CN111610245A - Chemical biosensor for detecting Tau protein of Alzheimer disease - Google Patents
Chemical biosensor for detecting Tau protein of Alzheimer disease Download PDFInfo
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Abstract
The invention discloses a chemical biosensor for detecting Tau protein of Alzheimer's disease, which comprises the following steps: preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P · xH 2O); the method has the advantages of signal amplification only by adding the redox mediator and the reducing agent into the electrolyte solution, simple operation, easy realization of detection of the target analyte with ultralow concentration, sensitive detection, low cost, better accuracy, anti-interference performance, specificity and selectivity, and capability of providing a simple and accurate detection method for early diagnosis of the Alzheimer disease.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a chemical biosensor for detecting Tau protein of Alzheimer disease.
Background
Two major pathologies developed in the brain by Alzheimer's Disease (AD) include neurofibrillary tangles composed of Tau protein and senile plaques containing amyloid beta (a β) protein, the most common form of dementia, with misfolding of the protein often occurring within a few years prior to extensive neurodegeneration. There are six Tau protein subtypes in the human brain, and their good dynamic equilibrium with microtubules is mainly regulated by phosphorylation of serine and threonine in healthy neurons, thereby promoting the polymerization and stabilization of microtubules in the nervous system. However, in the case of poor physiological conditions, since Tau protein has tens of phosphorylation sites, it may undergo abnormal hyperphosphorylation at different sites, failing to stabilize microtubules, causing neurofibrillary tangles and paired helical filaments to form, thereby causing AD dementia. Many studies have shown that the level of Tau protein in cerebrospinal fluid (CSF) increases slightly with age, but the level of Tau protein in CSF of AD patients increases significantly, indicating that Tau protein is a key biomarker for early diagnosis of AD.
Sang dran et al demonstrated a cutoff value of 4.3 pM for Tau protein in cerebrospinal fluid, which distinguishes healthy from AD patients with 92% sensitivity and 89% specificity. Since neurodegenerative diseases are irreversibly progressive, effective methods are needed to diagnose AD early before the disease is so severe that it cannot be cured. The Tau protein is used as a reliable disease marker for early diagnosis of AD, and a Tau protein detection method which is simple to operate and is economical and effective is needed to be developed, the content of the Tau protein in CSF is measured by conventional methods at present, such as enzyme-linked immunosorbent assay (ELISA) documents, Mass Spectrometry (MS) documents, Magnetic Resonance Imaging (MRI) documents or Positron Emission Tomography (PET) documents, and the like, however, the conventional methods are complex to operate, time-consuming and labor-consuming, and a chemical biosensor for detecting the Tau protein of the Alzheimer disease is provided for solving the problem.
Disclosure of Invention
The invention aims to provide a chemical biosensor for detecting Tau protein in Alzheimer's disease, which solves the problems that the conventional methods are complex to operate and time-consuming and labor-consuming, such as enzyme-linked immunosorbent assay (ELISA) documents, Mass Spectrometry (MS) documents, Magnetic Resonance Imaging (MRI) documents or Positron Emission Tomography (PET) documents and the like, which are used for measuring the content of Tau protein in CSF at present.
In order to achieve the purpose, the invention provides the following technical scheme: a chemical biosensor for the detection of Tau protein of alzheimer's disease comprising the steps of:
step 1: preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP);
step 2: activation of gold electrode: firstly, a gold electrode is sequentially coated on polishing cloth by using a metal material with the grain diameter of 1.0,0.3, 0.05 μm Al2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished;
and step 3: stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7 mu L of 1 mu MSA-ALP is dripped to react for 1 hour at 37 ℃ to obtain ALP/Apt/Tau381a/Ab/modified gold electrode;
and 4, step 4: DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM FcM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms and a pulse period of 0.2 s, and Tau was measured for changes in the intensity of the electrical signal381Carrying out quantitative analysis on the protein concentration;
and 5: and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The protein contents were 0.092 nM, 1.04 nM and 52.0 nM, respectively, and the electric potential was adjusted toThe result obtained by chemical detection is not much different from the reference value.
Preferably, in step 1, the water to be used is ultrapure water (Milli-Q, Millipore, 18.2 M.OMEGA.cm)-1) All reagents were purchased for use directly without further purification.
