阅读说明：本技术 一种P-tau检测免疫传感器及其制备和应用方法 (P-tau detection immunosensor and preparation and application methods thereof ) 是由 陈红霞 于 2021-09-23 设计创作，主要内容包括：本发明公开了一种P-tau检测免疫传感器的制备方法,包括步骤：(1)合成花状TiO-(2)；(2)将电极与杯芳烃衍生物溶液进行反应,使得杯芳烃衍生物附着在电极上；(3)将Anti-tau抗体添加到附着有杯芳烃衍生物的电极表面上；(4)将电极与超分子化合物溶液进行反应,为抗体提供固定基底。(The invention discloses a preparation method of a P-tau detection immunosensor, which comprises the following steps: (1) synthetic flower-like TiO 2 (ii) a (2) Reacting the electrode with the calixarene derivative solution to attach the calixarene derivative to the electrode; (3) adding Anti-tau antibody to the surface of the electrode to which the calixarene derivative is attached; (4) the electrodes are reacted with the solution of supramolecular compounds to provide a substrate for immobilization of the antibodies.)

1. A preparation method of a P-tau detection immunosensor is characterized by comprising the following steps:

(1) synthetic flower-like TiO₂；

(2) Reacting the sensor electrode with the calixarene derivative solution to attach the calixarene derivative to the electrode;

(3) adding Anti-tau antibody to the surface of the electrode to which the calixarene derivative is attached;

(4) the chip electrode reacts with the supramolecular compound solution to provide a fixed substrate for the antibody.

2. The method for preparing a P-tau detection immunosensor of claim 1, wherein step (1) comprises the steps of:

(101) adding tetrabutyl titanate into a mixture of hydrochloric acid and deionized water, and stirring for 1 h;

(102) transferring the mixture into an autoclave lined with polytetrafluoroethylene, and reacting at 160 ℃ for 3 hours;

after the reaction is finished, repeatedly washing the precipitate obtained by centrifugation with deionized water to remove unreacted reagents;

(103) finally, the precipitate was dried at 80 ℃ for 12h to obtain flower-like TiO₂And stored at 4 ℃ until use.

3. The method for preparing an immunosensor for P-tau detection according to claim 1, wherein the calixarene derivative solution in step (2) is 0.1mM of a Prolinker solution, the electrode is placed in the Prolinker solution for reaction for 8-12h, after the reaction is finished, the electrode is placed in a methanol solution for washing for 1h, and after the washing, the electrode is cleaned with ultrapure water and the water on the surface of the electrode is blown dry by nitrogen.

4. The method for preparing a P-tau detection immunosensor according to claim 1, wherein in the step (3), Anti-tau antibody is added to the surface of the electrode to which the Prolinker is attached at a concentration of 1 μ g/mL, and after incubation for 2 hours, the surface of the gold plate is washed with a buffer solution to elute the unbound antibody.

5. The method of preparing a P-tau detection immunosensor of claim 1, wherein in step (4), the chip is reacted with a BSA solution at a concentration of 0.01mg/mL for half an hour.

6. A P-tau detection immunosensor is characterized by using flower-shaped TiO₂As an enrichment material of Anti-tau antibody, a supramolecular compound is used as an antibody immobilization substrate.

7. The P-tau detection immunosensor of claim 6, wherein flower-like TiO₂The preparation method comprises the following steps:

(1) adding tetrabutyl titanate into a mixture of hydrochloric acid and deionized water, and stirring for 1 h;

(2) transferring the mixture into an autoclave lined with polytetrafluoroethylene, and reacting at 160 ℃ for 3 hours; after the reaction is finished, repeatedly washing the precipitate obtained by centrifugation with deionized water to remove unreacted reagents;

(3) finally, the precipitate was dried at 80 ℃ for 12h to give flower-like TiO2 and stored at 4 ℃ until use.

8. The P-tau detection immunosensor of claim 6, wherein the supramolecular compound is BSA.

9. A method of using a P-tau detection immunosensor as described in any one of claims 1-8, comprising the steps of: adding flower-shaped TiO into target solution₂The solution, then added to the electrode surface by centrifugation, and P-tau detected by electrochemical impedance induced electrochemical signal changes.

