CN105784801A - Method for detecting low density lipoprotein cholesterin through double-enzyme concerted catalysis silver deposition - Google Patents
Method for detecting low density lipoprotein cholesterin through double-enzyme concerted catalysis silver deposition Download PDFInfo
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- 229910052709 silver Inorganic materials 0.000 title claims abstract description 32
- 239000004332 silver Substances 0.000 title claims abstract description 32
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000008021 deposition Effects 0.000 title claims abstract description 24
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 21
- 230000002153 concerted effect Effects 0.000 title claims abstract description 19
- 108010007622 LDL Lipoproteins Proteins 0.000 title claims abstract description 15
- 102000007330 LDL Lipoproteins Human genes 0.000 title claims abstract description 15
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title abstract description 18
- HVYWMOMLDIMFJA-UHFFFAOYSA-N 3-cholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 HVYWMOMLDIMFJA-UHFFFAOYSA-N 0.000 title abstract 3
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 238000000151 deposition Methods 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 108010089254 Cholesterol oxidase Proteins 0.000 claims abstract description 8
- -1 gold ions Chemical class 0.000 claims abstract description 7
- 108010028554 LDL Cholesterol Proteins 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 238000013019 agitation Methods 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 24
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 11
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 10
- 102000006991 Apolipoprotein B-100 Human genes 0.000 claims description 9
- 108010008150 Apolipoprotein B-100 Proteins 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 9
- 238000003950 stripping voltammetry Methods 0.000 claims description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 claims description 6
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 claims description 6
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000005498 polishing Methods 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical class NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 claims description 5
- 241000209094 Oryza Species 0.000 claims description 5
- 235000007164 Oryza sativa Nutrition 0.000 claims description 5
- 235000013339 cereals Nutrition 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- 238000007654 immersion Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 3
- 238000004070 electrodeposition Methods 0.000 claims description 3
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 101710134784 Agnoprotein Proteins 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 239000010931 gold Substances 0.000 abstract description 7
- 235000012000 cholesterol Nutrition 0.000 abstract description 6
- 229910052737 gold Inorganic materials 0.000 abstract description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 5
- 238000006116 polymerization reaction Methods 0.000 abstract description 4
- 239000003638 chemical reducing agent Substances 0.000 abstract 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 abstract 1
- 102000007592 Apolipoproteins Human genes 0.000 abstract 1
- 108010071619 Apolipoproteins Proteins 0.000 abstract 1
- 108010055297 Sterol Esterase Proteins 0.000 abstract 1
- 102000000019 Sterol Esterase Human genes 0.000 abstract 1
- 238000004873 anchoring Methods 0.000 abstract 1
- 229920005597 polymer membrane Polymers 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
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- 201000001320 Atherosclerosis Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000008214 LDL Cholesterol Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
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- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
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- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- ZOSTZYMLOPBGQI-NKWVEPMBSA-N [[(2r)-2-[(1r)-1-(6-aminopurin-9-yl)-2-oxoethoxy]-3-oxopropoxy]-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound NC1=NC=NC2=C1N=CN2[C@H](O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C=O)C=O ZOSTZYMLOPBGQI-NKWVEPMBSA-N 0.000 description 1
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 description 1
- 229940043377 alpha-cyclodextrin Drugs 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 229960000633 dextran sulfate Drugs 0.000 description 1
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- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
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- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008012 small dense LDL Proteins 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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Classifications
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- 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
-
- 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
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting low density lipoprotein cholesterin through double-enzyme concerted catalysis silver deposition. The method comprises the following steps: firstly, forming a sulfydryl-containing poly-aniline phenthiol film on an electrode surface through an electric polymerization method; performing electroreduction on gold ions on the electrode surface through a constant potential deposition method, thereby forming nanometer gold; anchoring the nanometer gold on the electrode surface through sulfydryl on an electric polymer membrane; fixing an apolipoprotein apoB-100 antibody on the nanometer gold; and via identification of the apoB-100 antibody on the specificity of low density lipoprotein, capturing the low density lipoprotein on the electrode surface. Under the synergistic effect of two enzymes of cholesterol esterase and cholesterol oxidase, cholesterol in the low density lipoprotein is decomposed and a weak reducing agent H2O2 is generated; the reducing agent can reduce and deposit silver ions on the gold nanometer grain surface; lastly, a standard curve is drawn by detecting a dissolving volt-ampere current value of an elemental silver, and the detection for the low density lipoprotein cholesterin can be realized.
Description
Technical field
The invention belongs to technical field of biological, relate to a kind of double enzyme concerted catalysis deposition of silver detection low density lipoprotein
The method of protein cholesterol.
Background technology
Low-density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-C), is tremulous pulse
The main component of atherosclerotic plaque, is the chief-criminal causing atherosclerotic angiopathy.Clinical research is demonstrate,proved
Real, LDL-C raises and atherosclerosis, coronary heart disease sickness rate between being proportionate property.Therefore, accurate
Really measure the content of LDL-C in serum, for the medicals diagnosis on disease such as atherosclerosis, hypertension, coronary heart disease,
Prevent and treat significant.Detection method mainly has supercentrifugation, electrophoresis method, chemistry or immunoprecipitation
Methods etc., and the sedimentation method that clinical laboratory uses at present, its detection is had bigger by the content of Triglycerides in Serum
Interference.Recently, John J etc. reports a kind of homogeneous method detection LDL-C, its detection sensitivity and specificity
Be greatly improved (John J, Albers, Hal Kennedy, Santica M, Marcovina.Evaluation of
a new homogenous method for detection of small dense LDL cholesterol:Comparison
with the LDL cholesterol profile obtained by density gradient ultracetrifugation[J].
Clinica ChimicaActa.412.556-561(2011))；And cross limit base first-class and utilize following three kinds of reagent or examination
Any one of agent group: (1) sulphuric acid alpha-cyclodextrin, dextran sulfate, magnesium ion, polyoxyethylene-polyoxy
Polypropylene block copolyether reagent set；(2) amphoteric surfactant and have carboxyl or sulfonic aliphatic amine examination
Agent group；(3) polycationic agents.A kind of cholesterol sensor and cholesterol is constructed together with oxidoreductase
Quantitative approach (cross limit base one, Tang Chuan system, South Sea history youth. cholesterol sensor and the quantitative square of cholesterol
Method. China, patent of invention, authorize time: 2005.02.02, granted patent number: 00802824.9).These sides
Method instrument is expensive, it is complicated, time-consuming to operate and technology requires height, needs to set up a kind of quick, sensitive, behaviour
Make easy LDL-C detection method.
Summary of the invention
It is an object of the invention to provide a kind of double enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol
Method, solve tradition LDL-C detection method somewhat expensive, operate complicated, time-consuming and technology and require height
Problem.
The technical solution adopted in the present invention is to follow the steps below:
Step 1: pretreatment of glassy carbon electrode；
(1) