CN108226250A - A kind of incretion interferent electrochemical assay based on enzyme nano-reactor - Google Patents
A kind of incretion interferent electrochemical assay based on enzyme nano-reactor Download PDFInfo
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- CN108226250A CN108226250A CN201810027873.XA CN201810027873A CN108226250A CN 108226250 A CN108226250 A CN 108226250A CN 201810027873 A CN201810027873 A CN 201810027873A CN 108226250 A CN108226250 A CN 108226250A
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- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/36—Glass electrodes
Abstract
The present invention relates to a kind of incretion interferent electrochemical assays based on enzyme nano-reactor.The present invention is by the use of the silica of functionalization as template, it is acted on by hydrophobicity hydrophobicity and is mixed with graphene oxide, freezing, calcining obtains grapheme foam, while organized enzyme is fixed on grapheme foam (NGF) hole so as to build CYP3A4/PNGF enzyme reactors using Electrostatic Absorption and covalent bond method.Developing a kind of has the characteristics that at low cost, simple, convenient quick electrochemical methods.The controllable aperture of enzyme reactor that the present invention uses, the metabolism behavior of incretion interferent can be quickly and accurately measured by Amperometric, and method high sensitivity, stability are good, suitable for promoting and applying.
Description
Technical field
The invention belongs to analytical chemistry fields, and in particular to a kind of incretion interferent electrification based on enzyme nano-reactor
Learn detection method.
Background technology
Incretion interferent (endocrine disrupting chemicals, EDCs) is referred in intervention organism
Hormonal synthesis, secretion, conveying, combination, effect or decomposition maintain so as to influence the normality of organism, endanger reproduction, hair
It educates or the xenobiotics of behavior.EDCs is prevalent in pesticide, cosmetics, plastics, food, drug etc., common
There are bisphenol-A (BPA), phthalic acid ester, induced by alkyl hydroxybenzene, Polychlorinated biphenyls (PCBs) etc..Thus establish a kind of at low cost, operation letter
Single, conveniently detection incretion interferent method is particularly important.
Numerous studies show that CYP450S enzymes may activate certain EDCs, make its metabolite and internal receptor, protease
Wait large biological molecules that there is stronger binding force.The external temperature of common external CYP450, which incubates method, mainly has the hepatomicrosome warm in vitro
Incubate method, the external temperature of recombinant C YP450 enzymes incubates the external temperature of method, liver cell and incubates method etc., mainly by a certain amount of CYP substrates and preparation
Good hepatomicrosome or recombinant C YP450S enzymes or the liver cell temperature in the case where simulating physiological environment is incubated.All based on CYP450S enzymes
In In vitro metabolism method, either laboratory method or commercial run is required for the participation of electron donor coenzyme NADP 11, NADPH
It is expensive, not easy to maintain, it is uneconomical to carry out source electrode.To solve this problem, people start with electrochemical means and carry out pair
The in vitro study of CYP450s.But research has been found that directly is fixed on electrode surface by zymoprotein, easily leads to zymoprotein
Absorption deformation, therefore its Direct Electrochemistry is not easily accomplished.To solve this problem, people generally use nano combined material
The method of modified electrode is expected to maintain the bioactivity of zymoprotein.The albumen that Gilardi et al. forms CYP fusions CPR, leads to
The time-to-live of the active furans oxygroup oxidant of control CYP is spent to enhance catalytic activity.Mie et al. is devised with hydrophobic list
The Thiolation gold electrode of member, for fixing recombinant C YP450s, and demonstrates its electrocatalysis characteristic.Rusling et al. is by purifying
CYP450S enzymes and CPR construct 6 multilayers of PDDA/PSS (CYP450S1A2/CPR+b5) by layer assembly in PG electrode surfaces
Film can give electron transmission to CYP450S enzyme ferrohemes center again first by electron transmission to CPR by electrode by CPR, this
Electron transfer process is consistent in vivo with reality.But these work all generally existing substrate catalysis low yield, response signal is micro-
The phenomenon that weak.On the one hand the reason of causing more than phenomenon is since immobile interface is unfavorable for the holding of fixed enzyme conformation；It is another
Aspect is that two electronics for being catalyzed reaction and needing are obtained from electrode since immobile interface is unfavorable for enzyme active center.Therefore, it builds
Immobile interface with good biocompatibility, excellent electron transmission, for building high catalytic efficiency, highly sensitive activity
Enzyme electrochemical reactor is of great significance.