Technical Field

The invention relates to the field of medical instruments, in particular to a P-tau detection immunosensor and preparation and application methods thereof.

Background

Alzheimer's Disease (AD) is the leading cause of dementia and one of the world's greatest health care challenges and poses a serious family and social burden. In view of the fact that no specific medicine for treating AD exists at present, early diagnosis and early intervention lay the foundation for delaying the progress of AD, improving the life quality of patients and reducing the social burden. The use of Positron Emission Tomography (PET) for biomarkers in cerebrospinal fluid (CSF) has revolutionized the diagnostic work of AD. Core CSF biomarkers include Α β 42, total tau (T-tau) and phosphorylated tau (P-tau). A β 42 reduction and P-tau increase are thought to reflect A β and tau pathology in AD. Importantly, cerebrospinal fluid phosphorylated tau protein is more specific for AD than total tau protein and a β 42. CSF analysis and neuroimaging can determine the underlying pathophysiology at the earliest stages of certain neurodegenerative diseases, but do not have the scalability required for population screening. Therefore, blood-based biomarkers are becoming increasingly important for the diagnosis and prognosis of AD. From the future clinical practice, low cost routine blood examinations are expected to replace expensive PET scans.

Recent reports indicate that plasma p-tau₁₈₁AD dementia can be distinguished from amyloid beta-negative young and old people with unimpaired cognitive function and other neurodegenerative diseases. Other sites of P-tau, e.g. P-tau₁₉₉、 P-tau₂₁₇And P-tau₂₃₁The accuracy of diagnosis of AD is similar. Thus, plasma total P-tau is a more reliable plasma biomarker for AD. The method has important significance for the clinical detection of P-tau on the early diagnosis of AD, the determination of diagnosis and treatment scheme, the optimization of treatment dosage and the evaluation of drug efficacy.

At present, the detection method developed by P-tau is mainly an ELISA kit, but the existing method still cannot effectively distinguish patients with low P-tau expression from non-diseased people, cannot predict clinical results of most patients, and the sensitivity is still to be improved. The application of ELISA in serum P-tau detection is also limited by the defects of poor repeatability, susceptibility to interference of nonspecific protein to false positive, excessive interference factors in the detection process and the like. Furthermore, immunoassay of phosphorylated proteins still suffers from low abundance of phosphorylated proteins (P-proteins) and signal inhibition of a large number of non-P-proteins. Therefore, it is necessary to enrich the P protein prior to immunoassay. At present, means such as ultracentrifugation and the like are commonly used, however, because uncertain factors in the operation process are too many, the experimental result is possibly greatly influenced. So that the reliability and availability of the sample used for the detection cannot be fully guaranteed. For P protein enrichment, strategies have been developed, such as aptamer-based or antibody-based functionalized magnetic beads. Although the strategy based on functional magnetic beads is most widely applied to the enrichment of P protein, the method has the defects of complicated modification process of the phosphorylated antibody, low loading efficiency, easy damage of the activity of the antibody and the like. In addition, antibodies or aptamers developed for phosphorylation can typically specifically recognize a site. The corresponding antibody as a recognition molecule for enrichment and capture will not achieve full phosphorylation recognition. Multiple sites of P-tau, e.g. P-tau₁₉₉、P-tau₂₁₇And P-tau₂₃₁The diagnostic accuracy for AD was similar. The single specific recognition molecule can cause the omission of target protein, and reduce the accuracy of AD analysis. Therefore, the development of a rapid, sensitive and accurate method for detecting P-tau is a problem that researchers need to solve urgently.

Disclosure of Invention

In view of the above defects in the prior art, the technical problem to be solved by the present invention is to provide a rapid, sensitive and accurate P-tau detection immunosensor, and a preparation method and an application method thereof.

In order to achieve the above object, the present invention provides a method for preparing an immunosensor for P-tau detection, comprising the steps of:

(1) synthetic flower-like TiO₂；

(2) Putting the chip electrode into the calixarene derivative solution to attach the calixarene derivative to the chip electrode;

(3) adding Anti-tau antibody to the surface of the electrode to which the calixarene derivative is attached;

(4) the chip electrode is put into the supermolecular compound solution to provide a fixed substrate for the antibody.

