CN116609410B - Preparation method and application of paper-based electrochemical sensor for detecting Alzheimer's disease - Google Patents
Preparation method and application of paper-based electrochemical sensor for detecting Alzheimer's disease Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a paper-based electrochemical sensor for detecting Alzheimer's disease, comprising the steps of preparing a multi-channel paper-based electrode with three working electrodes by a vacuum suction filtration method, preparing SiO2-Au-Thi, incubating the SiO2-Au-Thi with a specific antibody of a target, and synthesizing a SiO2-Au-Thi-Ab compound; preparing an immunosensor on two of the paper-based electrodes by combining the SiO2-Au-Thi-Ab complex based on a sandwich strategy of a sandwich structure; the method has the advantages that ferrocene is modified on the other electrode of the paper-based electrode to serve as a reference signal, the CHI660e electrochemical workstation is utilized, innovative detection of Abeta o and Fetuin B is achieved, the method has the characteristics of being high in specificity, high in sensitivity and good in stability, and the method has higher sensitivity and wider linear range through optimization of synthesis conditions and incubation time, so that the requirements of Abeta o and Abeta f content measurement in brain tissues of whole animals can be completely met.
Description
Technical Field
The invention relates to a preparation method and application of a paper-based electrochemical sensor for detecting Alzheimer's disease.
Background
The world population is aging at an unprecedented rate, and therefore, more and more elderly people will need to deal with the problem of cognitive decline. Alzheimer's Disease (AD) is a common, progressive and fatal, age-related neurodegenerative disease, which is manifested mainly by progressive memory disorders, cognitive disorders, behavioral disorders, etc., early detection of AD is critical to impair its effects and to deepen our understanding of such diseases, and by early diagnosis, we can better understand the pathogenesis of AD, and plan and conduct treatment earlier.
Although the pathogenesis of AD is currently unknown, researchers generally believe that Senile Plaques (SP) formed by beta-amyloid (aβ) aggregation and neurofibrillary tangles (NFT) formed by phosphorylated tau aggregation are two key pathological hallmarks of AD, which can currently be detected using cerebrospinal fluid or imaging techniques, and PET imaging of aβ amyloid PET and tau-protein is the gold standard of amyloid and tau pathology in clinical trials. The lumbar puncture and collection of cerebrospinal fluid is a relatively more invasive method, and also, PET imaging is not suitable for weak elderly patients and is relatively expensive; thus, more and more recent studies have begun to divert the eye towards blood-based AD biomarkers. Detection of blood biomarkers is relatively simple, less invasive, cost-effective, readily available, and can be measured continuously as compared to cerebrospinal fluid.
Currently, many detection methods for aβo have been established, and various analysis techniques including electrochemical methods, fluorescence spectrophotometry, colorimetry and electrochemiluminescence have been established, and the quantification of feturin B is mostly performed by enzyme-linked immunosorbent assay (ELISA). Although these methods have obtained low detection Limit (LOD), special equipment is required, portability is poor, and immediate detection is difficult to realize, while the electrochemical sensor has the advantages of high sensitivity, good selectivity, simple operation, good reproducibility, low cost, strong real-time performance and the like, and on the basis, a preparation method and application of the paper-based electrochemical sensor for detecting alzheimer disease are needed to solve the problems.
Disclosure of Invention
The invention provides a preparation method and application of a paper-based electrochemical sensor for detecting Alzheimer's disease, which are used for solving the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method of making a paper-based electrochemical sensor for detecting alzheimer's disease comprising:
s1, preparing a multi-channel paper-based electrode with three working electrodes by a vacuum suction filtration method;
s2, preparing SiO2-Au-Thi, incubating the SiO2-Au-Thi with a target specific antibody, and synthesizing a SiO2-Au-Thi-Ab complex;
s3, preparing an immunosensor on two electrodes of a paper-based electrode by combining a sandwich strategy based on a sandwich structure and a SiO2-Au-Thi-Ab complex;
s4, ferrocene is modified on the other electrode of the paper-based electrode to serve as a reference signal.
Preferably, in step S1, the preparation of the multi-channel paper-based electrode is specifically:
firstly, filtering a carbon nano tube on cellulose paper to form a substrate for communicating a circuit;
secondly, carrying out suction filtration on AuNPs to form a layer of reflective gold film on the filter paper;
thirdly, coating Ag/AgCl conductive silver paste on the corresponding position to form a reference electrode, and putting the reference electrode into an oven for curing;
fourth, the successful preparation of paper-based electrodes was demonstrated by SEM observation of electrode surface morphology and investigation of the electrochemical performance of the electrode by CV.
