CN116818735A - Method for detecting alkaline phosphatase based on click chemistry and graphene oxide - Google Patents
Method for detecting alkaline phosphatase based on click chemistry and graphene oxide Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application relates to a method for detecting alkaline phosphatase based on click chemistry and graphene oxide, belonging to the technical field of biological analysis and detection. The method utilizes phosphorylated ascorbic acid to be hydrolyzed into ascorbic acid reduced copper ions by alkaline phosphatase, and catalyzes the generation of click chemical reaction so as to connect DNA short segments, and the fluorescence intensity of the DNA short segments is obviously changed under the action of graphene oxide before and after connection, so that a novel fluorescence method for detecting alkaline phosphatase is constructed. The method has good linear response within the range of 0-1U/mL, and the detection limit is 0.0036U/mL. The application has the characteristics of high specificity, good stability, low cost, simple operation and the like.
Description
Technical Field
The application belongs to the technical field of biological analysis and detection, and particularly relates to a method for detecting alkaline phosphatase based on click chemistry and graphene oxide.
Background
Alkaline phosphatase (Alkaline Phosphatase, ALP) is a metalloenzyme localized to multiple mammalian cells and organs and has a metal-containing active site, each monomer containing two Zn 2+ And one Mg 2+ . ALP can catalyze the hydrolysis of various phosphate monoesters and participate in various biological processes such as signal transduction, molecular transport, substance metabolism, and the like. ALP normal value in serum of adult is 40-190U/L, ALP activity level abnormality is closely related to cardiovascular disease, diabetes, cancer, alzheimer's disease, hypophosphoric acid deficiency, hemolysis, periodontitis, bone injury, liver dysfunction, etc., ALP is widely used as a biomarker in clinical diagnosis and treatment research of related diseases, so the detection of ALP has important significance to clinical diagnosis and biomedical research.
There is an urgent need in biomedical research to detect ALP in extracellular or intracellular and tissues and body fluids by a rapid and accurate method, and thus various bioanalytical methods have been used to detect ALP, such as an electroanalytical method, surface-enhanced Raman scattering, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, quartz crystal microbalance, field Effect Transistor (FET), colorimetry, magnetic resonance imaging, and fluorescence method. However, most of the above biological analysis methods have the defects of low sensitivity, poor anti-interference capability, complicated steps, large-scale instrument requirement, high cost and the like. In recent years, with the rapid development of fluorescent probe technology, the advantages of convenience, rapidness, high sensitivity and the like make the detection of ALP by a fluorescence method a research hotspot.
Click chemistry (Click chemistry) is a synthetic concept introduced by Nobel chemical prize acquirer K.B.Sharpless in 2001, the reaction can rapidly and reliably complete a chemical synthesis process through small unit splicing, and the method has the advantages of high yield, harmless byproducts, readily available raw material reagents, rapid synthesis reaction and the like, and is widely applied to various fields of chemical synthesis, drug development, biomedical materials and the like. The most classical representative reaction of click chemistry is Copper-Catalyzed azido cycloaddition (CuAAC) which has the characteristics of high efficiency, mild condition, high product yield, simple post-treatment and the like.
Graphene is regarded as the thinnest and most ideal novel two-dimensional nanomaterial in the world at present, and graphene and derivatives thereof are widely applied to fluorescence analysis methods at present, for example, graphene Quantum Dots (GQDs) have excellent fluorescence and can be used as fluorescent probes; graphene Oxide (GO) has been widely used as a fluorescence quencher in the construction of analytical methods because it has abundant oxygen functional groups (such as carboxyl and epoxy) and exhibits great dispersibility and solubility in aqueous solutions and other solvents, and can quench fluorescent groups effectively. Graphene oxide has good adsorption capacity and quenching effect, so that the reagent has a huge application prospect in fluorescence sensing, and particularly, graphene oxide has strong adsorption capacity to single-stranded DNA and weak double-stranded adsorption capacity.
In the application, the applicant provides a simple and convenient method for detecting ALP based on the characteristic of different adsorption capacities of click chemistry reaction connection DNA and graphene oxide on single-strand and double-strand DNA, and the method has the characteristics of mild reaction condition, strong anti-interference capacity, good stability, low cost and the like.