Further, the step (1) comprises the steps of:

(11) adding tetrabutyl titanate into a mixture of hydrochloric acid and deionized water, and stirring for 1 h;

(12) transferring the mixture into an autoclave lined with polytetrafluoroethylene, and reacting at 160 ℃ for 3 hours; after the reaction is finished, repeatedly washing the precipitate obtained by centrifugation with deionized water to remove unreacted reagents;

(13) finally, the precipitate was dried at 80 ℃ for 12h to give flower-like TiO2 and stored at 4 ℃ until use.

Further, the calixarene derivative solution in the step (2) is a 0.1mM Prolinker solution, the electrode is placed in the Prolinker solution for reaction for 8-12h, after the reaction is finished, the electrode is placed in a methanol solution for washing for 1h, and after washing, the electrode is cleaned by ultrapure water and the water on the surface of the electrode is dried by nitrogen.

Further, in step (3), Anti-tau antibody was added to the surface of the electrode to which the Prolinker was attached at a concentration of 1. mu.g/mL, and after incubation for 2 hours, the gold plate surface was washed with a buffer to elute the unbound antibody.

Further, in step (4), the chip was reacted with a BSA solution at a concentration of 0.01mg/mL for half an hour.

The invention also provides a P-tau detection immunosensor, which uses flower-shaped TiO₂As an enrichment material of Anti-tau antibody, a supramolecular compound is used as an antibody immobilization substrate.

Further, among them, flower-like TiO₂The preparation method comprises the following steps:

(1) adding tetrabutyl titanate into a mixture of hydrochloric acid and deionized water, and stirring for 1 h;

(2) transferring the mixture into an autoclave lined with polytetrafluoroethylene, and reacting at 160 ℃ for 3 hours; after the reaction is finished, repeatedly washing the precipitate obtained by centrifugation with deionized water to remove unreacted reagents;

(3) finally, the precipitate was dried at 80 ℃ for 12h to give flower-like TiO2 and stored at 4 ℃ until use.

Further, wherein the supramolecular compound is BSA.

The invention also provides an application method of the P-tau detection immunosensor, which comprises the following steps: adding flower-shaped TiO into target solution₂The solution, then added to the electrode surface by centrifugation, and P-tau detected by electrochemical impedance induced electrochemical signal changes.

The invention has the following beneficial effects:

1. the host-guest recognition of the supramolecular compound provides rich binding sites for the immobilized antibody and ensures the activity of the antibody;

2. flower-like TiO₂The target analytes are separated and enriched by the one-step method, the separation efficiency is high, the specificity is strong, and the target analytes can be used as signal amplification molecules for detection;

3. flower-like TiO₂The target complex has a significant enhancement effect on the electrochemical impedance signal;

4. the sensing mode provides practical clinical application value for developing a more direct and effective biosensor.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram of the P-tau detection principle of the present invention;

FIG. 2 is a representation of a synthetic flower TiO2 material in accordance with a preferred embodiment of the invention;

FIG. 3 is a graph of EIS induced by P-tau at different concentrations in a preferred embodiment of the invention;

FIG. 4 is a graph of the linear relationship between the resistance response and the logarithm of the P-tau concentration in a preferred embodiment of the invention;

FIG. 5 is a schematic diagram of a P-tau detection immunosensor in accordance with a preferred embodiment of the present invention;

FIG. 6 is a schematic diagram of the use of a P-tau detection immunosensor in a preferred embodiment of the present invention;

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Electrochemical Impedance Spectroscopy (EIS) is currently one of the most effective analytical methods because it can characterize the physical and chemical processes in the different modified layers, sample charge transfer at high frequencies and trace mass transfer at low frequencies. EIS is less lost than other electrochemical methods in measuring immune interactions because it is performed over a narrow range of applied potentials. Thus, EIS has attracted a great deal of attention in the field of biosensor design. EIS analysis of phosphorylated proteins still suffers from low abundance of phosphorylated proteins (P-proteins) and signal inhibition of a large number of non-P-proteins. Therefore, it is necessary to enrich for P protein prior to EIS analysis. Although the strategy based on functional magnetic beads is most widely used in P protein enrichment, the methodThe method has the defects of complicated modification process of the phosphorylated antibody, low loading efficiency, easy damage of the activity of the antibody and the like. In addition, antibodies or aptamers developed for phosphorylation can typically specifically recognize a site. The corresponding antibody as a recognition molecule for enrichment and capture will not achieve full phosphorylation recognition. Multiple sites of P-tau, e.g. P-tau₁₉₉、 P-tau₂₁₇And P-tau₂₃₁The diagnostic accuracy for AD was similar. Therefore, a single specific recognition molecule can cause missed detection of the target protein, and reduce the accuracy of the AD analysis.