Preferably, in step S2, the preparation of SiO2-Au-Thi is specifically:
firstly, synthesizing dendritic mesoporous SiO2 nano particles by an anion-assisted method;
secondly, performing amino functionalization on SiO2 by using APTES;
thirdly, through the reaction of-NH 2 on the surface of SiO2 and Au, auNPs are loaded on the surface of SiO2, and SiO2-Au is synthesized;
fourth, the preparation of SiO2-Au-Thi is achieved by successful loading of Thi through electrostatic interactions between negatively charged AuNPs and Thi.
Wherein the specific antibody of interest in step S2 is a specific antibody of aβo or Fetuin B.
Further preferably, the concentration of SiO2-Au-Thi in the SiO2-Au-Thi-Ab complex is 1.25mg/mL.
Preferably, in step S3, the immunosensor preparation is specifically:
firstly, antibodies corresponding to Abeta o and Fetuin B are dripped on two working electrodes of a paper-based electrode, and are dried in a refrigerator at 4 ℃ overnight;
secondly, dropwise adding BSA blocking buffer solution to block the residual active sites and eliminate nonspecific binding;
thirdly, dripping the incubated A beta o or Fetuin B standard solution on a corresponding working electrode, and incubating for 2 hours in a constant-temperature water tank;
fifthly, the SiO2-Au-Thi-Ab complex is dripped on the electrode, and incubated for 1.5h in a constant temperature water tank, so that the immunosensor is prepared.
Preferably, different concentrations of aβ monomers are incubated in a thermostatted water tank for 24h to form aβo.
Preferably, in step S4, fcHT is dropped onto the other working electrode of the paper-based electrode, and FcHT molecules are self-assembled onto the AuNPs layer using Au-S bonds, completing the preparation of the FcHT modified electrode.
Paper-based electrochemical sensor for detecting Alzheimer's disease, which is prepared by a preparation method of the paper-based electrochemical sensor for detecting Alzheimer's disease.
The application of the paper-based electrochemical sensor for detecting the Alzheimer's disease is that the paper-based electrochemical sensor for detecting the Alzheimer's disease is used for detecting the Aβo and the Fetuin B, the CHI660e electrochemical workstation is used for detecting the current signals of the FC and the Thi, and the concentration of the Aβo and the Fetuin B is quantified through the ratio of the two.
Compared with the prior art, the invention has the beneficial effects that: the invention is based on a sandwich method, combines a paper-based electrode and an immunosensor constructed by SiO2, realizes innovative detection of Abeta o and Fetuin B by utilizing a CHI660e electrochemical workstation, has strong specificity, high sensitivity and good stability, and is successfully applied to content determination of Abeta o and Fetuin B in a sample, wherein the immunosensor has higher sensitivity and wider linear range by optimizing synthesis conditions and incubation time, and can completely meet the requirements of Abeta o and Abeta f content determination in brain tissues of whole animals. In addition, the dendritic silicon dioxide nano particles have larger specific surface area, can load a large amount of AuNPs, amplify electrochemical signals, have good biocompatibility and have smaller toxicity to biomolecules such as antibodies.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is a schematic illustration of the preparation principle of a paper-based electrode of the present invention;
FIG. 2 is a scanning electron microscope image of the cellulose paper (A), suction filtered SWCNT (B) and AuNPs of the present invention;
FIG. 3 is a CV diagram (A) of a paper-based electrode at different sweep rates, a linear relationship (B) between peak current and sweep rate, and three working electrodes on the paper-based electrode at 1mM K 3 [Fe(CN) 6 ]CV diagram (C) of scanning at a scanning rate of 100 mV/s;
FIG. 4 is a cyclic voltammogram (A) and an enlarged view of the reduction peak (B) of the working electrode of the present invention in 0.05M H2SO 4;
FIG. 5 is a TEM image of SiO2 (A), siO2/NH2 (B) and SiO2-Au (C, D) of the present invention;
FIG. 6 is an SEM image of SiO2 (A, B) and SiO2-Au (C, D) of the present invention;
FIG. 7 is an infrared spectrum of SiO2, siO2/NH2 and SiO2-Au of the present invention;
FIG. 8 is a graph (A) of nitrogen adsorption and a graph (B) of pore size distribution of SiO2-Au of the present invention;
FIG. 9 is a graph of Square Wave Voltammetry (SWV) response of Abeta o and Fetuin B (A and C) at various concentrations on a sensor and corresponding linear graphs (B and D) according to the present invention;
FIG. 10 is a view of the sensor of the present invention looking at the selectivity of different substances;
FIG. 11 is a graph of Square Wave Voltammetry (SWV) response of the Abeta monomers, oligomers and fibrils of the invention on a sensor;
FIG. 12 is a schematic representation of the simultaneous detection of A.beta.o and Fetuin B by a paper-based electrode of the present invention.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Examples: a method of making a paper-based electrochemical sensor for detecting alzheimer's disease comprising:
preparing a multi-channel paper-based electrode with three working electrodes by a vacuum filtration method;
referring to fig. 1, first, carbon nanotubes are suction-filtered onto cellulose paper to form a substrate for connecting a circuit; the AuNPs was then suction filtered onto filter paper to form a reflective gold film for use as a working electrode, counter electrode and circuit connection. Finally, coating Ag/AgCl conductive silver paste on the corresponding reference position to form a reference electrode, and completing the preparation of the paper-based electrode;
in one implementation:
preparation of 10mM PBS buffer (pH 7.4): 0.01g of KCl, 0.01g of NaH2PO, 0.15g of NaCl and 0.155g of Na2HPO4.12H2O are respectively weighed and dissolved in a certain volume of deionized water, the pH value is regulated to 7.4 by HCl, the volume is fixed to 500.00mL, and the mixture is stored in a refrigerator at 4 ℃.