Disclosure of Invention
The application provides a method for detecting alkaline phosphatase based on click chemistry and graphene oxide, which aims to solve the problems of poor anti-interference capability, large instrument requirement, low sensitivity, poor stability, high cost and the like in the method for detecting alkaline phosphatase based on fluorescence in the prior art.
In order to solve the problems, the application provides the following technical scheme:
a method for detecting alkaline phosphatase based on click chemistry and graphene oxide, comprising the steps of:
step one: mixing the test samples with 2-phosphoric acid-Ascorbic Acid (AAP) respectively, and placing the mixture in a water bath for full reaction;
step two: respectively dissolving short single-stranded nucleic acid modified with azide groups, short single-stranded nucleic acid modified with alkynyl groups and nucleic acid serving as a connecting template and modified with fluorescent groups in pure water to prepare a nucleating acid solution;
step three: mixing the three nucleic acid solutions prepared in the second step to prepare a nucleic acid mixed solution, and then adding the reaction solution of the test sample reacted in the first step, AAP, copper ions and Tris-HCl buffer solution into the nucleic acid mixed solution to form a reaction system;
step four: adding pure water, graphene oxide and Tris-HCl buffer solution into the reaction system in the third step, and detecting the fluorescence intensity of the detection system after heating;
step five: detecting a sample to be detected of unknown alkaline phosphatase concentration by using a standard curve established by a sample with known alkaline phosphatase concentration and a corresponding fluorescence intensity value, obtaining fluorescence intensity by the sample to be detected according to the operation of the steps one to four, and calculating to obtain the concentration of the alkaline phosphatase contained in the sample to be detected.
The application has the working principle and beneficial effects that:
firstly, carrying out water bath on a test sample and phosphorylated ascorbic acid for a certain time at a proper reaction temperature of enzyme; simultaneously, respectively dissolving the short single-stranded nucleic acid modified with the azide group, the short single-stranded nucleic acid modified with the alkynyl group and the nucleic acid serving as a connecting template and modified with the fluorescent group in pure water to prepare a nucleating acid solution; then mixing the nucleic acid solution with a long-chain nucleic acid solution serving as a connecting template and modified with a fluorescent group to prepare a nucleic acid mixed solution, and adding a test sample, a phosphorylated ascorbic acid reaction solution, copper ions and a Tris (Tris-HCl) buffer solution into the nucleic acid mixed solution to form a reaction system; after reacting for a period of time at a proper temperature, mixing a reaction system, water, graphene oxide and Tris-HCl buffer solution to form a detection system, raising the temperature, and carrying out fluorescence detection on the intensity of the detection system; and drawing a standard curve by taking the known alkaline phosphatase concentration as an abscissa and the relative fluorescence intensity value as an ordinate to obtain a linear relation equation between the alkaline phosphatase concentration and the fluorescence intensity, and then calculating the concentration of the alkaline phosphatase in the corresponding test sample according to the fluorescence intensity of the test sample.
In the scheme, 2-phosphate-Ascorbic Acid (AAP) is phosphorylated ascorbic acid, in the presence of alkaline phosphatase (ALP), the AAP can be hydrolyzed into Ascorbic Acid (AA) by the ALP, and the AA has reducibility, so that cupric ions can be reduced into monovalent copper, thereby catalyzing the generation of a 'click chemistry' azidoalkynyl cycloaddition reaction (CuAAC), namely catalyzing the generation of cycloaddition reaction between adjacent azido and alkynyl, connecting gaps formed by two short single strands of DNA respectively marked with azido and alkynyl through chemical bonds, and then forming stable DNA double chains with long single strands of DNA marked with fluorescent groups, wherein the melting temperature of the stable DNA double chains is far higher than that between the two short single strands and the long single strand serving as a template; without ALP, AAP cannot hydrolyze to AA reduced copper ions, so that the CuAAC reaction will not occur; after the system is heated to a proper temperature, the DNA double chain with the notch is changed into three single chains due to the fact that CuAAC reaction does not occur, the double chain structure of the DNA double chain is stable due to the fact that the melting temperature is higher after the CuAAC reaction occurs, at the moment, the fluorescence quenching degree is different due to the fact that the adsorption capacity of graphene oxide to single-double chain DNA is different, the fluorescence intensity is weak due to the fact that the CuAAC reaction does not occur, the fluorescence intensity is positive and positive correlation exists between the fluorescence intensity and the ALP concentration in the system, and therefore the ALP concentration can be detected through the fluorescence intensity.