TiO₂Can form coordinate bonds with phosphate groups of P protein and stably combine with the phosphate groups, is not interfered by activity change of antibodies, and realizes high-selectivity enrichment. Flower-like TiO₂(F-TiO₂) The superstructure, namely the 3D micro-nano material with the hierarchical structure, has larger specific surface area, excellent biocompatibility and strong electron transfer capacity. F-TiO₂The large specific surface area of (a) contributes to more sensitive binding of the protein P to its surface. Good biocompatibility will keep the natural activity of the binding protein unchanged. In addition, the larger electron transfer inhibition capability enables the F-TiO2 to obtain a better signal amplification effect as an enrichment material, and the detection sensitivity is obviously improved.

Based on the mechanism, the invention designs a simple and sensitive immunosensor, and the level of total P-tau is quantified through electrochemical signal output. The sensor relates to F-TiO₂Coordinate binding to P-tau, and specific recognition by antibodies, as shown in FIG. 1. By F-TiO₂The successful construction of the mediated immunosensor realizes P-tau enrichment and target signal amplification by a one-step method. First, calixarene derivatives were used to direct Anti-tau to the gold electrode surface. The supramolecular compound provides abundant binding sites for immobilized antibodies through host-guest recognition and ensures Anti-tau activity through non-covalent interactions. F-TiO₂Will act as a signal amplification tag, its unique flower-like structure and high electron blocking capability results in a net change in charge transfer resistance (Rct). Furthermore, F-TiO in the sandwich method₂Not only provides hydrophilicity and can reduce nonspecific binding, but also ensures P-High sensitivity of tau binding. This allows the designed electrochemical biosensor to output an amplified impedance signal during target detection. Anti-tau and F-TiO₂Realizes dual specificity recognition of P-tau and ensures high selectivity of total P-tau detection. By the mode, more antigens are captured on the interface, the signal output is further enhanced, and the detection sensitivity is further increased.

In a preferred embodiment according to the invention, the flower-like TiO₂The electrochemical assay method and procedure for specific detection of P-tau is as follows:

(I) Synthesis of flower-like TiO₂：

(1) Adding tetrabutyl titanate (TBOT) into a mixture of 37% hydrochloric acid and deionized water, uniformly stirring, and continuing stirring for 1 h;

(2) the mixture was then transferred to an autoclave lined with polytetrafluoroethylene and reacted at 160 ℃ for 3 hours. After the reaction was completed, the precipitate obtained by centrifugation was repeatedly washed with deionized water to remove the unreacted reagent.

(3) Finally, the precipitate was dried at 80 ℃ for 12h to give F-TiO₂And stored at 4 ℃ for subsequent use.

The resulting F-TiO was characterized by ultraviolet-visible spectroscopy (Uv-vis), Dynamic Light Scattering (DLS), Zeta potential and Scanning Electron Microscopy (SEM), respectively₂And the material stability was analyzed by DLS. The material is characterized in figure 2.

(II) electrochemical method for measuring P-tau:

(1) first, 0.1mM of a Prolinker solution is prepared, and an electrode is placed in the Prolinker solution to react for 8-12 h. Prolinker is a commercially available calixarene derivative that can react with the chip surface by gold-sulfhydryl bonds, and its calixarene cavity can be used to anchor the antibody to the chip surface. After the reaction, the electrode was washed in methanol solution for one hour, and after washing, the electrode was washed with ultrapure water and the water on the surface of the electrode was blown dry with nitrogen.