Preparation of 30% Triton X-100 solution: 28.2mL of Triton X-100 and 72.8mLPBS are mixed and placed in a water bath at 37 ℃ for 2 hours, so that the Triton X-100 and the 72.8mLPBS are fully dissolved and uniformly mixed; the solution was diluted to 1% before use.
Preparation of 6. Mu.g/mL SWCNT solution: 2mg of SWCNT is weighed and dissolved in 4mL of PBS to obtain 0.5mg/mL of SWCNT mother liquor, which is thoroughly mixed by ultrasound and diluted to 6 μg/mL with 1% Triton X-100 solution when in use.
Synthesis of AuNPs: 50mL of a 0.01% HAuCl4 solution (w/v) was heated to boiling by a reflux apparatus; then, 5mL of 1% sodium citrate solution (w/v) was added rapidly with vigorous stirring; the solution was boiled for 30 minutes and then slowly cooled to room temperature to give AuNPs solution.
Preparation of polyethylene terephthalate template: using a polyethylene terephthalate (PET) film to prepare a template in an electrochemical device; firstly, directly attaching the PET film to a sealing film by utilizing the adhesive surface of the PET film to form a laminated film so as to promote adhesion with filter paper; the preparation of the top layer template and the bottom layer template is to cut the laminated film according to a pre-designed pattern by a laser cutting machine; the top template is round, has the same diameter as the filter paper and comprises two electrode patterns; each electrode consists of three working electrodes, a counter electrode and a reference electrode. The bottom mask is also circular, the same diameter as the filter paper, but without engraving inside;
preparation of paper-based electrode: firstly, laminating filter paper and a sealing film layer of a top template, flushing the laminated paper with a few milliliters of water, putting the laminated paper into a vacuum filtration system, and adding 10 milliliters of water for wetting; dispersing 6 mug/mL of single-walled carbon nanotube solution in 1% Triton X-100 solution, filtering 10mL of single-walled carbon nanotube solution onto filter paper under vacuum at a flow rate of 0.29mL cm < -2 > min < -1 >, washing 5 times under vacuum after the filtering is finished with 10mL of distilled water to remove Triton X-100, and carefully removing bubbles on the filter paper with a suction tube after each washing; then, the bottom template is placed under filter paper loaded with single-walled carbon nanotubes, 10mL of AuNPs solution is filtered onto the filter paper under vacuum at a flow rate of 0.77mLcm-2min-1, and then the filter paper with the electrode is dried at room temperature overnight; after complete drying, the bottom mask and the filter paper are laminated, and the appointed reference electrode area is coated with Ag/AgCl conductive silver paste, and the silver paste is put into a baking oven at 75 ℃ and heated for 30min;
the manufactured paper-based electrode is characterized by a Scanning Electron Microscope (SEM), electrochemical performance is detected by Cyclic Voltammetry (CV), and as shown in FIG. 2, a rough surface and a typical fiber network structure which are formed by cellulose paper fibers can be clearly observed from the surface of cellulose paper, which is beneficial to the penetration of carbon nanotubes on the cellulose surface, the conductivity of the electrode can be increased, the pore diameter is about 0.1-1 mu m, and the smaller pore diameter is beneficial to the interception of the carbon nanotubes on the cellulose surface, as shown in FIG. 2A; after the carbon nanotubes are suction-filtered, as shown in fig. 2B, the cellulose paper almost cannot see the original porous structure, is replaced by the tubular structure, and the carbon nanotubes are tightly stacked to form a film, which proves that the carbon nanotubes are covered on the surface of the cellulose paper, and as also shown in fig. 2C, the surface of the electrode after the AuNPs are suction-filtered is a layer of even and tight AuNPs, which ensures the connection of electrode lines and increases the conductivity.