Compared with the prior art, the method utilizes click chemistry to connect the reaction of nucleic acid and the quenching effect of graphene oxide, and has the advantages of mild reaction conditions, strong anti-interference capability, good stability and no need of large-scale instruments; the material reagent used in the application is commercially available, has low cost and stable property, does not need complex and complicated preparation process and pretreatment process, only needs simple solution mixing and solution incubation process, has simple operation, adopts long single chain DNA marked with widely applied fluorescent groups, has strong biocompatibility, basically no toxicity and simple detection process; meanwhile, the fluorescence enhancement mode is adopted, so that the possibility of false positive signals can be greatly reduced; the detection method has the characteristics of high specificity, good stability, low cost, simple operation and the like, has good linear response in the range of 0-1U/mL, and has the detection limit as low as 0.0036U/mL.
Preferably, the concentration of AAP in the first step is 0.5 mM-10 mM, the water bath reaction temperature is 20-50 ℃, and the reaction time is 10-50 min.
Preferably, the nucleic acid as the ligation template in the second step includes one long single strand having a Tm value of 40℃and two short single strands having a Tm value of 20 ℃.
Preferably, the concentration ratio between the short single-stranded nucleic acid modified with an azide group and the short single-stranded nucleic acid modified with an alkyne group and the nucleic acid as a ligation template in the step three is (1 to 6): (1-6): 1.
preferably, in the reaction system of the third step, the final concentration of the short single-stranded nucleic acid modified with the azide group is the same as the final concentration of the short single-stranded nucleic acid modified with the alkynyl group, and the final concentration is 10 nM-80 nM; the final concentration of nucleic acid as the ligation template and modified with a fluorescent group is 5nM to 30nM.
Preferably, in the reaction system of the third step, the concentration of copper ions is 5 mu M-20 mu M, the salt concentration of the Tris-HCl buffer solution is 10 mM-60 mM, and the pH is 6.6-8.2; the reaction time for forming the reaction system is 20 min-5 h, and the reaction temperature is 10-40 ℃.
Preferably, the graphene oxide in the third step is a fluorescence quencher, and the dosage of the graphene oxide is 2-20 mu L of graphene oxide with the concentration of 0.5 mg/mL.
Preferably, in the detection system of the step four, the salt concentration of the Tris-HCl buffer solution is 100 mM-500 mM, the pH value is 6.6-8.2, the fluorescence intensity detection time of the step four is 10 min-50 min, and the detection temperature is 20-50 ℃.
Preferably, the short single stranded nucleic acid modified with an azide group is a nucleotide sequence shown as 5'-GAT CTA AAT TCC AA-azide-3', 5 '-azide-TGG CAA CAG C-3', 5'-GAC GGG AAC T-azide-3', 5 '-azide-T ACA AGA CAC GG-3';
the short single-stranded nucleic acid modified with an alkynyl group is preferably a nucleotide sequence shown as 5 '-alkynyl-CAA ACT GAT AG-3', 5'-GGG AGC TAG AG-alkynyl-3', 5 '-alkynyl-ACA AGA CAC G-3', 5'-CTG ACG GGA AG-alkynyl-3'.
Preferably, the nucleic acid as a ligation template is specifically one or more of the following nucleotide sequence structures:
5’-FAM-ATT CTA TCA GTT TCT TGG AAT TTA GCG A-3’、5’-CATATA TGT TGC CAC TCT AGC GGC CGT GG-TAMRA-3’、5’-FAM-CCG TGC CGT GTC TTG TAG TTC CCG TCG AAT CG-3’、5’-TAMRA-CCT CTA CGT GTC TTG TAC TTC GGA TCA GAG AGG-3’。
drawings
FIG. 1 is a graph showing fluorescence spectrum of a detection system in the case of adding 1U/mL ALP and not adding ALP to the reaction system in example 1 of the present application;
FIG. 2 is a response curve of the application for example 2 with different concentrations of ALP;
FIG. 3 is a graph showing the specificity of ALP detection in example 3 of the present application.