Preferably, in step 1, Tau381Protein and its antibody were purified with 1 × PBS (137 mM NaCl; 2.7 mM KCl; 8.1mM Na)2HPO4; 1.8 mM KH2PO4) Diluted to the desired concentration, Apt and SA-ALP were diluted with 1 × B2 (10 mM Tris-HCl;50 mM NaCl; 10 mM KCl; 10 mM MgCl)2) Dilute to the desired experimental concentration.
Preferably, in step 2, Cyclic Voltammetry (CV) is performed on CHI660A, and scanning is performed at a scanning speed of 0.1V/S in a potential range of-0.2 to 1.6V by continuous cyclic voltammetry.
Preferably, in step 3, the gold electrode obtained is finally soaked in a solution containing 20 mM Tris-HCl (pH =8.0), 5mM AAP, 10 mM MgCl2The mixed solution of (3) was reacted at room temperature for 0.5 hour.
Preferably, in step 3, the principle is as follows: firstly, modifying sulfydryl in MPA on a gold electrode through Au-S bond, then reacting amino in an antibody with carboxyl on the gold electrode modified with MPA to obtain the gold electrode modified by the antibody, and adding target protein Tau381Apt/Tau formation following aptamer binding381the/Ab sandwich structure, modified by SA-ALP streptavidin which interacts with biotin on the aptamer, finally results in an alkaline phosphatase modified gold electrode which, when immersed in a solution of alkaline phosphatase substrate AAP, catalytically hydrolyzes the AAP to L-Ascorbic Acid (AA), i.e. undergoes an enzymatic reaction, but the insulating self-assembled monolayer (SAMs) of linear thiol molecules may result in a slow electron transfer between the electrode and the enzyme or enzyme product, i.e. in an enzyme amplification system the enzyme and enzyme product itself usually do not directly exchange electrons with the SAMs covered electrode, so that the ` outer sphere to inner sphere ` ECC redox cycle triggered by the enzyme product is used to amplify electrochemical signals, FcM, AAP and TCEP respectivelyAs redox mediators, enzyme substrates and reducing agents, the ECC redox cycle process is: (1) FcM is stable in air and can be reduced and regenerated by ALP enzyme product AA after electrooxidation; (2) TCEP can reduce AA oxidation product dehydroascorbic acid (DAA) and regenerate AA rapidly, and TCEP is not in contact with FcM+(FcM oxidized form) and thus in the presence of the reducing agent TCEP, an electrochemical-chemical redox cycle is formed, amplifying the electrical oxidation signal of FcM, it is noted that neither AA nor TCEP in the system is oxidized over the potential sweep, and AAP is chosen as an enzyme substrate for ALP due to its low cost, water solubility and high potential.
Preferably, in step 3, the optimal Ab concentration is 0.05 mg.mL-190 min was chosen as the optimal reaction time for Apt.
Preferably, in step 3, after dropping 10. mu.L of 5. mu.M modified biotin aptamer (Apt-biotin), the reaction is carried out at 37 ℃ for 1.5 hours.
Preferably, in step 4, Tau is381The presence of the compound can form an ALP/Apt/Tau/Ab/sandwich type compound on the surface of an electrode, further generate electrochemical-chemical redox cycle amplification reaction guided by enzymatic reaction, and finally generate a remarkably enhanced FcM oxidation electric signal at 1.0 × 10-3nM ~ 1.0×102Enhancement of DPV Strength with Tau in nM range381The logarithm of the concentration has a good linear relationship.
Compared with the prior art, the invention has the beneficial effects that: the invention solves the problem that the content of Tau protein in CSF is measured by the conventional method at present by setting the process flows of preparing reagent and instrument, activating the gold electrode, gradually modifying the gold electrode, measuring DPV and detecting the actual sample, such as enzyme-linked immunosorbent assay (ELISA) literature, Mass Spectrometry (MS) literature, Magnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET) literature, however, the conventional methods have the problems of complicated operation, time and labor waste, the chemical biosensor for detecting the Alzheimer disease Tau protein has the advantages that signal amplification can be realized only by adding the redox mediator and the reducing agent into the electrolyte solution, the operation is simple, the detection of the ultralow-concentration target analyte is easy to realize, the detection is sensitive and low in cost, the accuracy, the anti-interference performance, the specificity and the selectivity are better, and a simple and accurate detection method can be provided for early diagnosis of the Alzheimer disease.