Referring to fig. 3, the electrochemical performance of the paper-based electrode was evaluated by electron transfer rate of potassium ferricyanide. First, cyclic Voltammetry (CV) measurements were performed on 1mM K3[ Fe (CN) 6] in 0.1M KCl at a scan rate of 10-400mV/s, as shown in FIGS. 3A and 3B, a reversible electrochemical response was observed, and there was a linear relationship between peak current and scan rate, indicating that the redox reaction in the system was diffusion limited. Immediately after cyclic voltammetry measurements were performed on all three working electrodes on the same paper base electrode, as shown in fig. 3C, a reversible redox reaction was observed with overlapping reduction and oxidation peak potentials (epc=0.101V and Epa =0.199V), the peak-to-peak spacing of the redox peaks was 98mV, indicating a high degree of reversibility of the electrodes, and the CV patterns generated by the three working electrodes were highly coincident, demonstrating very similar surface areas between the working electrodes;
referring to FIG. 4, as shown in FIG. 4A, by scanning the working electrode at a scanning rate of 100mV/s in 0.05M H2SO4 to measure its specific surface area, typical oxidation peaks of gold and reduction peaks of gold oxide can be observed at 1.1V and 0.8V, and the electrochemical area of the working electrode is calculated by the reduction peaks, finding that the specific surface area of the working electrode is 0.287cm 2 Is greater than 0.125cm of the design area of the electrode 2 The average surface roughness was 2.3, which can be attributed to the rough surface of the filter paper.
Preparation of SiO2-Au-Thi: incubating SiO2-Au-Thi with a target specific antibody to synthesize a SiO2-Au-Thi-Ab complex;
in one embodiment:
preparation of 10mM Tris-HCl buffer solution (pH 7.4): 0.61g of Tris is weighed and dissolved in a certain volume of deionized water, the pH is regulated to 7.4 by HCl, the volume is regulated to 500.00mL, and the mixture is stored in a refrigerator at 4 ℃.
Synthesis of SiO2/NH 2: 68mg of TEA was weighed into a three-necked flask containing 25mL of water, and after stirring at 80℃for 30 minutes, 380mg of CTAB and 168mg of sodium salicylate were added and the stirring was continued for 1 hour; then, 4mL of TEOS was added to the above solution, and the mixture was stirred slowly at 80℃for 4 hours; centrifuging the product after the reaction is finished, washing the product with ethanol for 3 times, and removing unreacted substrate; the product was then extracted 3 times with 3mL of 37% hydrochloric acid and 50mL of absolute methanol at 60 ℃ for 6 hours each to purify the product; centrifuging the product after the reaction is finished, washing the product with ethanol for 3 times, and finally dispersing SiO2 in 100mL of ethanol solution; 2.5mL of ammonia water and 1mL of APTES are added into the SiO2 ethanol solution, and the mixture is refluxed and stirred for 24 hours at 80 ℃; the final product was collected by centrifugation, washed 3 times with ethanol and dispersed in 50mL of ethanol to give SiO2/NH2.
Preparation of SiO2-Au-Thi: taking 1mL of SiO2/NH2 ethanol solution, centrifuging for 5 minutes at 8000 rotating speed, removing supernatant, adding AuNPs solution, and performing ultrasonic treatment for 10 minutes to obtain uniform solution; collecting the SiO2-Au complex by centrifugation and washing with deionized water for 1 time to remove excessive AuNPs, thereby obtaining SiO2-Au; adding 1mL of saturated Thionine (THi) into the precipitate, stirring at room temperature for 24 hours, centrifuging at 8000 rotation speed after the reaction is finished to remove unbound THi until the supernatant is transparent, and re-suspending with PBS to obtain a suspension which is a SiO2-Au-Thi complex and is placed in a refrigerator at 4 ℃ when not in use;
synthesis of SiO 2-Au-Thi-Ab: to the SiO2-Au-Thi complex was added 10. Mu.L of AB or FB antibody diluted 3-fold with antibody dilution, and incubated at 150r/min for 4 hours in a shaker at 25 ℃. Centrifuging at 8000 rpm, collecting precipitate, washing, and suspending in 132 μl Tris-HCl buffer solution again to obtain suspension as SiO2-Au-Thi-Ab biocomposite; placing in a refrigerator at 4 ℃ for standby; the SiO2-Au-Thi-Ab was characterized by a Transmission Electron Microscope (TEM), a Fourier transform infrared spectrum (FT-IR), an X-ray photoelectron spectrum (XPS), an Element Distribution (EDS), and the like.