Detailed Description
The following is a further detailed description of the embodiments:
example 1: a method for detecting alkaline phosphatase based on click chemistry and graphene oxide, comprising the steps of:
step one: 1U/mL alkaline phosphatase (ALP) and 4mM AAP were reacted in a 37℃water bath for 30min;
step two: the short single-stranded nucleic acid modified with azide group as shown in SEQ ID No. 1 (5 '-CTA AAT TCC AA-azide-3'), the short single-stranded nucleic acid modified with alkyne group as shown in SEQ ID No. 5 (5 '-alkyne-GAA ACT GAT AG-3'), and the nucleic acid as a ligation template as shown in SEQ ID No. 9 (5 '-FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-3'), were dissolved in pure water, respectively, and prepared into nucleic acid solutions with a final concentration of 200 nM;
step three: according to the following steps: 2:1, mixing a short single-stranded nucleic acid solution modified with an azide group, a short single-stranded nucleic acid solution modified with an alkyne group and a nucleic acid solution of a long-chain nucleic acid modified with a fluorescent group as a ligation template to prepare a mixed solution, then adding the reaction solution of step one, 5. Mu.M copper ions and Tris-HCl buffer solution (40 mM Tris-HCl (30 mM NaCl, pH 7.4)) to the mixed solution to form a reaction system, wherein the final concentration of the short single-stranded nucleic acid solution modified with an azide group and the short single-stranded nucleic acid solution modified with an alkyne group is 20nM, the final concentration of the nucleic acid solution as a ligation template is 10nM, and placing the reaction system in a water bath at 20 ℃ to react for 3 hours, respectively.
Step four: to the reaction system, 3. Mu.L of 0.5mg/mL graphene oxide, pure water, tris-HCl buffer solution (20 mM Tris-HCl (400mM NaCl,pH 7.4)) was added, and after heating to 35℃for 10 minutes, the fluorescence intensity of the detection system under the heating condition was detected by a fluorescence spectrometer.
FIG. 1 is a graph showing fluorescence spectra of a detection system in example 1 of the present application, wherein 1U/mL of ALP is added to the reaction system and no ALP is added, FIG. 1 is a graph showing that the fluorescence intensity does not change significantly when no ALP is added, FIG. 1 is a graph showing that the fluorescence intensity is enhanced after 1U/mL of ALP is added to the reaction system, and FIG. 1 is a graph showing that curves 2 and 3 can well illustrate that the detection method of the present application can be used to detect ALP by monitoring the reaction of nucleic acid ligation by reducing copper ions by hydrolyzing phosphate groups by ALP.
Example 2A method for detecting alkaline phosphatase based on click chemistry and graphene oxide, comprising the steps of:
step one: ALP (0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4, 5U/mL) at different concentrations and 4mM AAP were reacted in a water bath at 37℃for 30min;
step two: dissolving short single-stranded nucleic acid modified with azide group as shown in SEQ ID No. 1 (5 '-CTA AAT TCC AA-azide-3'), short single-stranded nucleic acid modified with alkynyl group as shown in SEQ ID No. 5 (5 '-alkynyl-GAA ACT GAT AG-3') and long-stranded nucleic acid modified with fluorophore as a connecting template as shown in SEQ ID No. 9 (5 '-FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-Dabcyl-3'), respectively in pure water to prepare nucleic acid solutions with concentrations of 200 nM;
step three: according to the following steps: 2:1, mixing the short single-stranded nucleic acid solution modified with an azide group, the short single-stranded nucleic acid solution modified with an alkyne group and the long-chain nucleic acid solution modified with a fluorescent group as a ligation template to prepare a mixed solution, and then adding the reaction mixed solution of step one, 5. Mu.M copper ions and Tris-HCl buffer solution (40 mM Tris-HCl (30 mM NaCl, pH 7.4)) buffer solution to the mixed solution to form a reaction system, wherein the final concentration of the short single-stranded nucleic acid solution modified with an azide group and the short single-stranded nucleic acid solution modified with an alkyne group is 20nM, and the final concentration of the long-chain nucleic acid solution modified with a fluorescent group as a ligation template is 10nM, and incubating at 20℃for 3h.
