form a suspension, which is then sprayed on the surface of the nuclear pore membrane by a spray gun, and a hydrophobic film is formed after drying and curing, thereby obtaining a functionalized nuclear pore membrane.
[0015] Further, the mixing scheme is to mix by dry grinding and then ultrasonic dissolution, and then take the supernatant.
[0016] Further, the particle size of the hydrophobic modified SiO2 in the emulsion is in the range of 50-80 nm.
[0017] In the third aspect, it is found in the current research on Alzheimer's disease that sialic acid can specifically bind to Aβ oligomers during the deposition of Aβ protein, and further form toxic protein fibers on the surface of cells, so the product uses sialic acid as a recognition unit for specific recognition of Aβ oligomers on the gold nanoflower.
[0018] Further, during the modification of sialic acid, esterification may occur between sialic acids, and the hydroxyl group to be replaced has similar chemical properties to the hydroxyl group on the branched chain. Therefore, a thiol group is introduced at the C1 position to prevent the hydroxyl group at other positions except C1 from being replaced and internalized, while reducing the influence of end group effects on the hydrogen bond of sialic acid. Therefore, the sialic acid is methyl acetylated, the hydroxyl group is converted into an acetyl group, and the carboxyl group is converted into a methyl acetate group, and the sialic acid is protected to obtain acetylated sialic acid methyl ester.
[0019] Further, the methyl acetylation conditions are that the sialic acid and the cation exchange resin are added to methanol under stirring at room temperature for 6 h, then filtered, and the filtrate is rotary evaporated to a solid to obtain sialic acid methyl ester. The sialic acid methyl ester and acetic anhydride are dissolved in pyridine under ice water bath conditions, and the reaction is carried out at room temperature for 24 h. Then, HCl solution is added dropwise to adjust the pH to 1-3. The reaction finished solution is extracted with ethyl acetate. The organic phase is washed with saturated sodium bicarbonate solution until the pH is 7, and then rotary evaporated to a solid.
[0020] Further, the concentration of the HCl solution is 1 mol / L.
[0021] The sialic acid is a cyclohexane with O atom as a heteroatom in structure, and has an end group effect. First, acetylated sialic acid methyl ester is added to a pyridine solution containing excess anhydrous hydrofluoric acid to replace -OAc on the anomeric carbon with -F to obtain fluorine-substituted acetylated sialic acid methyl ester, which is dried by rotary evaporation. Subsequently, thioacetic acid is added to a dichloromethane solution, which is activated by boron trifluoride ether to obtain a thiol acetic acid salt solution with good selectivity. The fluorine-substituted acetylated sialic acid methyl ester is added to the thiol acetic acid salt solution to replace the fluorine group with a thiol acetic acid group to obtain thiol acetylated sialic acid methyl ester.
[0022] Further, the concentration of the pyridine solution of anhydrous hydrofluoric acid is 3-5 mol / L.
[0023] Further, the molar amount of thioacetic acid is 2 mmol, the molar amount of boron trifluoride ether is 2.5 mmol, the volume of the dichloromethane solution is 40 mL, and the activation time is 30 min.
[0024] Further, the concentration of the thiol acetic acid salt solution is 0.5 mol / L, the volume is 40 mL, and the amount of fluorine-substituted acetylated sialic acid methyl ester is 2 mmol.
[0025] The specific binding of Aβ oligomers to sialic acid is a hydrogen bond, so it is necessary to deprotect the thiol acetylated sialic acid methyl ester. The methyl ester is hydrolyzed, and the acetyl group is converted back to a hydroxyl group to restore the hydrogen bond donor of sialic acid, and the thiol acetic acid group is converted to a thiol group. To this end, the thiol acetylated sialic acid methyl ester with the anomeric carbon is dissolved in a sodium methoxide solution in methanol, stirred uniformly, and then a cation exchange resin is added to adjust the pH to 7. After the reaction is completed, the remaining solution is filtered and dried by rotary evaporation to obtain thiolated sialic acid. Finally, the thiolated sialic acid is dissolved in ultrapure water, and a nuclear pore membrane with gold nanoflowers is placed in the solution for 30 min, and the treated thiolated sialic acid is connected to the gold nanoflowers through a gold-sulfur bond. After the reaction is completed, the thiolated sialic acid on the nuclear pore membrane that fails to be modified on the gold nanoflowers is washed clean with ultrapure water to obtain a nuclear pore membrane nanion channel with thiolated sialic acid on the anomeric carbon as a specific recognition unit and as a switch. Subsequently, an excess amount of bovine serum albumin is added to block the active sites for 20 min, and after the reaction is completed, the nuclear pore membrane is washed and then dried under vacuum.