Referring to fig. 5 to 6, as shown in fig. 5A and 6A, the synthesized dendritic mesoporous silica is observed to be spherical under an electron microscope, has an average particle diameter of about 270nm, has a pore diameter of about 30nm, and has good dispersibility; after amino functionalization of SiO2, as shown in TEM fig. 5B and SEM fig. 6B, the morphology, size, and pore size of SiO2 hardly changed. The AuNPs with uniform size are synthesized by reducing sodium citrate, the AuNPs are directly loaded on SiO2 by ultrasound through metal ligand affinity between SiO2/NH2 and the AuNPs, as shown in FIGS. 5C, D and 6C, D, the AuNPs with spherical shape can be obviously observed on the SiO2 after the AuNPs are loaded, and the AuNPs are loaded on the SiO2 with larger pore diameter, the SiO2 size is slightly enlarged, and the pore diameter is obviously reduced, thus proving successful preparation of SiO2-Au;
referring to FIG. 7, FT-IR diagrams of SiO2, siO2/NH2, and SiO2-Au are shown; the absorption peak of the spectrum at 1087cm-1 was attributable to the stretching vibration of Si-O, and the infrared spectra of SiO2/NH2 and SiO2-Au showed an infrared absorption peak of amino group at 1543cm-1, whereby the functionalization of amino group on the SiO2 surface could be confirmed. The infrared spectrum of SiO2-Au shows a distinct absorption peak at 3468cm < -1 >, which corresponds to the stretching vibration of the O-H bond in the carboxyl group of AuNPs; meanwhile, the stretching vibration peak of C=O bond in AuNPs carboxyl appears at 1648cm < -1 >, and the results provide strong evidence for successful formation of SiO2-Au by using AuNPs and SiO 2;
the N2 adsorption-desorption isothermal curves of the products obtained in each step in the SiO2-Au synthesis process are characterized, as shown in FIGS. 8A and 8B, siO2 has uniform and large pore channels, and the calculated Brunauer-Emmett-Teller (BET) specific surface area and total pore volume of the SiO2 are 521.41m respectively 2 /g and 2.04cm 3 Per g, the specific surface area and the total pore volume of SiO2/NH2 obtained after amination are reduced to 299.96m respectively due to the introduction of amino groups 2 Per g and 1.41cm 3 Per g, the specific surface area and the total pore volume respectively drop sharply to 94.03m after loading with AuNPs 2 Per g and 0.46cm 3 /g, which indicates that the SiO2 channels are effectively filled by AuNPs;
finally, the potential change in the layer-by-layer modification process of the SiO2-Au-Thi-Ab is characterized, the surface of the SiO2 is negatively charged to be-18.9 mV, and the surface group is silicon hydroxyl. After the SiO2 is subjected to amino functionalization, the surface of the SiO2/NH2 is positively charged to be 6.65mV, because the surface charge is reversed from negative charge to positive charge due to the protonation reaction on the surface of the SiO2, the successful modification of the amino group on the surface of the SiO2 is shown; the SiO2-Au surface is negatively charged to-25.3 mV, because SiO2 carries a large amount of negatively charged AuNPs; when the positive and negative AuNPs combine by electrostatic interaction, the charge is reversed from negative to positive due to neutralization of the positive and negative charges, at 12.3mV, indicating a successful loading of Thi.
Preparing an immunosensor on two of the paper-based electrodes by combining the SiO2-Au-Thi-Ab complex based on a sandwich strategy of a sandwich structure; and modifying ferrocene on the other electrode of the paper-based electrode as a reference signal.
In one embodiment:
1mM Ca2+, na+, cu2+, fe2+ solution preparation: 5.33mg NaCl,1.06mg CaCO3,1.268mg FeCl2, 23.10mg CuSO4, respectively, are precisely weighed and dissolved in deionized water to obtain 1mM Ca2+, na+, cu2+, fe2+ solution, and are preserved at room temperature.
Preparation of 10. Mu.M amino acid solutions: 1.28mg of glutamic acid, 1.046mg of cysteine, 1.025mg of threonine, 0.8mg of serine and 1.0mg of valine are weighed and dissolved in deionized water solution to obtain 10 mu M of each amino acid solution, and the amino acid solution is placed in a refrigerator at 4 ℃ for later use.
Preparation of 10. Mu.M dopamine solution: accurately weighing 1.32mg of dopamine, adding 2mL of deionized water for full dissolution, and storing in a refrigerator at 4 ℃ for preparation.
Preparation of 10. Mu.M ascorbic acid solution: 1.76mg of ascorbic acid is precisely weighed and dissolved in 100mL of deionized water to obtain 100 mu M ascorbic acid solution, and the solution is placed in a refrigerator at 4 ℃ for storage, is prepared and used at present, and is diluted to 10 mu M when in use.