At present, it is increasingly attracted attention for the detection of incretion interferent based on structure enzyme reactor.Tang et al. systems
Standby TiO2/CdSe@CdS/ glucose oxidases construct a kind of Photoelectrochemistrbiosensor biosensor and come measure for glucose, Li
Et al. synthesis photosensitive TNA materials measure H2O2 contents, Liu et al. people measures testosterone etc. using macropore silicon foam.This side
Method realizes the structure of enzyme nano-reactor and the sensing of object simultaneously, simplifies operating procedure, and with preferable choosing
Selecting property and sensitivity.
The undisclosed skill that incretion interferent is detected based on the electrochemical methods for preparing enzyme nano-reactor of the prior art
Art scheme.
Invention content
Enzyme nano-reactor made of template is based on the object of the present invention is to provide one kind for detecting endocrine disruption
Simple, cheap, the effectively and rapidly electrochemical detection method of object.The method by the use of the controllable grapheme foam of size as
Enzyme nano-reactor, and the catalyst present invention being metabolized using organized enzyme as incretion interferent is asked in the above-mentioned technology of solution
Technical solution is as follows used by topic：
A kind of incretion interferent electrochemical detection method based on enzyme nano-reactor, the method specifically includes as follows
Step：
(1) it using the silica of different-grain diameter functionalization as template, is acted on by hydrophobicity-hydrophobicity by itself and oxidation
Graphene mixes:Graphite oxide powder is distributed in water simultaneously ultrasound 4h first, then centrifuges 30min under the conditions of 3000rmp and obtains
To the graphene oxide water solution of a concentration of 0.5mg/mL-1mg/mL, then according to volume ratio 1:3 ratios are by the dioxy of functionalization
SiClx and graphene oxide water solution mix 12h and obtain composite material at room temperature；
(2) composite material obtained into step (1) carries out -20 DEG C of freeze-dryings, freeze-drying, is then forged in inert gas
It burns, then is washed with the hydrofluoric acid of 5wt%, obtain grapheme foam；
(3) grapheme foam obtained into step (2) is water-dispersible, by the hydrochloric acid DOPA for adding in a concentration of 1mg/mL
Amine aqueous solution carries out functional modification to grapheme foam：It is 20 according to volume ratio：1 ratio is added in into Dopamine hydrochloride solution
Grapheme foam obtains the PNGF nanocomposites of a concentration of 0.5mg/mL；
(4) the PNGF nanocomposites obtained in a certain amount of step (3) are taken, and are put into as enzyme nano-reactor
In centrifuge tube, then organized enzyme is added in inward, and mixing on the oscillator, drop in for use on glass-carbon electrode；Utilize three-electrode system
I.e. amber silk electrode is auxiliary electrode, and saturated calomel electrode is reference electrode, and glass-carbon electrode is working electrode, a concentration of 0.1M, pH
=7.0 phosphate buffer adds in incretion interferent as electrolyte into electrolyte every time by timing-current method,
For detecting incretion interferent.
The synthetic method of the silica of functionalization described in the step (1) is：First pass through the synthesis of the colloidal sol seed law
Silicon oxide pellets；Then by the use of DMDMS as modifying agent, hydrochloric acid is as catalyst, according to volume ratio 1:100 ratio will be modified
Agent and hydrochloric acid are added in dilution, i.e., 15M hydrochloric acid, 0.6g polyethers F108,0.875mLDMDMS are respectively added to 75mL concentration
Methylated silicon dioxide solution is obtained in aqueous solution for the silicon oxide pellets of 1mg/mL, then ammonium hydroxide neutralizes the titanium dioxide that methylates
The silica of the functionalization described in step (1) is made in silicon solution.The silica of colloidal sol seed law synthesis different-grain diameter is small
Ball is i.e. using originating monodispersity micelle as seed, then by method offer silica physically or chemically, on seed
Synchronous growth, so as to obtain the silica of monodispersity.
The incretion interferent is nifedipine either testosterone or oestrone or progesterone.
The aperture of the silica of functionalization is 60-250nm, preferably 60nm in the step (1).
Inert gas in the step (2) is argon gas.