Detailed Description
The present invention will now be described in more detail by way of examples, which are given by way of illustration only and are not intended to limit the scope of the present invention in any way.
The invention provides a technical scheme that: a chemical biosensor for the detection of Tau protein of alzheimer's disease comprising the steps of:
step 1: preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP);
step 2: activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished;
and step 3: stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7 mu L of 1 mu MSA-ALP is dripped to react for 1 hour at 37 ℃ to obtain ALP/Apt/Tau381a/Ab/modified gold electrode;
and 4, step 4: DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM FcM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms and a pulse period of 0.2 s, and Tau was measured for changes in the intensity of the electrical signal381Carrying out quantitative analysis on the protein concentration;
and 5: and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The protein contents were 0.092 nM, 1.04 nM and 52.0 nM, respectively, and the results obtained by electrochemical assay and ginsengThe difference in the reference value is not large.
The first embodiment is as follows:
preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP); activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished; stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then, carboxyl groups on the surface of the gold electrode were activated with a mixed solution of 0.2M EDC and 0.1M NHS (prepared with 20 mM MES, pH = 6.5), and after the above steps, 7. mu.L of Ab was dropped onto the MPA-modified gold electrode and reacted at 4 ℃ for 12 hoursThen, obtaining an Ab modified gold electrode, and blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7. mu.L of 1. mu.M SA-ALP was added dropwise and reacted at 37 ℃ for 1 hour to obtain ALP/Apt/Tau381a/Ab/modified gold electrode; DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM FcM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms and a pulse period of 0.2 s, and Tau was measured for changes in the intensity of the electrical signal381Carrying out quantitative analysis on the protein concentration; and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
Example two:
in the first embodiment, the following steps are added:
in step 1, an aptamer of Tau protein is purchased from Shanghai bioengineering technology, Inc., and the aptamer Apt sequence is: 5 '-Biotin-GCGGAGCGTGGCAGG-3', Tau381Proteins and their antibodies (Ab) were purchased from Abcam, Bovine Serum Albumin (BSA) and alkaline phosphatase-labeled streptavidin (SA-ALP) were purchased from Shanghai Biotechnology, Inc., and the water used was ultrapure water (Milli-Q, Millipore, 18.2 M.OMEGA.cm.)-1) All reagents were purchased for direct use without further purification, Tau381Proteins and antibodies thereto were purified using 1 × PBS (137 mM NaCl; 2.7 mM KCl; 8.1mM Na)2HPO4; 1.8 mM KH2PO4) Diluted to the desired concentration, Apt and SA-ALP were diluted with 1 × B2 (10 mM Tris-HCl;50 mM NaCl; 10 mM KCl; 10 mM MgCl)2) Diluting to the required experimental concentration, and greatly facilitating the manufacture of users.
Preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP); activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished; stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activated with 0.2M EDC, 0.1M NHS mixed solution (formulated with 20 mM MES, pH = 6.5)After the above steps, 7 μ L of Ab is dripped on the gold electrode modified with MPA to react for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then 10 μ M BSA solution is used for sealing redundant active sites on the gold electrode; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7. mu.L of 1. mu.M SA-ALP was added dropwise and reacted at 37 ℃ for 1 hour to obtain ALP/Apt/Tau381a/Ab/modified gold electrode; DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM MFCM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms, and a pulse period of 0.2 s, and Tau was measured according to the variation of the intensity of the electrical signal measured381Carrying out quantitative analysis on the protein concentration; and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
Example three:
in the second embodiment, the following steps are added:
in step 2, Cyclic Voltammetry (CV) is performed on CHI660A, and continuous cyclic voltammetry is used for scanning in a potential range of-0.2-1.6V at a scanning speed of 0.1V/S, so that smooth measurement is guaranteed.
Preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP); activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished; stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7. mu.L of 1. mu.M SA-ALP was added dropwise and reacted at 37 ℃ for 1 hour to obtain ALP/Apt/Tau381a/Ab/modified gold electrode; DPV measurement: DPV measurementThe measurement is carried out in a mixed solution containing 20 mM Tris-HCl (pH =8.0), 2 mM MFCM, 5mM TCEP, and the sweep is carried out by differential pulse voltammetry, wherein the sweep potential is in the range of-0.1 to 0.6V, the pulse amplitude is 50 mV, the pulse width is 50 ms, and the pulse period is 0.2 s, and Tau is measured according to the change of the intensity of an electric signal381Carrying out quantitative analysis on the protein concentration; and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
Example four:
in the third embodiment, the following steps are added:
in step 3, the resulting gold electrode was finally soaked in a solution containing 20 mM Tris-HCl (pH =8.0), 5mM AAP, 10 mM MgCl2The mixed solution of (A) is reacted for 0.5 hour at room temperature, and the principle is as follows: firstly, modifying sulfydryl in MPA on a gold electrode through Au-S bond, then reacting amino in an antibody with carboxyl on the gold electrode modified with MPA to obtain the gold electrode modified by the antibody, and adding target protein Tau381Apt/Tau formation following aptamer binding381the/Ab sandwich structure, modified with streptavidin in SA-ALP, interacts with biotin on the aptamer to finally obtain a gold electrode modified with alkaline phosphatase, and soaking the electrode in a solution of alkaline phosphatase substrate AAP catalyzes the hydrolysis of AAP to L-Ascorbic Acid (AA), i.e. an enzymatic reaction, but the insulating self-assembled monolayers (SAMs) of linear thiol molecules may result in slow electron transfer between the electrode and the enzyme or enzyme product, i.e. in an enzyme amplification system, the enzyme and enzyme product themselves will not normally exchange electrons directly with the electrode with SAMs coating, since SAMs-coated electrodes are modified with streptavidinHere, the "outer-sphere to inner-sphere" ECC redox cycle triggered by the enzyme product is used to amplify the electrochemical signal, FcM, AAP and TCEP are used as redox mediator, enzyme substrate and reducing agent, respectively, the ECC redox cycle process is: (1) FcM is stable in air and can be reduced and regenerated by ALP enzyme product AA after electrooxidation; (2) TCEP can reduce AA oxidation product dehydroascorbic acid (DAA) and regenerate AA rapidly, and TCEP is not in contact with FcM+(FcM oxidized form) reaction, thus in the presence of reducing agent TCEP, electrochemical-chemical redox cycle process is formed, the electrical oxidation signal of FcM is amplified, it is noted that neither AA nor TCEP in the system is oxidized in the potential scanning range, and in addition, because AAP is low cost, easy to be dissolved in water and high potential, it is selected as enzyme substrate of ALP, the optimal Ab concentration is 0.05 mg.mL-1And selecting 90 min as the optimal reaction time of Apt, dropwise adding 10 mu L of 5 mu M modified biotin (Apt-biotin), and reacting at 37 ℃ for 1.5 hours, so that the error in the operation process is reduced, and the operation accuracy is guaranteed.
Preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the apparatus comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultrapure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical stationOther materials include aptamers to Tau protein, Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP); activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished; stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7. mu.L of 1. mu.M SA-ALP was added dropwise and reacted at 37 ℃ for 1 hour to obtain ALP/Apt/Tau381a/Ab/modified gold electrode; DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM MFCM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms, and a pulse period of 0.2 s, and Tau was measured according to the variation of the intensity of the electrical signal measured381Carrying out quantitative analysis on the protein concentration; and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and then measuring the triplicate with the biosensorTau in artificial cerebrospinal fluid samples381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
Example five:
in the fourth example, the following steps were added:
in step 4, Tau381The presence of the compound can form an ALP/Apt/Tau/Ab/sandwich type compound on the surface of an electrode, further generate electrochemical-chemical redox cycle amplification reaction guided by enzymatic reaction, and finally generate a remarkably enhanced FcM oxidation electric signal at 1.0 × 10-3nM ~ 1.0×102Enhancement of DPV Strength with Tau in nM range381The logarithm of the concentration has a good linear relation, and the detection accuracy is guaranteed.
Preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP); activation of gold electrode: firstly, sequentially using A with the grain sizes of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrodel2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode is scanned in 0.5M sulfuric acid solution by using continuous cyclic voltammetry until a stable and repeatable cyclic voltammetry peak is obtained, which indicates that the activation process of the gold electrode is finished; stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7. mu.L of 1. mu.M SA-ALP was added dropwise and reacted at 37 ℃ for 1 hour to obtain ALP/Apt/Tau381a/Ab/modified gold electrode; DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM MFCM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms, and a pulse period of 0.2 s, and Tau was measured according to the variation of the intensity of the electrical signal measured381Carrying out quantitative analysis on the protein concentration; and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A chemical biosensor for the detection of Tau protein of alzheimer's disease, characterized by: the method comprises the following steps:
step 1: preparation of reagents and instruments: the reagent comprises 3-mercaptopropionic acid (MPA) (C)3H6O2S), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (C)8H17N3HCl), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (C)9H15O6P. HCl), N-hydroxysuccinimide (NHS) (C)4H5NO3) 2- (N-morpholino) ethanesulfonic acid Monohydrate (MES) (C)6H13NO4S), ferrocene methanol (FcM) (C)11H12FeO), L-ascorbic acid 2-phosphoric acid magnesium salt hemihydrate hydrate (AAP) (C)6H6Mg1.5O9P. xH 2O), potassium ferrocyanide (K)4Fe(CN)6) Potassium ferricyanide (K)3Fe(CN)6) Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl)2) Ethanol (C)2H6O), the instrument comprises a gold electrode, an electronic balance, a liquid-moving gun, an ultra-pure water system, a vortex mixer, a super constant-temperature mixer and an electrochemical station, and other materials comprise an aptamer of Tau protein and Tau381Protein and its antibody (Ab), Bovine Serum Albumin (BSA), and alkaline phosphatase-labeled streptavidin (SA-ALP);
step 2: activation of gold electrode: firstly, using Al with the grain diameters of 1.0, 0.3 and 0.05 mu m on polishing cloth for a gold electrode2O3Polishing and grinding the powder; then ultrasonic cleaning is carried out for 2 min by using ethanol and ultrapure water respectively; finally, the gold electrode was scanned in a 0.5M sulfuric acid solution using continuous cyclic voltammetry until stable and reproducible cyclic voltammetry peaks were obtained, indicating goldCompleting the electrode activation process;
and step 3: stepwise modification of gold electrodes: firstly, immediately soaking a gold electrode subjected to electrochemical cleaning in a 10 mM MPA ethanol solution to carry out light-shielding reaction overnight, and modifying MPA on the surface of the gold electrode through the Au-S bond self-assembly effect; then activating carboxyl on the surface of the gold electrode by using 0.2M EDC and 0.1M NHS mixed solution (20 mM MES and pH =6.5 configuration), dripping 7 mu L of Ab on the gold electrode of the modified MPA after the steps, reacting for 12 hours at 4 ℃ to obtain an Ab modified gold electrode, and then blocking redundant active sites on the gold electrode by using 10 mu M BSA solution; followed by dropwise addition of 7. mu.L of Tau at different concentrations381After the protein solution reacts for 30 minutes at room temperature, 10 mu L of 5 mu M aptamer (Apt-biotin) for modifying biotin is dripped; finally, 7 mu L of 1 mu MSA-ALP is dripped to react for 1 hour at 37 ℃ to obtain ALP/Apt/Tau381a/Ab/modified gold electrode;
and 4, step 4: DPV measurement: DPV measurements were performed in a mixture containing 20 mM Tris-HCl (pH =8.0), 2 mM FcM, 5mM TCEP, scanned by differential pulse voltammetry at a potential ranging from-0.1 to 0.6V, with a pulse amplitude of 50 mV, a pulse width of 50 ms and a pulse period of 0.2 s, and Tau was measured for changes in the intensity of the electrical signal381Carrying out quantitative analysis on the protein concentration;
and 5: and (3) actual sample detection: to verify that the biosensor can be used for detection in complex matrices, we applied the biosensor to detect Tau in artificial cerebrospinal fluid381Protein content, artificial cerebrospinal fluid (aCSF) was prepared from 150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl 2.2H2O, 0.8 mM Mg Cl 2.6H2O and 1mM phosphate, and three different concentrations of Tau were added to the aCSF381Protein standard solution, and measuring Tau in the three artificial cerebrospinal fluid samples by the biosensor381The contents of the proteins are respectively 0.092 nM, 1.04 nM and 52.0 nM, and the difference between the results obtained by electrochemical detection and the reference value is not great.
2. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: said at the step1, the water to be used was ultrapure water (Milli-Q, Millipore, 18.2 M.OMEGA.cm)-1) All reagents were purchased for use directly without further purification.
3. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 1, Tau is381Proteins and antibodies thereto were treated with 1 × PBS (137 mM NaCl; 2.7 mM KCl; 8.1mM Na)2HPO4; 1.8 mM KH2PO4) Diluted to the desired concentration, Apt and SA-ALP were diluted with 1 × B2 (10 mM Tris-HCl;50 mM NaCl; 10 mM KCl; 10 mM MgCl)2) Dilute to the desired experimental concentration.
4. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 2, Cyclic Voltammetry (CV) was performed on CHI660A, and scanning was performed at a sweep rate of 0.1V/S in a potential range of-0.2 to 1.6V by continuous cyclic voltammetry.
5. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 3, the resulting gold electrode was finally soaked in a solution containing 20 mM Tris-HCl (pH =8.0), 5mM AAP, 10 mM MgCl2The mixed solution of (3) was reacted at room temperature for 0.5 hour.
6. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in the step 3, the principle is as follows: firstly, modifying sulfydryl in MPA on a gold electrode through Au-S bond, then reacting amino in an antibody with carboxyl on the gold electrode modified with MPA to obtain the gold electrode modified by the antibody, and adding target protein Tau381Apt/Tau formation following aptamer binding381the/Ab sandwich structure is characterized in that streptavidin is modified in SA-ALP and can interact with biotin on an aptamer to finally obtain a gold electrode modified with alkaline phosphataseSoaking the electrode in a solution of the substrate AAP for alkaline phosphatase will catalytically hydrolyze AAP to L-Ascorbic Acid (AA), i.e. an enzymatic reaction proceeds, however, the insulating self-assembled monolayer of linear thiol molecules (SAMs) may result in a slow electron transfer between the electrode and the enzyme or enzyme product, i.e. in an enzyme amplification system the enzyme and enzyme product itself will not normally exchange electrons directly with the electrode with the SAMs covering, thus an "outer sphere to inner sphere" ECC redox cycle triggered by the enzyme product is used to amplify the electrochemical signal, FcM, AAP and TCEP are used as redox mediators, enzyme substrates and reductants, respectively, the ECC redox cycle process being: (1) FcM is stable in air and can be reduced and regenerated by ALP enzyme product AA after electrooxidation; (2) TCEP can reduce AA oxidation product dehydroascorbic acid (DAA) and regenerate AA rapidly, and TCEP is not in contact with FcM+(FcM oxidized form) and thus in the presence of the reducing agent TCEP, an electrochemical-chemical redox cycle is formed, amplifying the electrical oxidation signal of FcM, it is noted that neither AA nor TCEP in the system is oxidized over the potential sweep, and AAP is chosen as an enzyme substrate for ALP due to its low cost, water solubility and high potential.
7. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 3, the optimal Ab concentration is 0.05 mg.mL-190 min was chosen as the optimal reaction time for Apt.
8. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 3, 10. mu.L of 5. mu.M aptamer (Apt-biotin) modified biotin was added dropwise and the mixture was reacted at 37 ℃ for 1.5 hours.
9. The chemical biosensor for detecting Tau protein of alzheimer's disease according to claim 1, wherein: in step 4, Tau is381Will exist in the electrode tableForming ALP/Apt/Tau/Ab/sandwich type complex, generating electrochemical-chemical redox cycle amplification reaction guided by enzymatic reaction, and generating significantly enhanced FcM oxidation electric signal at 1.0 × 10-3nM ~ 1.0×102Enhancement of DPV Strength with Tau in nM range381The logarithm of the concentration has a good linear relationship.
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