[0026] Further, the concentration of the thiolated sialic acid aqueous solution is 0.1-0.5 mol / L, and the concentration of the sodium methoxide solution in methanol is 2-5 mol / L, and the two solutions are mixed for reaction.
[0027] Further, the stirring time is 6 h.
[0028] Further, the reaction time for loading functionalized sialic acid on the nuclear pore membrane is 2-12 h, the washing liquid is ultrapure water, and the drying environment is 40°C under vacuum conditions.
[0029] The reaction time for further gold-sulfur bond formation is 20-60 min.
[0030] The blocking time of gold nanoflower by bovine serum albumin is 20 min.
[0031] Compared with the prior art, the present application has the following advantages:
[0032] The present application uses the fixed pore size of nuclear pore membrane, and the fixed reaction time forms gold nanoflower with uniform size at the small hole end of the nanopore channel of the nuclear pore membrane, and the sialic acid as the specific binding recognition unit is fixed on the carrier on the nuclear pore membrane through gold-sulfur bond.
[0033] The recognition unit of the present application only uses hydrogen bond, changes the ion flow through the nanopore channel of the nuclear pore membrane by changing the effective pore size of the nuclear pore membrane, and does not participate in chemical reaction, without considering the influence on molecules after applying voltage.
[0034] The present application uses hydrophobic modified SiO2 nanoparticles to hinder the fibrillation of Aβ protein on the surface of the nuclear pore membrane, avoids the false positive results caused by the aggregation of Aβ protein at low concentration, and greatly improves the accuracy of detection. BRIEF DESCRIPTION OF DRAWINGS
[0035] Figure 1 It is the cross-sectional view and top view of the ion flow channel structure of the present application;
[0036] Figure 2 It is a schematic diagram of the formation of ion flow channel restriction of sialic acid and Aβ oligomer;
[0037] Figure 3 It is a schematic diagram of the thiolization process of sialic acid in Example 2;
[0038] Figure 4 It is the I-V curve of Example 1 and Example 3 based on the different modification degrees of conical nuclear pore membrane;
[0039] Figure 5 It is the I-V curve of Example 4 based on the present application under the condition of different concentrations of Aβ oligomer;
[0040] Figure 6 It is the linear regression equation of Example 4 based on the present application based on ΔI-lgC of Aβ oligomer;
[0041] Figure 7 It is the stability test of the porous membrane sensor for detecting Aβ oligomer in Example 5;
[0042] Figure 8 It is the I-V curve of the specific detection of the sensor for Aβ oligomer in Example 6. DETAILED DESCRIPTION
[0043] The technical solutions of the embodiments of the application are clearly and completely illustrated below with reference to the drawings, and the features and advantages of the application are further described:
[0044] Example 1
[0045] Gold nanoflower modified hydrophobic nuclear pore membrane:
[0046] Preparation of gold nanoflower modified conical hydrophobic nuclear pore membrane: the conical nuclear pore membrane is cut to a suitable size and fixed in an electrolytic cell. A 0.02 mol / L solution of NAa3Au(SO3)2 is preferentially added to the large pore end, and a 0.03 mol / L NaBH4 solution is added to the small pore end after standing for 1 min. The gold nanoflower modified nuclear pore membrane is formed by diffusion on both sides of the nuclear pore membrane through a one-step redox method.
[0047] Tetraethyl orthosilicate, hexamethyl disilazane and ethanol are reacted in a molar ratio of 1:2.5:60 under stirring at 40°C in a water bath for 25 min to obtain hydrophobically modified SiO2 nanoparticles. γ-Aminopropyl triethoxysilane is used as a coupling agent and is thoroughly ground and mixed with the hydrophobically modified SiO2 nanoparticles to form a suspension, and then the upper clear liquid is taken. The surface of the large pore end of the conical nuclear pore membrane is sprayed using an airbrush, and then it is dried in a vacuum drying oven at 40°C to form a hydrophobic film.