(2) The electrode with the Prolinker attached was then mounted to the electrochemical workstation instrument. Then, 100. mu.L of Anti-tau antibody at a concentration of 1. mu.g/mL was added to the surface of the electrode to which the Prolinker was attached. After 2 hours of incubation, the gold plate surface was washed with buffer to elute the unbound antibody.

(3) To avoid non-specific adsorption on the chip surface, the chip was reacted with a BSA (bovine serum albumin) solution at a concentration of 0.01mg/mL for half an hour.

(4) Then, F-TiO was added to 100. mu.L of the target solution₂Solution due to TiO₂Coordinate binding ability to P-tau, which forms the corresponding complex, is then added to the electrode surface by centrifugation. And detecting the change of electrochemical signals caused by different concentrations of P-tau through electrochemical impedance.

Different concentrations of P-tau were tested as described above.

The P-tau concentrations used for the test were: 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100 ng/ml.

And (3) testing conditions are as follows: electrochemical impedance measurements at different concentrations of P-tau were performed at room temperature using an electrochemical workstation (AutolabPGSTAT128Nsystem) with 10min as incubation time.

See figures 3 and 4. FIG. 3 is a graph of EIS induced by different concentrations of P-tau. FIG. 4 is a linear relationship between the resistance response and the log of P-tau concentration.

As can be seen in fig. 3 and 4: as the concentration of P-tau increases, the resulting electrochemical signal is also greater. So that sensitive quantitative analysis can be performed thereon.

Referring to the drawings, fig. 2 is a characterization of a material, specifically: (A) and the inset shows the synthesis of F-TiO₂SEM image of (d). (B) F-TiO₂Ultraviolet-visible spectrum of (a). (C) F-TiO₂DLS and Zeta potential of (a). (D) F-TiO₂Stability at different time conditions (n-3). As can be seen from the figure, F-TiO₂The overall shape of (a) is a uniformly dispersed flower-like spherical structure. The particle size is 350 +/-50 nm, the dispersibility is good, and the shape is clear. The peak around 325nm in the UV-vis absorption spectrum is considered to be F-TiO₂Characteristic absorption peak of (1). F-TiO₂Has an equivalent hydrodynamic diameter of 401 + -15.4 nm. The zeta potential was 23.2. + -. 0.51 mV. The above results demonstrate that F-TiO₂Successful synthesis of the compound;F-TiO₂after 20 days at 4 ℃, the particle size change was only 7.59%, indicating that the synthesized nanoparticles had good stability. Demonstration of F-TiO₂Has better stability and can be used for subsequent tests.

FIGS. 3 and 4 show the linear dependence of different P-tau concentrations on the change in impedance. As can be seen from the figure: as the concentration of P-tau increases, the impedance change caused by the concentration of P-tau also increases, and a better linear correlation between the concentration of P-tau and the impedance change appears. The linear equation is: y is 93.153+333.51 XlgCP-tau, the linear correlation coefficient is 0.98, the detection limit is 25.1pg/mL, and the detection requirement is met.

As shown in fig. 5 and 6, the invention also provides a P-tau detection immunosensor, which comprises a pair of gold electrodes 2 arranged on a PI substrate 1, wherein the gold electrodes 2 are conducted to a detection cell 4. The gold electrode 2 is treated by the adhesion of a Prolinker solution and Anti-tau antibody and the reaction of a BSA solution in advance, and then F-TiO is dripped into the detection cell 4₂Drying the solution at 120 deg.C for 10min to obtain flower-like TiO in detection cell 4₂The deposited film 3 is stored for later use. During detection, the target solution 7 is dripped into the detection pool 4, so that the TiO is enabled to be₂Binds to P-tau and the sensor is then electrochemically detected as described above.

The invention utilizes flower-shaped TiO₂Highly specific coordinate binding of P-tau, dual selectivity with Anti-tau in combination, and by F-TiO₂The P-tau complex signal amplifies the impedance value. The method has high specificity and selectivity, high sensitivity, and capability of detecting trace amount of P-tau. In addition, the method is used for bi-selectively combining P-tau, so that the problem of false positive caused by single antigen-antibody specificity recognition is solved, and the detection result is high-efficiency and reliable. The constructed simple electrochemical immunosensor with high specificity and double selection provides a more convenient, rapid and accurate method for detecting total P-tau in blood.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.