The preparation method of Dopamine hydrochloride solution in the step (3) is：By Tris-HCl buffer solutions and dopamine
The Dopamine hydrochloride solution that pH value is 6-9 is hybridly prepared into, the pH is preferably 8.5.Functional modification in the step (3)
Dopamine hydrochloride solution concentration is 0.5mg/mL-2mg/mL, preferably 0.5mg/mL.
Organized enzyme is CYP3A4 in the step (4).
It is 10 μ L to add in the step (4) as the PNGF nanocomposites of enzyme nano-reactor, endocrine to be measured
A concentration of 5mM of chaff interferent.A concentration of 10mg/mL of organized enzyme is added in, the preferably amount of organized enzyme is 5 μ L.
The Km of testosterone during electrochemical detection method detection testosterone of the present inventionappValue is 110.70 μM.
More specifically, a kind of incretion interferent Electrochemical Detection side based on enzyme nano-reactor of the present invention
Method includes the following steps：
(1) using the grain size of the silica template of functionalization be 60-250nm as template, pass through hydrophobicity-hydrophobicity and make
It is mixed with by it with graphene oxide；
(2) mixed liquor obtained into step (1) carries out -20 DEG C of freeze-dryings, is freeze-dried, and is calcined in inert gas, uses
The hydrofluoric acid mold cleaning plate of 5wt% obtains grapheme foam；
(3) grapheme foam obtained into step (2) is water-dispersible, molten by the Dopamine hydrochloride for adding in various concentration
Liquid carries out functional modification to grapheme foam and obtains PNGF nanocomposites.
(4) the PNGF nanocomposites obtained into step (3) take the material of 10ul to be put into as enzyme nano-reactor
In A centrifuge tubes, the organized enzyme of a concentration of 10mg/mL is added in inward, mixing on oscillator drops in for use on glass-carbon electrode, is used for
Detectable concentration is the incretion interferent of 5mM.
The present invention is acted on by hydrophobicity-hydrophobicity by oxygen by the use of the silica of different-grain diameter functionalization as template
Graphite alkene is mixed with, and freezing, calcining obtains different pore size and uniform grapheme foam.Dopamine hydrochloride is one
Amphiprotic substance, in pH>Negatively charged when 4, due to Dopamine hydrochloride meeting autohemagglutination synthesis poly-dopamine, poly-dopamine has Superhydrophilic
Property, therefore by π-π effects can with grapheme foam is compound obtains hydrophilic PNGF nano-reactors.Simultaneously using quiet
Organized enzyme is fixed in grapheme foam (NGF) hole anti-so as to build CYP3A4/PNGF enzyme nanometers by Electro Sorb and covalent bond method
Answer device.It can be consequently used for the Sensitive Detection to incretion interferents such as nifedipine, testosterone, oestrone, progesterone.
Grapheme foam used by technical solutions according to the invention, since its solubility property is good, as organized enzyme
Nano-reactor can effectively ensure that the catalytic activity of enzyme using confinement cavity effect, more fully simplify operation step
Suddenly, a step of enzyme nano-reactor structure and object sensing is realized.In addition, the present invention is based on electrochemical methods biographies
Sense, visualization are suitable for high-throughput detection, and the grapheme foam of Template synthesis is more stable, is interfered by extraneous factor
It is small, available for the detection of incretion interferent in practical systems, suitable for promoting and applying.
Description of the drawings
Fig. 1 (A), Fig. 1 (B) are respectively functionalized SiO 2 (grain size 60nm) Template synthesis grapheme foam (aperture
66nm or so) field emission scanning electron microscope figure and transmission electron microscope picture, Fig. 1 (C), Fig. 1 (D) be functionalized SiO 2 (grain size
250nm) the field emission scanning electron microscope figure and transmission electron microscope picture of Template synthesis grapheme foam (aperture 260nm or so)；
The grapheme foam nano-reactor that it is respectively different pore size that Fig. 2, which is, curve a is aperture 66nm graphenes in Fig. 2
Curve b is that curve c is not add in stone in aperture 250nm grapheme foams nano-reactor, Fig. 2 in foam nano-reactor, Fig. 2
Black alkene foam nano-reactor adds in the cyclic voltammogram that organized enzyme (10mg/mL) forms enzyme nano-reactor.