[0048] Example 2
[0049] The thiolation process of sialic acid is as follows Figure 3 :
[0050] S1. Methyl acetylation protection of sialic acid:
[0051] Methyl acetylation protection of sialic acid: 10 g of sialic acid T1 is dissolved in 200 mL of a methanol solution by adding 4 g of sodium methoxide and 2 g of cation exchange resin, and stirring for 24 h. After 24 h, it is observed whether the solution is clear. If the solution is not clear, the mixture is placed in a 30°C water bath until the solution is clear. Then, the solution is filtered, and the filtrate is rotary evaporated to dryness to obtain sialic acid methyl ester T2. The rotary evaporation product is dissolved in an appropriate amount of pyridine until it is completely dissolved, and then it is placed in an ice water bath. When the solution temperature drops to 0°C, 5 mL of acetic anhydride is added dropwise, and the reaction is carried out for 12 h to obtain acetylated sialic acid methyl ester T3. The reaction completed solution is extracted with ethyl acetate. After washing the organic phase with saturated sodium bicarbonate solution to pH = 7, it is rotary evaporated to a solid.
[0052] S2. Activation of sialic acid by sulfur atom:
[0053] 4 mol / L anhydrous HF in pyridine solution was used to treat acetylated sialic acid methyl ester T3 for 30 min to obtain fluoride T4. After activating thioacetic acid with boron trifluoride ether, fluoride T4 was added to react with boron trifluoride in the presence of excess thioacetic acid for 12 h to obtain thioacetylated sialic acid methyl ester T5 with good selectivity.
[0054] S3. Deprotection of sialic acid:
[0055] 200 mg of thioacetylated sialic acid was dissolved in 20 mL of a methanol / water mixed solution (v / v = 1:1), and then 1 mol / L sodium methoxide solution was added to adjust the pH value to 9. After stirring at room temperature for 24 h, cation exchange resin was added and stirred until the pH value was about 6.5-7.5. After rotary evaporation of the solvent under reduced pressure, solid powder T6, i.e. thiolated sialic acid, was obtained by freeze-drying.
[0056] Example 3
[0057] Connecting thiolated sialic acid to gold nanoflowers:
[0058] Thiolated sialic acid was dissolved in ultrapure water, and the nuclear pore membrane with gold nanoflowers was placed in the solution for 30 min. Thiolated sialic acid was connected to the gold nanoflowers through gold-sulfur bonds. After the reaction, the nuclear pore membrane was washed with ultrapure water three times to remove the thiolated sialic acid that was not modified on the gold nanoflowers. Then, the nuclear pore membrane was placed in a 5 g / L aqueous solution of bovine serum albumin for 20 min. After the reaction, the nuclear pore membrane was washed with ultrapure water three times, and then excess bovine serum albumin was added to block the active sites. After standing for 20 min, the nuclear pore membrane was washed, and then placed in a vacuum box and dried at a temperature of 40°C for 6 h.
[0059] Application examples
[0060] Example 4
[0061] To study the response of the nanochannel modified with sialic acid on the small pore end of the conical nuclear pore membrane with gold nanoflowers as the substrate. The nuclear pore membrane was fixed in an electrolytic cell, 1×PBS solution was added and then incubated for 30 min. Ag / AgCl electrodes were inserted into the electrolytic cell and placed on both sides of the nuclear pore membrane. A voltage of -0.5-0.5 was applied to the nuclear pore membrane under different conditions using an electrochemical workstation. For example, Figure 4 The nuclear pore membrane modified with only gold nanoflowers and further sprayed with hydrophobically modified SiO2 nanoparticles and further connected with thiolated sialic acid was detected, and the results showed that the modified hydrophobically modified SiO2 and sialic acid had a slight effect on the ion flux of the nuclear pore membrane, further proving that the combination of sialic acid and Aβ oligomers would further reduce the ion flux. β-Amyloid (1-42), human oligomers were configured into solutions with different concentrations, such as Figure 5The change ratio of ion current under the voltage of 0.5V was used to quantitatively analyze the ion current change. It can be seen that the ion current signal gradually decreases with the increase of the concentration of the detected β-Amyloid (1-42), human oligomer. As shown in Figure 6 I0 represents the current at-0.5V when the concentration of β-Amyloid (1-42), human is 0, and I is the current value at-0.5V under different concentrations of β-Amyloid (1-42), human. In summary, when the concentration of β-Amyloid (1-42), human is in the range of 10 -12 -10 -8 g / L, the ion current signal difference increases linearly with the exponential increase of the concentration of β-Amyloid (1-42), human oligomer.