Preparation of Aβ monomer, oligomer and fiber solution: first, A.beta.powder was dissolved in HFIP to prepare a monomer stock solution of 1.0 mg/mL. Ultrasonic treatment was carried out at 25℃and stored as stock solution in a-20℃refrigerator. Prior to use, the stock solution was evaporated by nitrogen flow to remove HFIP solvent. Subsequently, 100. Mu.L of DMSO was added for sufficient dissolution to give the A.beta.monomer. The stock solution was diluted to different concentrations with 20mM Tris-HCl buffer to give an A.beta.monomer solution. And placing the A beta monomer solution in a shaking table at 37 ℃ for incubation for 24 hours to obtain the A beta oligomer. And (5) continuing to incubate for three days to obtain Abeta fibrils, and storing at 4 ℃.
Preparation of 2% chloral hydrate: about 0.5g of chloral hydrate is weighed and dissolved in 25mL of deionized water, and the chloral hydrate is fully dissolved by ultrasonic treatment, so that the obtained colorless transparent solution is placed in a refrigerator at 4 ℃ for storage.
Preparation of artificial cerebrospinal fluid aCSF solution: 7.3634g NaCl,0.1789g KCl,0.6804g KH2PO4,0.1728g MgCl2,0.1221g CaCl2,2.3102g NaHCO3 and 0.7102g Na2SO4 are respectively weighed precisely and dissolved in a certain volume of deionized water, the pH is regulated to 7.4 by using 1M HCl, the volume is fixed to 1000.00mL, and the mixture is preserved at room temperature.
Preparation of 0.1M PBST buffer (pH 7.4): respectively precisely weighing 2.9794g NaCl,0.2255g NaH2PO4,3.0769g Na2HPO4. 12H2O,0.2195g KCl, dissolving in distilled water of a certain volume, adjusting pH to 7.4 with 1M HCl, adding 0.05% Tween-20, constant volume to 1000.00mL, and preserving at room temperature.
Preparation of 1% bsa solution: 1g of BSA is precisely weighed, 100mL of deionized water is added, 0.05% Tween-20 is added after the BSA is fully dissolved, and the BSA is placed in a refrigerator at 4 ℃ for storage and is prepared for use.
Preparation of 1mM K3[ Fe (CN) 6] solution: 16.46mg of K3[ Fe (CN) 6] is precisely weighed, added into 50mL of 0.1M KCl, stirred to be fully dissolved, and stored in a dark place for preparation.
Antibodies corresponding to 5 μ L A βo and Fetuin B (AB and FB, three times diluted with antibody dilution) were applied drop-wise to paper-based electrodes and allowed to dry overnight in a refrigerator at 4 ℃. After sufficient drying, washing was repeated with PBS and PBST washes to remove unbound antibody. Thereafter, 10. Mu.L of BSA blocking buffer was added dropwise to block the remaining active sites, eliminating non-specific binding. Repeatedly and alternately washing with PBS and PBST, and storing at 4deg.C.
Different concentrations of aβ monomers were incubated in a 37 ℃ water bath for 24h to form aβo. 10 mu L A beta o and Fetuin B are respectively dripped on the corresponding working electrodes, cultured for 2 hours in a constant temperature water tank at 37 ℃, and repeatedly and alternately washed by PBS and PBST to remove unbound Abeta o and Fetuin B. Finally, 5. Mu.L of the prepared SiO2-Au-Thi-AB and SiO2-Au-Thi-FB biocomposites were dripped on the electrode, incubated in a constant temperature water tank at 37℃for 1.5h to interact with their corresponding antigens, and washed alternately with PBS and PBST for three cycles to remove unbound biocomposites. And drying at room temperature to finish the preparation process of the immunosensor. The sensor should be stored in a refrigerator at 4 ℃ when not in use.
Preparing an FC functionalized electrode: while incubating the two working electrodes used as the sensor with SiO2-Au-Thi-Ab, 10. Mu.L of FcHT was dropped on the working electrode of the unmodified antibody, fcHT molecules were self-assembled onto the AuNPs layer by Au-S bond, thoroughly washed with deionized water, and unbound FcHT was removed to give FcHT modified electrode.
Paper-based electrochemical sensor for detecting Alzheimer's disease, and the paper-based electrochemical sensor prepared by the method.
The application of the paper-based electrochemical sensor for detecting Alzheimer's disease, the detection of Aβo and Fetuin B based on the paper-based electrochemical sensor, referring to FIG. 12, and the detection of the current signals of FC and Thi by using the CHI660e electrochemical workstation, the concentration of Aβo and Fetuin B is quantified by the ratio between the two:
in one embodiment: determination of Aβo and Fetuin B content in blood and brain tissue of AD transgenic mice by the paper-based electrochemical sensor obtained as described above.