Fig. 3 is that aperture is that 66nm nano-reactors are metabolized incretion interferent with organized enzyme formation enzyme nano-reactor
Dynamics research：The test condition of curve a is anaerobic in figure, and the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 are as electricity
Solve liquid；The test condition of curve b is under aerobic conditions, the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 are as electricity in figure
Solve liquid；The test condition of curve c is aerobic in figure, the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 as electrolyte,
Add in the substrate of 25ul a concentration of 5mM in toward electrolyte, the test condition of curve d is aerobic, 5mL a concentration of 0.1M, pH in figure
=7.0 PBS buffer solutions as electrolyte, toward electrolyte in add in the substrate of a concentration of 5mM of 50ul.
Specific embodiment
Technical solutions according to the invention are further described in detail below by specific embodiment, but it is necessary to
It points out that following embodiment is served only for the description to invention content, does not form limiting the scope of the invention.
Embodiment 1：
(1) using the silica that grain size is 60nm functionalization as template, by hydrophobicity-hydrophobicity effect by itself and oxygen
Graphite alkene mixing (preparation method of silicon oxide pellets be TEOs hydrolysis, condensation process i.e.：By 80mL ethyl alcohol, 4.85mL
Water, 3.6mL ammonium hydroxide are added in flask, in the case where being stirred continuously, are progressively heated at certain temperature, are then quickly added into TEOS and second
Mixed liquor (the volume ratio 0.3875 of alcohol:1), reaction 5h is that can obtain the silicon oxide pellets that grain size is 60nm)；
The synthetic method of the silica of functionalization is：15M hydrochloric acid, 0.6g polyethers F108,0.875mLDMDMS are distinguished
It is added in the aqueous solution of the silicon oxide pellets of a concentration of 1mg/mL of 75mL and obtains methylated silicon dioxide solution, then in ammonium hydroxide
With methylated silicon dioxide solution, the silica of the functionalization is made.
Using the silica of functionalization as template, it is mixed with graphene oxide by hydrophobicity-hydrophobicity effect:
Graphite oxide powder is distributed in water and ultrasound 4h first, then under the conditions of 3000rmp centrifugation 30min obtain it is a concentration of
The graphene oxide water solution of 0.5mg/mL-1mg/mL, then according to volume ratio 1:3 ratios are by the silica and oxygen of functionalization
Graphite aqueous solution mixes 12h and obtains composite material at room temperature；
(2) composite material obtained into step (1) carries out -20 DEG C of freeze-dryings, freeze-drying, calcines, uses in inert gas
The hydrofluoric acid mold cleaning plate of 5wt% obtains grapheme foam；By using field emission scanning electron microscope and transmission electron microscope to manufactured graphite
Alkene foam carries out pattern test (Fig. 1 a).
(3) grapheme foam obtained into step (2) is water-dispersible, by the hydrochloric acid DOPA for adding in a concentration of 1mg/mL
Amine aqueous solution carries out functional modification to grapheme foam：It is 20 according to volume ratio：1 ratio is added in into Dopamine hydrochloride solution
Grapheme foam obtains the PNGF nanocomposites of a concentration of 0.5mg/mL；
(4) the PNGF nanocomposites obtained in a certain amount of step (3) are taken, centrifuge tube is put into as enzyme nano-reactor
In, add in organized enzyme inward, mixing on oscillator drops in for use on glass-carbon electrode；It is using three-electrode system, that is, amber silk electrode
Auxiliary electrode, saturated calomel electrode are reference electrode, and glass-carbon electrode is working electrode, the PBS of electrolyte 0.1M, pH=7.0
Buffer solution carries out cyclic voltammetry under speed and oxygen free condition in certain sweep.The glass-carbon electrode prepared again into step (4)
The upper incretion interferent testosterone for adding in various concentration studies its dynamics.
It is methylated silicon dioxide (60nm) Template synthesis grapheme foam (aperture that Fig. 1 (A), Fig. 1 (B), which are respectively,
66nm or so) field emission scanning electron microscope figure and transmission electron microscope picture；Fig. 1 (C), Fig. 1 (D) are respectively methylated silicon dioxide
The field emission scanning electron microscope figure and transmission electron microscope picture of (250nm) Template synthesis grapheme foam (aperture 260nm or so), from field
The pore size for emitting scanning electron microscope it can be seen from the figure that synthesizing graphite alkene foam is uniform, this and transmission electron microscope picture result phase one
It causes.These phenomenons illustrate that grapheme foam nano-reactor structure is feasible, and a confinement can be provided for organized enzyme
The environment of cavity, advantageously ensures that enzyme activity.Therefore, the detection that this technical solution can be used for incretion interferent is demonstrated.
