CN103712983A - Magnetic graphene enzyme-mimicking property-based acetylcholine visual-detection method - Google Patents

Magnetic graphene enzyme-mimicking property-based acetylcholine visual-detection method Download PDF

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CN103712983A
CN103712983A CN201310718874.6A CN201310718874A CN103712983A CN 103712983 A CN103712983 A CN 103712983A CN 201310718874 A CN201310718874 A CN 201310718874A CN 103712983 A CN103712983 A CN 103712983A
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acetylcholine
rgo
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王坤
杨兴旺
钱静
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Jiangsu University
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Abstract

The invention relates to a magnetic graphene enzyme-mimicking property-based acetylcholine visual-detection method, belonging to the technical field of biosensing. A hydrothermal method is used for synthesizing Fe3O4/rGO by one step, H2O2 (hydrogen peroxide) is catalyzed to oxidize a substrate TMB (tetramethylabenzidine) to generate blue by utilizing the peroxidase-like property of the Fe3O4/rGO, and the characteristic absorption intensity of an oxidation product of the TMB at the wavelength of 652nm is detected to realize the detection of hydrogen peroxide; furthermore, the H2O2 can be catalyzed to oxidize and convert the colorless TMB into a corresponding blue product based on the Fe3O4/rGO, and the blue product is combined with acetyl choline to decompose H2O2 in the presence of both acetylcholinesterase and choline oxidase, and a novel acetylcholine visual-sensing method is established. The invention aims to provide an acetylcholine colorimetric detection method, and according to the method, the operation is convenient and flexible, the detection cost is low, the sensitivity is high, and instruments and equipment are simple.

Description

Acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene
Technical field
The present invention relates to a kind of structure to acetylcholine visible detection method, relate to technically materialogy, photoanalytical chemistry, the fields such as bio-sensing.
Background technology
Acetylcholine (Acetylcholine, Ach) is between a kind of neuron or the neurotransmitter of carrying out information transmission between neuron and effector, the peripheral nervous system that is mainly the present in biosome central nervous system of unifying.In people's brain tissue, have a large amount of acetylcholines, but declining can appear along with the increase at age in the content of acetylcholine.Normal old man declines 30% when young, and old dementia patients decline is even more serious, can reach 70%～80%.When acetyl choline content is lower than certain level in brain, will increase the probability of suffering from Alzheimer disease.Alzheimer disease is a kind of lethal nerve degenerative diseases of chronic development, clinical manifestation is cognitive and memory function is constantly degenerated, activity of daily living is faded, and is attended by various psychotic symptoms and behavior disorder, to patient, can cause great puzzlement.Equally, the generation of the disease such as parkinsonism and multiple sclerosis also has substantial connection with acetyl choline content.Research shows, the food that is rich in acetylcholine is highly beneficial with the patient of the unbalance diseases related of acetylcholine to suffering from Alzheimer disease etc.Therefore, acetylcholine, as the very important a kind of nutriment of human body, has been added in the middle of food to such as milk and health food etc. widely.Therefore realize significant to fields such as clinical analysis, food industry, feed addictives to the quantitative detection of acetylcholine.
At present, the method for quantitative measurement acetyl choline content is broadly divided into two large classes: bioassay method and physical chemistry determination method.Biologicall test ratio juris is based on some biological tissue, the sensitive reaction of acetylcholine concentration to be realized.As frog rectus aabdominis etc., under the effect of low concentration acetylcholine, can shrink, and the amplitude of tissue contracts within the specific limits with the proportional example of concentration of acetylcholine.Therefore can realize the mensuration to acetylcholine concentration according to the amplitude of contraction of muscle.But the sensitivity of bioassay method and specificity are poor, as the reaction that acetylcholine produces different specimens can be disturbed by relevant ester class.Thereby nearly decades, the development of the bioassay method of acetylcholine does not have made marked progress.Physical chemistry is measured ratio juris to be had a lot, and concrete assay method has multiple.Traditional chromatographic process detects and is widely studied for acetyl choline content, as gas chromatography and flame ionic detector coupling method (GC – FID) and Gao Xiao Ye Xiang – electrochemical detector coupling (HLPC – ED) method and the ionogenic mass spectrometer coupling of Ye Xiang Se Pu – charged spray method (LC/ESI – MS/MS) etc., wherein GC – FID is applied to the quantitative detection of acetylcholine the earliest.GC – FID method is determined the content of acetylcholine by detecting tertiary amine compounds content, detectability can reach 0.02 nM.Traditional chromatography reality with in all have that complicated operation, sample preparation are time-consuming, sensitivity and specificity not, the shortcoming such as apparatus expensive.In recent years, the method that some are lower to main equipment dependence, detects as fluorescence method and chemoluminescence method etc. is used to realize acetylcholine.Yet, the shortcoming such as sensors with auxiliary electrode were expense is high, the shelf-life is short and preparation process is tediously long, in view of the existing limitation of these detection methods, development is simple, quick, economical, effectively acetylcholine new detecting method has become extremely urgent important subject.Colorimetric detection is an importance in analysis science field, and its key is transformed into change color detection event exactly.Colorimetric detection is compared with traditional detection very large advantage, especially at aspects such as operability, economy and practicality.Colourimetry can be passed through naked eyes Direct Recognition reaction solution change color, does not need costliness or complicated instrument, operate very easyly, so can be applicable to on-the site analysis and care diagnostic.