Step four: to the reaction system, 3. Mu.L of 0.5mg/mL graphene oxide, water and Tris-HCl buffer solution (20 mM Tris-HCl (400mM NaCl,pH 7.4)) were added, and after heating to 35℃for 10 minutes, the fluorescence intensity of the detection system under the heating condition was detected by a fluorescence spectrometer.
Step five: drawing a standard curve by taking the known ALP concentration as an abscissa and the corresponding fluorescence intensity value as an ordinate to obtain a curve relationship diagram between the copper ion concentration and the fluorescence intensity, wherein in the interval of 0-1U/mL of the ALP concentration, the concentration of alkaline phosphatase and the fluorescence intensity show good linear relationship, and the linear equation y=570359.59181x+39975.20021 is met (y represents the fluorescence intensity of a detection system, x represents the ALP concentration in the detection system in U/mL), R is met 2 =0.997, the limit of detection of copper ions in water by the present method was calculated to be about 0.0036U/mL by (3 δ/S).
FIG. 2 is a response curve of the application for example 2 with different concentrations of ALP. FIG. 2 illustrates that from 0 to 1U/mL, the fluorescence intensity of the detection system increases with increasing ALP concentration, and the fluorescence intensity of the system gradually reaches the plateau when the ALP concentration reaches 1U/mL, illustrating that the efficiency/rate of the ligation reaction increases with increasing ALP; in addition, the detection system has good linear relation with fluorescence intensity and smaller data deviation in the range of 0-1U/mL, which shows that the method has good reproducibility in the aspect of detecting ALP.
Example 3: a method for detecting alkaline phosphatase based on click chemistry and graphene oxide comprises the following steps,
step one: reacting different kinds of proteins (ALP, bovine serum albumin BSA, thrombin, trypsin, alpha-glucosidase, papain, wherein the concentration of ALP is 1U/mL, the concentration of other proteins is 10U/mL, BSA is 0.1 g/mL), 4mM AAP in water bath at 37deg.C for 30min;
step two: a short single-stranded nucleic acid modified with an azide group as shown in SEQ ID No. 1 (5 '-CTA AAT TCC AA-azide-3'), a short single-stranded nucleic acid modified with an alkyne group as shown in SEQ ID No. 5 (5 '-alkyne-GAA ACT GAT AG-3'), and a long-stranded nucleic acid modified with a fluorophore as a ligation template as shown in SEQ ID No. 9 (5 '-FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-3'), were dissolved in pure water to prepare nucleic acid solutions each having a concentration of 200 nM.
Step three: according to the following steps: 2:1, mixing a short single-stranded nucleic acid solution modified with an azide group, a short single-stranded nucleic acid solution modified with an alkyne group and a long-chain nucleic acid solution modified with a fluorescent group as a ligation template, to form a nucleic acid mixture solution, then adding the reaction solution of step one, copper ions, water and Tris-HCl buffer solution (40 mM Tris-HCl (30 mM NaCl, pH 7.4)) to the nucleic acid mixture solution, to form a reaction system in which the final concentration of the short single-stranded nucleic acid solution modified with an azide group and the short single-stranded nucleic acid solution modified with an alkyne group is 20nM, the final concentration of the long-chain nucleic acid solution modified with a fluorescent group as a ligation template is 10nM, and incubating the reaction system at 20℃for 3h.
Step four: to the reaction system, 3. Mu.L of 0.5mg/mL graphene oxide, water and Tris-HCl buffer solution (20 mM Tris-HCl (400mM NaCl,pH 7.8)) were added, and after heating to 35℃for 10 minutes, the fluorescence intensity of the detection system under the heating condition was detected by a fluorescence spectrometer.
FIG. 3 is a graph showing the specificity of ALP detection in example 3 of the present application. Wherein I is the fluorescence intensity of the detection system after incubation, I 0 Is the fluorescence intensity before incubation, (I-I) 0 )/I 0 Is a multiple representing the increase in fluorescence intensity after incubation of the detection system; FIG. 3 shows that after incubation, the detection system containing 1U/mL ALP has increased fluorescence by about 17-fold, while each contains 10-fold of the interference of ALP (10U/mL)The fluorescence intensity of the protein detection system is not obviously changed, which indicates that the presence of the interfering protein in the detection system does not influence the detection of ALP.