The grapheme foam nano-reactor that it is respectively different pore size that Fig. 2, which is, curve a is aperture 66nm graphenes in Fig. 2
Curve b is that curve c is not add in stone in aperture 250nm grapheme foams nano-reactor, Fig. 2 in foam nano-reactor, Fig. 2
Black alkene foam nano-reactor adds in the cyclic voltammogram that organized enzyme (10mg/mL) forms enzyme nano-reactor.Thus figure can be with
Learn the CYP3A4 in assembling 66nm grapheme foams hole Direct Electrochemistry research can by cyclic voltammetry (CV) into
Row (curve a) in Fig. 2.In PBS (0.1M, pH=7.0) electrolyte solution of deoxygenation, the CV curves of CYP3A4/PNGF/GCE
In have a pair of stablize, symmetrical redox peaks of peak shape, redox spike potential is respectively -0.380V and -0.355V
(vsAg/AgCl) (curve c) in Fig. 2.And do not have then for CYP3A4/GCE or CYP3A4/PNGF/GCE (aperture 260nm)
There is (curve b and c in Fig. 2) in apparent redox peaks, this demonstrate that CYP3A4/PNGF electrochemical responses are because of CYP3A4
Electroactive site and PNGF electrodes between direct electron transfer, that is to say, that be assembled in CYP3A4 in PNGF have compared with
Good electro-chemical activity.
Fig. 3 is that aperture is that 66nm nano-reactors are metabolized incretion interferent with organized enzyme formation enzyme nano-reactor
Dynamics research：Curve a test conditions are anaerobic in figure, and the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 are as electrolysis
Liquid；Curve b test conditions are under aerobic conditions, the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 are as electrolysis in figure
Liquid；Curve c test conditions are aerobic in figure, and the PBS buffer solutions of 5mL a concentration of 0.1M, pH=7.0 are as electrolyte, toward electricity
Solve the testosterone that 25ul a concentration of 5mM are added in liquid, curve d test conditions are aerobic in figure, 5mL a concentration of 0.1M, pH=7.0's
PBS buffer solutions as electrolyte, toward electrolyte in add in the testosterone of a concentration of 5mM of 50ul.
Fig. 3 curves a and b are the CV comparison diagrams of CYP3A4/PNGF/GCE under anaerobic and aerobic conditions.It can be seen by figure
Go out, the presence of oxygen makes reduction currents of the CYP3A4/PNGF/GCE at -0.376V significantly increase.Thus illustrate CYP3A4/
The upper increased reduction currents of PNGF/GCE be based on oxygen molecule and reduced form ferrous iron CYP it is quick with reference to and generate.The process by
Electronics in electrode drive, first electrode is transferred to reductase CPR, is then then transferred to CYP3A4 ferrohemes center.And PNGF
Excellent electron transmission performance, bio-compatibility and bigger serface has played important work to the realization of the electronic transfer process
With.Further, testosterone is selected as model substrates, for verifying that CYP3A4 is to the catalytic activity of substrate on modified electrode.By Fig. 3
Middle curve c, d can be seen that with the addition of substrate testosterone, and reduction currents of the CYP3A4/PNGF/GCE at -0.376V is apparent
Increase.Thus the CYP3A4 that explanation is supported in PNGF holes has substrate the catalytic activity of electrochemistry driving.
This illustrates under aerobic conditions, and substrate testosterone is added in electrolyte solution, can cause CYP3A4 reduction peaks in PNGF
The increase of electric current, with the increase of testosterone addition, reduction peak current is consequently increased (Fig. 3), illustrates the CYP3A4 in PNGF
There is the driving catalytic action of apparent electrochemistry to the metabolism of testosterone, by testosterone metabolism into 6 beta-hydroxy testosterones.
CYP3A4/PNGF/GCE can characterize the current-responsive of testosterone being continuously added to time current curve.