Nano material has small-size effect, surface effect and quantum size effect, shows the performances such as special optics, electricity and magnetic, in fluorescence analysis, catalytic reaction and bio-sensing, is widely used.Along with the development of nano material, can make the nano material of solution generation change color more and more by catalytic substrate.For example: golden nanometer particle has unique optical property that depends on size, shape and structure, its gathering or the variation that divides again breaking up to cause colloidal solution color, be therefore widely used in colorimetric sensing; Some have the metal oxide nanoparticles of mimetic enzyme catalysis activity, oxidation reaction that can catalysis peroxidase substrate and produce change color.Colorimetric detection can utilize these change color to be applied to biologic medical diagnosis and environmental monitoring.
Fe 3o 4there is mimetic enzyme catalysis activity, can catalysis H 2o 2oxidation peroxidase chromogenic substrate 3,3 ', 5,5 ’ – tetramethyl benzidine (TMB), make detection system produce one by colourless to blue chromogenic reaction.Acetylcholine can produce hydrogen peroxide (H under the dual catalytic of acetylcholinesterase and choline oxidase simultaneously 2o 2), this is based on Fe 3o 4the foundation of the acetylcholine colorimetric detection method of the imitative enzymatic property of nano material provides possibility.Compare Fe with peroxidase 3o 4have obvious advantage as high in stability, preparation is simple, low price etc., and itself there is magnetic and be easy to separated recovery and meet Green Chemistry theory.But Fe 3o 4the characteristic of easily reuniting, has limited the application of its character to a certain extent.The present invention utilizes diglycol solvent-thermal method to synthesize to have the magnetic reduced graphene (Fe of better dispersiveness 3o 4/ rGO) compound, and the H that utilizes acetylcholine dual-enzyme system catalysis acetylcholine to produce 2o 2for oxygenant, set up Fe 3o 4the development properties of/rGO catalysis chromogenic substrate TMB and the corresponding relation of acetylcholine concentration, realized easy, economy, fast detecting to acetylcholine.
Summary of the invention
technical matters:the object of the invention is for above-mentioned technical matters, provide a kind of based on Fe 3o 4the acetylcholine visible detection method of the imitative enzymatic property of/Graphene.First, adopt diglycol solvent-thermal method to synthesize the Fe with better dispersiveness 3o 4/ rGO, further verifies H 2o 2concentration is to based on Fe 3o 4/ rGO catalysis H 2o 2the impact of the colorimetric detection of oxidation TMB colour developing character; Then, acetylcholinesterase and choline oxidase energy catalysis acetylcholine are decomposed to the H producing 2o 2with Fe 3o 4/ rGO colorimetric detection H 2o 2combine, set up the corresponding relation of acetylcholine concentration and absorbance; Finally realize the concentration determination to acetylcholine.