Example 4: a method for detecting alkaline phosphatase based on click chemistry and graphene oxide comprises the following steps,
step one: the actual sample is serum, centrifugal machine 10000rpm is used for centrifugation for 10min, and supernatant fluid is taken and stored at low temperature for standby. The diluted serum is added with three different concentrations of ALP, namely low (200 mU/mL), medium (400 mU/mL) and high (800 mU/mL), and 4mM phosphorylated ascorbic acid is reacted for 30min in a water bath at 37 ℃;
step two: preparing a nucleic acid solution with a concentration of 200nM by dissolving a short single-stranded nucleic acid with an azide group modified as shown in SEQ ID No. 1 (5 '-CTA AAT TCC AA-azide-3'), a short single-stranded nucleic acid with an alkyne group modified as shown in SEQ ID No. 5 (5 '-alkynyl-GAA ACT GAT AG-3'), and a long-stranded nucleic acid with a fluorophore group modified as a ligation template as shown in SEQ ID No. 9 (5 '-FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-3'), respectively, and dissolving in pure water;
step three: according to the following steps: 2:1, mixing a short single-stranded nucleic acid solution modified with an azide group, a short single-stranded nucleic acid solution modified with an alkyne group and a long-chain nucleic acid solution modified with a fluorescent group as a ligation template, to form a nucleic acid mixture solution, then adding the reaction solution of step one, 5. Mu.M copper ions and Tris-HCl buffer solution (40 mM Tris-HCl (30 mM NaCl, pH 7.4)) to the nucleic acid mixture solution, to form a reaction system in which the final concentration of the short single-stranded nucleic acid solution modified with an azide group and the short single-stranded nucleic acid solution modified with an alkyne group is 20nM, the final concentration of the nucleic acid solution as a ligation template for a long-chain nucleic acid ligation template modified with a fluorescent group is 10nM, and incubating the reaction system at 20℃for 3 hours.
Step four: to the reaction system, 3. Mu.L of 0.5mg/mL graphene oxide, water and Tris-HCl buffer solution (40 mM Tris-HCl (400mM NaCl,pH 7.8)) were added, and after heating to 35℃for 10 minutes, the fluorescence intensity of the detection system under the heating condition was detected by a fluorescence spectrometer.
Step five: fluorescence intensity results were recorded and recovery and Relative Standard Deviation (RSD) were calculated.
Experimental results show that all recovery rates are in the range of 99% -103%. The method has proved potential for application in complex sample detection.
Example 5: a method for detecting alkaline phosphatase based on click chemistry and graphene oxide comprises the following steps,
step one: the actual sample is saliva, the saliva is centrifuged for 10min at 10000rpm by a centrifuge, and the supernatant is taken and stored at low temperature for standby. Adding diluted saliva into ALP with three different concentrations of low (200 mU/mL), medium (400 mU/mL) and high (800 mU/mL), and reacting 4mM phosphorylated ascorbic acid in water bath at 37 ℃ for 30min;
step two: preparing a nucleic acid solution with a concentration of 200nM by dissolving a short single-stranded nucleic acid with an azide group modified as shown in SEQ ID No. 1 (5 '-CTA AAT TCC AA-azide-3'), a short single-stranded nucleic acid with an alkyne group modified as shown in SEQ ID No. 5 (5 '-alkynyl-GAA ACT GAT AG-3'), and a long-stranded nucleic acid with a fluorophore group modified as a ligation template as shown in SEQ ID No. 9 (5 '-FAM-TCG CTA TCA GTT TCT TGG AAT TTA GCG A-3'), respectively, and dissolving in pure water;
step three: according to the following steps: 2:1, mixing a short single-stranded nucleic acid solution modified with an azide group, a short single-stranded nucleic acid solution modified with an alkyne group and a long-chain nucleic acid solution modified with a fluorescent group as a ligation template, to form a nucleic acid mixture solution, then adding the reaction solution of step one, 5. Mu.M copper ions and Tris-HCl buffer solution (40 mM Tris-HCl (30 mM NaCl, pH 7.4)) to the nucleic acid mixture solution, to form a reaction system in which the final concentration of the short single-stranded nucleic acid solution modified with an azide group and the short single-stranded nucleic acid solution modified with an alkyne group is 20nM, the final concentration of the nucleic acid solution as a ligation template for a long-chain nucleic acid ligation template modified with a fluorescent group is 10nM, and incubating the reaction system at 20℃for 3 hours.