As shown in Figure 4 A, after testosterone is added to 0.1M pH 7.0PBS buffer solutions, CYP3A4/PNGF/GCE locates in -0.376V
Reduction current quickly increase, and a stationary value is quickly reached within 3s.What Fig. 4 B were provided is the stable state at -0.48V places
The curved line relation of electric current and concentration of substrate.Further, as shown in Figure 4 C, make the double reciprocal curve of steady-state current and concentration of substrate,
Equation of linear regression is obtained as Y (nA-1)=0.048+5.348X (mM-1)(R2=0.997) is according to Michaelis-Menten side
Journey:
In formula Iss be add in substrate after steady-state current, C be bulk solution in substrate concentration, ImaxSatisfy for concentration of substrate
With when maximum current, KmappFor apparent K_m.In the system, average I is acquiredmax(being equivalent to Vmax) and KmappPoint
It Wei not 0.245 μ A and 110.70 μM.This further proves that PNGF as an enzyme nano-reactor, is provided well for organized enzyme
Biological microenvironment, maintain the bioactivity of organized enzyme well.
Embodiment 2：
(1) be the silica of 250nm functionalization as template using grain size, by hydrophobicity-hydrophobicity effect by its with
Graphene oxide mixes；The preparation of silicon oxide pellets be TEOs hydrolysis, condensation process i.e.：Take 10mL seeded dispersions liquid,
70mL ethyl alcohol, 13mL water, 7.5mL ammonium hydroxide are added in flask, after stirring evenly at room temperature, are then added in constant pressure funnel
TEOs and the mixed liquor of ethyl alcohol (volume ratio 1:10) 5h, is reacted, this process, which is constantly repeated four times, can obtain grain size as 250nm
Silicon oxide pellets
The synthetic method of the silica of functionalization is：By 15M hydrochloric acid, 0.6g polyethers F108,0.875mL DMDMS difference
It is added in the aqueous solution of the silicon oxide pellets of a concentration of 1mg/mL of 75mL and obtains methylated silicon dioxide solution, then in ammonium hydroxide
With methylated silicon dioxide solution, the silica of the functionalization is made.
Using the silica of functionalization as template, it is mixed with graphene oxide by hydrophobicity-hydrophobicity effect:
Graphite oxide powder is distributed in water and ultrasound 4h first, then under the conditions of 3000rmp centrifugation 30min obtain it is a concentration of
The graphene oxide water solution of 0.5mg/mL-1mg/mL, then according to volume ratio 1:3 ratios are by the silica and oxygen of functionalization
Graphite aqueous solution mixes 12h and obtains composite material at room temperature；
(2) composite material obtained into step (1) carries out -20 DEG C of freeze-dryings, freeze-drying, calcines, uses in inert gas
The hydrofluoric acid mold cleaning plate of 5wt% obtains grapheme foam；By using field emission scanning electron microscope and transmission electron microscope to manufactured graphite
Alkene foam carries out pattern test (Fig. 1 b).
(3) grapheme foam obtained into step (2) is water-dispersible, by the hydrochloric acid DOPA for adding in a concentration of 1mg/mL
Amine aqueous solution carries out functional modification to grapheme foam：It is 20 according to volume ratio：1 ratio is added in into Dopamine hydrochloride solution
Grapheme foam obtains the PNGF nanocomposites of a concentration of 0.5mg/mL；
(4) the PNGF nanocomposites obtained into step (3), take a certain amount of material to be put as enzyme nano-reactor
Enter in A centrifuge tubes, add in organized enzyme inward, mixing on oscillator drops in for use on glass-carbon electrode.Utilize three-electrode system, that is, amber
Silk electrode is auxiliary electrode, and saturated calomel electrode is reference electrode, and glass-carbon electrode is working electrode, and electrolyte is a concentration of
The PBS buffer solutions of 0.1M, pH=7.0 carry out cyclic voltammetry under speed and oxygen free condition in certain sweep.Pass through addition again
The incretion interferent testosterone of various concentration studies its dynamics.
Although above having made detailed description to invention with a general description of the specific embodiments, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.