Purchase of AD transgenic mice and treatment of brain tissue samples: APP/PS1 transgenic AD mice 3 were purchased from Jiangsu Huazhenxin medicine Co., ltd; thereafter, mice were subjected to blood sampling: before the operation, an ice plate, a capillary tube, an anti-coagulation EP tube, an EP tube, and an ice bin for placing the EP tube and containing crushed ice are prepared. Firstly, anesthetizing a mouse, taking 100 mu L of blood from any orbital venous plexus of the mouse after anesthesia, rotationally penetrating a right hand-held capillary from the inner canthus of the eye, and inserting the eyeground blood into an EP tube containing EDTA;
brain tissue sampling was performed on mice: scissors, tweezers and the like are treated with alcohol in advance, the whole process is operated on an ice plate, and the fetched tissues are temporarily stored in crushed ice. Mice were cervical sacrificed under anesthesia and their cranium was cut with surgical tissue scissors. The mouse skull was then removed along the mouse skull midline with surgical scissors, the mouse brain was completely exposed, the fascia covering the brain surface was carefully removed with forceps, and the entire brain was then completely removed with forceps. After the brain was removed, the cerebellum was carefully removed with forceps and peeled off from the surface along the location of the hippocampus, the crescent-shaped tissue free from the surrounding tissue was seen as the hippocampus, which was carefully and completely removed, placed in a 2mL EP tube which had been weighed in advance and labeled, and then the EP tube with the hippocampus was placed in an ice box. Then, a sufficient amount of cortex was taken from the brain surface of the mice, also placed in a 2mL EP tube. The weight of the EP tube after the tissue was weighed, the weight of the individual EP tube weighed in advance was subtracted, and a previously prepared Tris-HCl (pH 7.4) solution was added in a proportion of 25mg/mL (1 mL of 50mM Tris-HCl was added per 25mg of tissue), and the mixture was homogenized well in crushed ice by a homogenizer (homogenization was continued after 30 seconds of each homogenization and 10 seconds of suspension). After homogenization, centrifugation is carried out at a rotation speed of 5000r/min at 4 ℃, supernatant is left for later use, and when not used, the supernatant is stored in a refrigerator at-80 ℃, and before use, thawing is carried out in the refrigerator at 4 ℃.
Synthesis conditions: the concentration of SiO2-Au-Thi in the SiO2-Au-Thi-Ab complex is 1.25mg/m;
incubation time: dripping the incubated A beta o or Fetuin B standard solution on a corresponding working electrode, and incubating for 2 hours in a constant-temperature water tank; the SiO2-Au-Thi-Ab complex is dripped on the electrode, and incubated for 1.5h in a constant temperature water tank;
based on the conditions and the incubation time, the analysis performances of the two immunosensors, such as the linear range, LOD, selectivity, stability, reproducibility and the like of the electrochemical detection of the Aβo and the Fetuin B, are respectively examined; fig. 9 is a graph of SWV response curves (fig. 9A and 9C) and their corresponding linear regression curves (fig. 9B and 9D) for two sensors at different concentrations aβo and Fetuin B. As can be seen from the trend in the graph, as the concentrations of the added Abeta o and Fetuin B gradually increase, the reduction peak current value of Thi also increases at-0.28V, and the current value of FC remains stable. The calculation shows that in the range of 0.1-40ng/mL, the logarithm of the concentration of Abeta o and Fetuin B and the ratio of FC and Thi reduction peak current are in good linear relation, and the linear regression equation is as follows:
Aβo:ΔI＝1.54lgC(ng/mL)+5.1；
Fetuin:ΔI＝2.49lgC(ng/mL)+5.72；
the linear correlation coefficients were 0.999 and 0.998, respectively. Under the condition that the signal-to-noise ratio is more than or equal to 3, the detection Limits (LOD) of the method on Abeta o and Fetuin B are respectively 0.01 and 0.008ng/mL.
The anti-interference capability of the sensor is examined:
considering the complexity of biological samples, before the sensing system is applied to an actual sample, whether various metal ions and amino acids possibly existing in the sensing system influence the detection performance of the sensor or not needs to be examined, and the selectivity of the system is examined through interference experiments and competition experiments; in the embodiment, several metal ions of Na+, fe2+, ca2+, cu2+, valine, cysteine, serine, glutamic acid, threonine, ascorbic acid and dopamine are selected; measuring SWV signals of the interfering substances, respectively, in the same detection manner as Abeta o, and then comparing differences between the SWV signals and Abeta o signals, and observing signal changes of a mixture of the interfering substances and Abeta o; as can be seen from fig. 10A, the response current of the interfering substance is significantly weaker than the response of aβo; this is due to the lack of specific recognition elements of these substances on the electrode surface, so that they cannot bind to the electrode; whereas as shown in fig. 10B, the electrochemical signal of the mixture is almost the same as aβo alone; the above results confirm that the constructed electrochemical sensor is capable of specifically responding and detecting aβo or Fetuin B, and thus can be applied to the measurement in an actual sample; in addition, by researching the influence of Abeta monomers, abeta o and Abeta f on results; as shown in fig. 11, the current values generated by the aβ monomers and the aβf are obviously lower than those generated by the aβo, and the SWV response of the aβf is slightly higher than that of the aβ monomers, probably because part of the aβo is not converted into fiber filaments during incubation, so that the fiber filaments are recognized by antibodies on the electrodes and are fixed on the surfaces of the electrodes, and the results prove that the method is accurate and reliable and can be used for measuring actual samples.