technical scheme:a kind of based on Fe 3o 4the acetylcholine colorimetric detection method of the imitative enzymatic property of/rGO: first, with graphene oxide, FeCl 24H 2o and FeCl 36H 2o is presoma, adopts and has synthesized the Fe with better dispersiveness with diglycol solvent-thermal method 3o 4/ rGO, further probes into Fe 3o 4the chromogenic reaction of/rGO catalysis and H 2o 2the corresponding relation of concentration; Then, under the dual-enzyme system catalysis of acetylcholinesterase and choline oxidase, acetylcholine decomposes generation H 2o 2; Finally, in the solution after enzymic catalytic reaction, directly add Fe 3o 4/ rGO, the H that catalysis acetylcholine decomposes 2o 2oxidation TMB produces chromogenic reaction, sets up the linear relationship of acetylcholine concentration and solution colour absorbance, realizes the colorimetric detection of acetylcholine.Concrete steps are:
(1) Fe 3o 4the preparation of/rGO: first by FeCl 24H 2o and FeCl 36H 2o joins in diglycol (DEG) solution that contains graphene oxide (GO), wherein FeCl 24H 2o, FeCl 36H 2the addition ratio of O and diglycol (DEG) solution is: 0.5:1:10 mmol/ mmol/mL, and graphene oxide (GO) is 5:10 mg/mL with the amount ratio of diglycol (DEG); And under 90 oC, stir 30 min; Then dropwise add the DEG solution containing 0.8 M NaAc, diglycol (DEG) solution that wherein contains graphene oxide (GO) is 1:1 with the volume ratio that contains the diglycol (DEG) of NaAc; Stir after 10 min, mixed liquor is transferred in reactor, heat 6 h at 200 ℃; Finally, by product cool to room temperature, with ethanol washing, be placed in 50 ℃ of vacuum drying ovens for several times and dry.
(2) H 2o 2the corresponding relation of concentration and reaction system absorbance: the hac buffer (ABS that first 965 μ L is contained to 0.5 mg TMB, 0.2 M, pH 4.0) H of respectively with 10 μ L variable concentrations (0,0.01,0.02,0.1,0.2,1,10,20,40 mM) 2o 2mix; Then add respectively 25 μ L 2 mgmL – 1fe 3o 4/ rGO, incubation 15 min at room temperature after mixing.Finally, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and measure respectively and record containing variable concentrations H under 652 nm wavelength 2o 2the absorbance of reaction solution, drawing standard curve.
(3) enzymic catalytic reaction of acetylcholine: the phosphate buffer solution (pH 7.4 for PBS, 10 mM) of the choline oxidase (ChOx) of acetylcholinesterase (AchE) the He0.5 unit that 200 μ L are contained to 0.5 unit respectively from different amounts (0,5 * 10 – 5, 10 – 4, 5 * 10 – 4, 10 – 3, 10 – 2, 10 – 1, 1,10 μ mol) acetylcholine mix, incubation reaction 30 min at 37 ℃.
(4) corresponding relation of acetylcholine concentration and absorbance: 800 μ L are containing 0.5 mg TMB and 50 μ g Fe 3o 4the ABS of/rGO, joins respectively in the solution after step (3) incubation and mixes, at room temperature incubation 30 min.Then, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and under 652 nm wavelength, measure the absorbance of reaction solution.Finally, set up corresponding relation the drawing standard curve of acetylcholine concentration and absorbance.
The described DEG solution containing GO is the uniform solution of preparing under continuous ultrasound 2 h conditions.
Described Fe 3o 4/ rGO magnetic nanoparticle mean grain size is 10 nm.
The described H that sets up 2o 2the corresponding relation of concentration and absorbance refers to H 2o 2under the catalysis of compound substance, oxidation substrates TMB produces blueness, and its characteristic absorption wavelength, at 652 nm, is measured absorbance under this wavelength, obtains the H of variable concentrations 2o 2and the relation between absorbance, by data processing drawing standard curve.
Described PBS(10 mM, pH 7.4) and 37 ℃ of incubation 30 min be pH value (7.4) and the body temperature (37 ° of C) under simulation people concrete conditions in the establishment of a specific crime, make acetylcholinesterase and choline oxidase reach optimum activity and make catalytic reaction reach equilibrium state.
The acetylcholine that the foundation of described acetylcholine concentration and the corresponding relation of absorbance and Specification Curve of Increasing refer to concentration known produces choline under acetylcholinesterase and choline oxidase catalysis, the H that choline produces again under the catalysis of choline oxidase 2o 2, utilize compound substance catalysis H 2o 2oxidation TMB produces blue, at its characteristic absorption wavelength 652 nm places, measures absorbance, obtains acetylcholine and the relation between absorbance the drawing standard curve of variable concentrations.
beneficial effect:the present invention utilizes one step hydro thermal method to synthesize Fe 3o 4/ rGO, the strategy of desmoenzyme catalysis and colorimetric detection, has set up a kind of colorimetric detection method of acetylcholine, and its characteristic and advantage are expressed as follows:
(1) at Fe 3o 4in the preparation of/rGO, adopt tirethylene glycol as solvent, the Fe of acquisition 3o 4particle diameter is minimum and be evenly distributed on rGO surface.