Claims (8)
1. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor, which is characterized in that specifically include as follows
Step：
(1) it using the silica of different-grain diameter functionalization as template, is acted on by hydrophobicity-hydrophobicity by itself and graphite oxide
Alkene mixes:Graphite oxide powder is distributed in water and ultrasound 4h first, then under the conditions of 3000rmp centrifugation 30min obtain it is dense
The graphene oxide water solution for 0.5mg/mL is spent, then according to volume ratio 1:3 ratios are by the silica of functionalization and oxidation
Graphene aqueous solution mixes 12h and obtains composite material at room temperature；
(2) composite material obtained into step (1) carries out -20 DEG C of freeze-dryings, freeze-drying, is then calcined in inert gas,
It is washed again with the hydrofluoric acid of 5wt%, obtains grapheme foam；
(3) grapheme foam obtained into step (2) is water-dispersible, molten by the Dopamine hydrochloride for adding in a concentration of 1mg/mL
Liquid carries out functional modification to grapheme foam：It is 20 according to volume ratio：1 ratio adds in graphite into Dopamine hydrochloride solution
Alkene foam obtains the PNGF nanocomposites of a concentration of 0.5mg/mL；
(4) the PNGF nanocomposites obtained in a certain amount of step (3) are taken, and centrifugation is put into as enzyme nano-reactor
Guan Zhong, then organized enzyme is added in inward, and mixing on the oscillator, it drops in for use on glass-carbon electrode；Utilize three-electrode system, that is, amber
Silk electrode is auxiliary electrode, and saturated calomel electrode is reference electrode, and glass-carbon electrode is working electrode, a concentration of 0.1M, pH=7.0
Phosphate buffer as electrolyte, incretion interferent is added in into electrolyte by timing-current method every time, for examining
Survey incretion interferent.
2. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is that the aperture of the silica of functionalization is 60-250nm in the step (1).
3. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is that the synthetic method of the silica of the functionalization described in the step (1) is：First pass through colloidal sol seed law synthesis two
Silica bead；Then by the use of DMDMS as modifying agent, hydrochloric acid as catalyst, by 15M hydrochloric acid, 0.6g polyethers F108,
0.875mLDMDMS is respectively added to obtain the dioxy that methylates in the aqueous solution of the silicon oxide pellets of a concentration of 1mg/mL of 75mL
SiClx solution, then the silica of the functionalization described in step (1) is made with methylated silicon dioxide solution in ammonium hydroxide.
4. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is that the inert gas in the step (2) is argon gas.
5. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is that the preparation method of the Dopamine hydrochloride solution in the step (3) is：Tris-HCl buffer solutions are mixed with dopamine
The Dopamine hydrochloride solution for being configured to that pH value is 6-9 is closed, the pH is preferably 8.5.
6. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is that it is 10 μ L to be added in the step (4) as the material of enzyme nano-reactor.
7. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is, a concentration of 10mg/mL of organized enzyme is added in the step (4), the amount of organized enzyme is 5 μ L.
8. a kind of incretion interferent electrochemical assay based on enzyme nano-reactor according to claim 1, special
Sign is, a concentration of 5mM of incretion interferent to be measured in the step (4).
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106422816A (en) * | 2016-09-22 | 2017-02-22 | 华中科技大学 | Preparation method of graphene foam-polydopamine composite membrane, product prepared with preparation method and application of product |
| CN106881069A (en) * | 2017-03-09 | 2017-06-23 | 吉林师范大学 | A kind of preparation method and application of temperature response type europium ion trace composite membrane |
-
2018
- 2018-01-11 CN CN201810027873.XA patent/CN108226250A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106422816A (en) * | 2016-09-22 | 2017-02-22 | 华中科技大学 | Preparation method of graphene foam-polydopamine composite membrane, product prepared with preparation method and application of product |
| CN106881069A (en) * | 2017-03-09 | 2017-06-23 | 吉林师范大学 | A kind of preparation method and application of temperature response type europium ion trace composite membrane |
Non-Patent Citations (3)
| Title |
|---|
| XIAODAN HUANG等: "Functional Nanoporous Graphene Foams with Controlled Pore Sizes", 《ADVANCE MATERIALS》 * |
| XIAONI FANG等: "Polydopamine Grafted Porous Graphene as Biocompatible Nanoreactor for Efficient Identification of Membrane Proteins", 《ACS APPLIED MATERIALS AND INTERFACES》 * |
| XUAN XU等: "Fast steroid hormone metabolism assays with electrochemical liver microsomal bioreactor based on polydopamine encapsulated gold-graphene nanocomposite", 《ELECTROCHIMICA ACTA》 * |
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