Finally, it should be noted that: the foregoing is merely a preferred example of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method of making a paper-based electrochemical sensor for detecting alzheimer's disease comprising:
s1, preparing a multi-channel paper-based electrode with three working electrodes by a vacuum suction filtration method;
s2, preparing silicon dioxide-gold-thionine SiO2-Au-Thi, and incubating the SiO2-Au-Thi with a target specific antibody to synthesize a SiO2-Au-Thi-Ab complex;
wherein the specific antibody of the target is a specific antibody of Abeta o or Fetuin B;
s3, preparing an immunosensor on two electrodes of a paper-based electrode by combining a sandwich strategy based on a sandwich structure and a SiO2-Au-Thi-Ab complex; the immunosensor is prepared specifically as follows:
firstly, antibodies corresponding to Abeta o and Fetuin B are dripped on two working electrodes of a paper-based electrode, and are dried in a refrigerator at 4 ℃ overnight;
secondly, dropwise adding BSA blocking buffer solution to block the residual active sites and eliminate nonspecific binding;
thirdly, dripping the incubated A beta o or Fetuin B standard solution on a corresponding working electrode, and incubating for 2 hours in a constant-temperature water tank;
fifthly, dripping the SiO2-Au-Thi-Ab complex on an electrode, and incubating for 1.5h in a constant temperature water tank to finish the preparation of the immunosensor;
s4, modifying ferrocene Fc on the other electrode of the paper-based electrode as a reference signal.
2. The method for manufacturing a paper-based electrochemical sensor for detecting alzheimer's disease according to claim 1, characterized in that: in step S1, the preparation of the multi-channel paper-based electrode is specifically:
firstly, filtering a carbon nano tube on cellulose paper to form a substrate for communicating a circuit;
secondly, carrying out suction filtration on AuNPs to form a layer of reflective gold film on the filter paper;
thirdly, coating Ag/AgCl conductive silver paste on the corresponding position to form a reference electrode, and putting the reference electrode into an oven for curing;
fourth, the successful preparation of paper-based electrodes was demonstrated by SEM observation of electrode surface morphology and investigation of the electrochemical performance of the electrode by CV.
3. The method for manufacturing a paper-based electrochemical sensor for detecting alzheimer's disease according to claim 1, characterized in that: in the step S2, the preparation of SiO2-Au-Thi is specifically as follows:
firstly, synthesizing dendritic mesoporous SiO2 nano particles by an anion-assisted method;
secondly, performing amino functionalization on SiO2 by using APTES;
thirdly, through the reaction of-NH 2 on the surface of SiO2 and Au, auNPs are loaded on the surface of SiO2, and SiO2-Au is synthesized;
fourth, the preparation of SiO2-Au-Thi is achieved by successful loading of Thi through electrostatic interactions between negatively charged AuNPs and Thi.
4. A method of manufacturing a paper-based electrochemical sensor for the detection of alzheimer's disease according to claim 3, characterized in that: the concentration of SiO2-Au-Thi in the SiO2-Au-Thi-Ab complex was 1.25mg/mL.
5. The method for manufacturing a paper-based electrochemical sensor for detecting alzheimer's disease according to claim 1, characterized in that: different concentrations of aβ monomers were incubated in a thermostated water tank for 24h to form aβo.
6. The method for manufacturing a paper-based electrochemical sensor for detecting alzheimer's disease according to claim 1, characterized in that: in step S4, fc is dripped on the other working electrode of the paper-based electrode, and the Fc molecule is self-assembled onto the AuNPs layer by using Au-S bond, thereby completing the preparation of the Fc modified electrode.
7. A paper-based electrochemical sensor for detecting alzheimer's disease, characterized in that: a paper-based electrochemical sensor prepared by the method for preparing a paper-based electrochemical sensor for detecting alzheimer's disease according to any one of claims 1-4.
8. An application of a paper-based electrochemical sensor for detecting alzheimer's disease, which is characterized in that: the detection of aβo and feturin B based on the paper-based electrochemical sensor for detecting alzheimer's disease according to claim 7, and the detection of the current signals of Fc and Thi using the CHI660e electrochemical workstation, the concentration of aβo and feturin B being quantified by the ratio between them.
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