(2) by Graphene and Fe 3o 4/ rGO is compound, Fe 3o 4the dispersiveness of nano particle is greatly improved, and catalytic effect significantly strengthens, H 2o 2response concentration be low to moderate 0.1 μ M.
(3) the method has been used the chromogenic substrate TMB of develop the color sensitiveer and non-carcinogenesis, the Fe of use 3o 4/ rGO nano composite material has magnetic, can, by physical method separated recycling very easily, meet Green Chemistry theory.
(4) the method utilizes acetylcholinesterase and choline oxidase dual-enzyme system catalytic decomposition acetylcholine to produce H 2o 2with H 2o 2colorimetric detection combines, and has realized the colorimetric detection to acetylcholine.
(5) by two-step approach, both guaranteed the activity of acetylcholinesterase, passed through H again 2o 2detection realized the Sensitive Detection to acetylcholine.
(6) compare the feature such as it is easier flexibly that the colorimetric determination method of constructed acetylcholine has operation, and instrument and equipment is simpler, and testing cost is cheap with traditional detection method.
Accompanying drawing explanation
Fig. 1 is Fe 3o 4and Fe (A) 3o 4/ rGO(B) TEM figure;
Fig. 2 is H 2o 2the corresponding relation of concentration and absorbance (embedded figure is typical curve);
Fig. 3 is the corresponding relation (embedded figure is typical curve) of acetylcholine concentration and absorbance.
Embodiment
embodiment mono-:fe 3o 4/ rGO preparation and to H 2 o 2 colorimetric detection
(1) Fe 3o 4the preparation of/rGO: first by 0.5 mmol FeCl 24H 2o and 1 mmol FeCl 36H 2o joins in the DEG solution that 10 mL contain 5 mg GO, and under 90 oC, stirs 30 min; Then dropwise add DEG solution 10 mL containing 0.8 M NaAc, stir after 10 min, mixed liquor is transferred in reactor, under 200 oC, heat 6 h; Finally, by product cool to room temperature, with ethanol washing, be placed in 50 ℃ of vacuum drying ovens for several times and dry, obtain Fe 3o 4/ rGO as shown in Figure 1.
(2) H 2o 2the response relation of concentration and absorbance: the H of ABS respectively with the 10 μ L variable concentrations (0,0.01,0.02,0.1,0.2,1,10,20,40 mM) that first 965 μ L contained to 0.5 mg TMB 2o 2mix; Then add respectively 25 μ L 2 mgmL – 1fe 3o 4/ rGO, incubation 15 min at room temperature after mixing.Finally, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and under 652 nm wavelength, measure respectively and record the absorbance of variable concentrations reaction solution, drawing standard curve is as shown in Figure 2.
embodiment bis-: the detection of acetylcholine and Specification Curve of Increasing
(1) enzymic catalytic reaction of acetylcholine: the PBS of the choline oxidase (ChOx) of acetylcholinesterase (AchE) the He0.5 unit that 200 μ L are contained to 0.5 unit respectively from different amounts (0,5 * 10 – 5, 10 – 4, 5 * 10 – 4, 10 – 3, 10 – 2, 10 – 1, 1,10 μ mol) acetylcholine mix, at 37 ℃, incubation reaction 30 min.
(2) corresponding relation of acetylcholine concentration and absorbance: 800 μ L are containing 0.5 mg TMB and 50 μ g Fe 3o 4the ABS of/rGO, joins respectively in the solution after step (3) incubation, mixes, at room temperature incubation 30 min.Then, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and under 652 nm wavelength, measure the absorbance of reaction solution.Finally, set up the corresponding relation of acetylcholine concentration and absorbance, and drawing standard curve as shown in Figure 3.

Claims (7)

1. based on magnetic graphite alkene, imitate the acetylcholine visible detection method of enzymatic property, according to following step, carry out: first, with graphene oxide, FeCl 24H 2o and FeCl 36H 2o is presoma, adopts and has synthesized the Fe with better dispersiveness with diglycol solvent-thermal method 3o 4/ rGO, further probes into Fe 3o 4the chromogenic reaction of/rGO catalysis and H 2o 2the corresponding relation of concentration; Then, under the dual-enzyme system catalysis of acetylcholinesterase and choline oxidase, acetylcholine decomposes generation H 2o 2; Finally, in the solution after enzymic catalytic reaction, directly add Fe 3o 4/ rGO, the H that catalysis acetylcholine decomposes 2o 2oxidation TMB produces chromogenic reaction, sets up the linear relationship of acetylcholine concentration and solution colour absorbance, realizes the colorimetric detection of acetylcholine.
2. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 1, concrete preparation process is as follows:
(1) Fe 3o 4the preparation of/rGO: first by FeCl 24H 2o and FeCl 36H 2o joins in the diethylene glycol solution that contains graphene oxide, wherein FeCl 24H 2o, FeCl 36H 2the addition ratio of O and diethylene glycol solution is: 0.5:1:10 mmol/ mmol/mL, and the amount ratio of graphene oxide and diglycol is 5:10 mg/mL; And under 90 oC, stir 30 min; Then dropwise add the diethylene glycol solution containing 0.8 M NaAc, the diethylene glycol solution that wherein contains graphene oxide is 1:1 with the volume ratio that contains the diglycol of NaAc; Stir after 10 min, mixed liquor is transferred in reactor, heat 6 h at 200 ℃; Finally, by product cool to room temperature, with ethanol washing, be placed in 50 ℃ of vacuum drying ovens for several times and dry;
(2) H 2o 2the corresponding relation of concentration and reaction system absorbance: the hac buffer ABS that first 965 μ L is contained to 0.5 mg TMB, 0.2 M, pH 4.0, the H of respectively with 10 μ L variable concentrations 0,0.01,0.02,0.1,0.2,1,10,20,40 mM 2o 2mix; Then add respectively 25 μ L 2 mgmL – 1fe 3o 4/ rGO, incubation 15 min at room temperature after mixing; Finally, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and measure respectively and record containing variable concentrations H under 652 nm wavelength 2o 2the absorbance of reaction solution, drawing standard curve;
(3) enzymic catalytic reaction of acetylcholine: the phosphate buffer solution PBS of the choline oxidase of the acetylcholinesterase He0.5 unit that 200 μ L are contained to 0.5 unit, 10 mM, pH 7.4 respectively from different amounts 0,5 * 10 – 5, 10 – 4, 5 * 10 – 4, 10 – 3, 10 – 2, 10 – 1, 1,10 μ mol acetylcholine mix, incubation reaction 30 min at 37 ℃;
(4) corresponding relation of acetylcholine concentration and absorbance: 800 μ L are containing 0.5 mg TMB and 50 μ g Fe 3o 4the ABS of/rGO, joins respectively in the solution after step (3) incubation and mixes, at room temperature incubation 30 min; Then, by externally-applied magnetic field (magnet), remove the Fe in reaction system 3o 4/ rGO, the observation change color of taking pictures, and under 652 nm wavelength, measure the absorbance of reaction solution; Finally, set up corresponding relation the drawing standard curve of acetylcholine concentration and absorbance.
3. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 2, is characterized in that: the DEG solution containing GO in step (1) is the uniform solution of preparing under continuous ultrasound 2 h conditions.
4. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 2, is characterized in that: described Fe 3o 4/ rGO magnetic nanoparticle mean grain size is 10 nm.
5. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 2, is characterized in that: the described H that sets up 2o 2the corresponding relation of concentration and absorbance refers to H 2o 2under the catalysis of compound substance, oxidation substrates TMB produces blueness, and its characteristic absorption wavelength, at 652 nm, is measured absorbance under this wavelength, obtains the H of variable concentrations 2o 2and the relation between absorbance, by data processing drawing standard curve.
6. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 2, it is characterized in that: PBS 10 mM that step (3) is described, pH 7.4 and 37 ℃ of incubation 30 min are pH value 7.4 and the 37 ° of C of body temperature under simulation people concrete conditions in the establishment of a specific crime, make acetylcholinesterase and choline oxidase reach optimum activity and make catalytic reaction reach equilibrium state.
7. the acetylcholine visible detection method based on the imitative enzymatic property of magnetic graphite alkene according to claim 2, it is characterized in that: the foundation of described acetylcholine concentration and the corresponding relation of absorbance and the acetylcholine that Specification Curve of Increasing refers to concentration known produce choline, the H that choline produces again under the catalysis of choline oxidase under acetylcholinesterase and choline oxidase catalysis 2o 2, utilize compound substance catalysis H 2o 2oxidation TMB produces blue, at its characteristic absorption wavelength 652 nm places, measures absorbance, obtains acetylcholine and the relation between absorbance the drawing standard curve of variable concentrations.
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CN110567953B (en) * 2019-10-12 2022-07-01 山西师范大学 Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof

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