[0062] Example 5
[0063] To study the stability of the nuclear pore membrane detection, 8 pieces of conical nuclear pore membranes modified with thiolated sialic acid were taken for detection of 10 -10 g / L β-Amyloid (1-42), human oligomer solution. The detection results are shown in Figure 7 , which prove that after standard treatment, the conical nuclear pore membrane modified with thiolated sialic acid has a relatively stable detection result for Aβ oligomer.
[0064] Example 6
[0065] To study the specificity of the conical nuclear pore membrane modified with thiolated sialic acid, two pieces of modified conical nuclear pore membranes were fixed in the electrolytic cell, and 10 -10 g / L β-Amyloid (1-42), human solution and 10 -10 g / L P-tau217 protein solution were added respectively, as Figure 8 The change ratio of ion current under the voltage of 0.5V was used to quantitatively analyze the ion current change. It can be seen that the conical nuclear pore membrane modified with thiolated sialic acid has good specificity.
Claims
1. A method for preparing a porous membrane electrochemical biosensor for detecting Aβ oligomers, characterized in that: The specific steps are as follows: S1. After fixing the conical nucleopore membrane in the electrolytic cell, Na3Au(SO3)2 solution is added to the large pore end and allowed to stand. Then, NaBH4 solution is added to the small pore end to generate ring-shaped gold nanoflowers through redox reaction. S2, spraying hydrophobically modified SiO2 nanoparticles on the surface of the large pore end of the conical nuclear pore membrane to form a hydrophobically modified membrane; S3, adding a cation exchange resin to a methanol solution of sialic acid to convert it into sialic acid methyl ester, acetylating the sialic acid methyl ester with acetic anhydride, and then adding an anhydrous HF pyridine solution to fluorinate the anomeric carbon to obtain the final product, fluorine-substituted acetylated sialic acid methyl ester; S4, activating thioacetic acid with boron trifluoride etherate, and then mixing with fluorine-substituted acetylated sialic acid methyl ester to replace the fluoride ion with a thiol acetyl group to obtain thiol acetylated sialic acid methyl ester; S5. Adding a methanol solution of sodium methoxide to the thiol acetylated sialic acid methyl ester, and then adding an ion exchange resin to adjust the pH to 6.5-7.5, thereby hydrolyzing and removing the protection of the acetyl group and the methyl ester of the thiol acetylated sialic acid methyl ester, and converting the thiol acetyl group into a sulfhydryl group to obtain sulfhydrylated sialic acid; S6. Thiolated sialic acid was connected to the gold nanoflowers of the conical nuclear pore membrane through gold-sulfur bonds, and then the active sites were blocked with bovine serum albumin.
2. The method for preparing a porous membrane electrochemical biosensor for detecting Aβ oligomers according to claim 1, characterized in that: The preparation process of gold nanoflowers is as follows: immerse the conical nucleus pore membrane in a solution with a Na3Au(SO3)2 concentration of 1mmol-0.05mol / L and a NaBH4 concentration of 1mmol-0.05mol / L, and the reaction time is 5-120min.
3. The method for preparing a porous membrane electrochemical biosensor for detecting Aβ oligomers according to claim 1, characterized in that: The preparation process of the hydrophobic material membrane is as follows: tetraethyl orthosilicate, hexamethyldisilazane and ethanol are mixed in a molar ratio of 1:2.5:60, the reaction temperature is 40°C, and hydrophobically modified SiO2 nanoparticles are obtained. The coupling agent is γ-aminopropyltriethoxysilane.
4. The method for preparing a porous membrane electrochemical biosensor for detecting Aβ oligomers according to claim 1, characterized in that: The invention relates to a method for preparing fluorine-substituted acetylated sialic acid methyl ester. The concentration of anhydrous HF in a pyridine solution is 3-5 mol / L.
5. The method for preparing a porous membrane electrochemical biosensor for detecting Aβ oligomers according to claim 1, characterized in that: The preparation method of thiol acetylated sialic acid methyl ester comprises the following reaction conditions for activating thioacetic acid: dissolving 0.05 mol / L thioacetic acid and 0.0625 mol / L boron trifluoride etherate in dichloromethane.