Step four: to the reaction system, 3. Mu.L of 0.5mg/mL graphene oxide, water and Tris-HCl buffer solution (20 mM Tris-HCl (400mM NaCl,pH 7.8)) were added, and after heating to 35℃for 10 minutes, the fluorescence intensity of the detection system under the heating condition was detected by a fluorescence spectrometer.
Step five: fluorescence intensity results were recorded and recovery and Relative Standard Deviation (RSD) were calculated.
Experimental results show that all recovery rates are in the range of 98% -103%. The method has proved potential for application in complex sample detection.
The method utilizes click chemistry to connect the reaction of nucleic acid and the quenching effect of graphene oxide, and has the advantages of mild reaction conditions, strong anti-interference capability, good stability and high sensitivity; the material reagent used in the application is commercially available, has low cost and stable property, does not need complex and complicated preparation process and pretreatment process, only needs simple solution mixing and solution incubation process, has simple operation, adopts long single chain DNA marked with widely applied fluorescent groups, has strong biocompatibility, basically no toxicity and simple detection process; meanwhile, the fluorescence enhancement mode is adopted, so that the possibility of false positive signals can be greatly reduced; the detection method has good stability and high specificity, has good linear response within the range of 0-1U/mL, and has a linear equation of y=570359.59181x+39975.20021 and a detection limit as low as 0.0036U/mL.
It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the technical spirit of the application, and these should also be considered as the scope of the application, which does not affect the effect of the application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. A method for detecting alkaline phosphatase based on click chemistry and graphene oxide, comprising the steps of:
step one: mixing the test samples with 2-phosphoric acid-Ascorbic Acid (AAP) respectively, and placing the mixture in a water bath for full reaction;
step two: respectively dissolving short single-stranded nucleic acid modified with azide groups, short single-stranded nucleic acid modified with alkynyl groups and nucleic acid serving as a connecting template and modified with fluorescent groups in pure water to prepare a nucleating acid solution;
step three: mixing the three nucleic acid solutions prepared in the second step to prepare a nucleic acid mixed solution, and then adding the reaction solution of the test sample reacted in the first step, AAP, copper ions and Tris-HCl buffer solution into the nucleic acid mixed solution to form a reaction system;
step four: adding pure water, graphene oxide and Tris-HCl buffer solution into the reaction system in the third step, and detecting the fluorescence intensity of the detection system after heating;
step five: detecting a sample to be detected of unknown alkaline phosphatase concentration by using a standard curve established by a sample with known alkaline phosphatase concentration and a corresponding fluorescence intensity value, obtaining fluorescence intensity by the sample to be detected according to the operation of the steps one to four, and calculating to obtain the concentration of the alkaline phosphatase contained in the sample to be detected.
2. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 1, wherein the concentration of AAP in the first step is 0.5mM to 10mM, the water bath reaction temperature is 20 ℃ to 50 ℃, and the reaction time is 10min to 50min.
3. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 2, wherein the nucleic acid as the ligation template in the second step includes one long single strand having a Tm value of 30 to 50 ℃ and two short single strands having a Tm value of 15 to 25 ℃.
4. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 3, wherein the concentration ratio between the short single-stranded nucleic acid modified with an azide group, the short single-stranded nucleic acid modified with an alkyne group, and the nucleic acid as a ligation template in the step three is (1 to 6): (1-6): 1.
5. the method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 4, wherein in the third reaction system, the final concentration of the short single-stranded nucleic acid modified with an azide group is the same as the final concentration of the short single-stranded nucleic acid modified with an alkyne group, and the final concentration is 10nM to 80nM; the final concentration of nucleic acid as the ligation template and modified with a fluorescent group is 5nM to 30nM.
6. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 5, wherein in the reaction system of the third step, the copper ion concentration is 5 μΜ -20 μΜ, the salt concentration of Tris-HCl buffer solution is 10 mM-60 mM, and the ph is 6.6-8.2; the reaction time for forming the reaction system is 20 min-5 h, and the reaction temperature is 10-40 ℃.
7. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 6, wherein the graphene oxide in the third step is a fluorescence quencher, and the amount of the graphene oxide is 2 μl to 20 μl with a concentration of 0.5 mg/mL.
8. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 7, wherein in the detection system of the fourth step, the salt concentration of the Tris-HCl buffer solution is 100 mM-500 mM, the pH is 6.6-8.2, the detection time of the fluorescence intensity of the fourth step is 10 min-50 min, and the detection temperature is 20 ℃ to 50 ℃.
9. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to claim 8, wherein the short single-stranded nucleic acid modified with an azide group is a nucleotide sequence shown as 5'-GAT CTA AAT TCC AA-azide-3', 5 '-azide-TGG CAA CAG C-3', 5'-GAC GGG AAC T-azide-3', 5 '-azide-T ACA AGA CAC GG-3'; the short single-stranded nucleic acid modified with an alkynyl group is preferably a nucleotide sequence shown as 5 '-alkynyl-CAA ACT GAT AG-3', 5'-GGG AGC TAG AG-alkynyl-3', 5 '-alkynyl-ACA AGA CAC G-3', 5'-CTG ACG GGA AG-alkynyl-3'.
10. The method for detecting alkaline phosphatase based on click chemistry and graphene oxide according to any one of claims 1 to 9, wherein the nucleic acid as a ligation template is specifically one or more of the following nucleotide sequence structures: 5'-FAM-ATT CTA TCA GTT TCT TGG AAT TTA GCG A-3', 5'-CATATA TGT TGC CAC TCT AGC GGC CGT GG-TAMRA-3', 5'-FAM-CCG TGC CGT GTC TTG TAG TTC CCG TCG AAT CG-3', 5'-TAMRA-CCT CTA CGT GTC TTG TAC TTC GGA TCA GAG AGG-3'.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007092A (en) * | 2014-05-19 | 2014-08-27 | 中国科学院长春应用化学研究所 | Fluorescence detection method for copper ion based on click chemistry |
| CN104792753A (en) * | 2015-04-07 | 2015-07-22 | 上海大学 | Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction |
| CN109576342A (en) * | 2018-11-21 | 2019-04-05 | 暨南大学 | A kind of fluorescence chemical method for detection of alkaline phosphatase and application |
| CN110108685A (en) * | 2019-05-09 | 2019-08-09 | 南宁师范大学 | Using graphene quantum dot as the method for fluorescence probe detection of alkaline phosphatase concentration |
| CN115165833A (en) * | 2022-07-08 | 2022-10-11 | 遵义医科大学 | Method for detecting copper ions based on click chemistry and graphene oxide |
-
2023
- 2023-07-03 CN CN202310806883.4A patent/CN116818735A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104007092A (en) * | 2014-05-19 | 2014-08-27 | 中国科学院长春应用化学研究所 | Fluorescence detection method for copper ion based on click chemistry |
| CN104792753A (en) * | 2015-04-07 | 2015-07-22 | 上海大学 | Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction |
| CN109576342A (en) * | 2018-11-21 | 2019-04-05 | 暨南大学 | A kind of fluorescence chemical method for detection of alkaline phosphatase and application |
| CN110108685A (en) * | 2019-05-09 | 2019-08-09 | 南宁师范大学 | Using graphene quantum dot as the method for fluorescence probe detection of alkaline phosphatase concentration |
| CN115165833A (en) * | 2022-07-08 | 2022-10-11 | 遵义医科大学 | Method for detecting copper ions based on click chemistry and graphene oxide |
Non-Patent Citations (1)
| Title |
|---|
| MENGMENG ZHAO ET AL.: "A sensitive fluorescence biosensor for alkaline phosphatase activity based on the Cu(II)-dependent DNAzyme", ANALYTICA CHIMICA ACTA, vol. 948, 31 October 2016 (2016-10-31), pages 98 - 103 * |
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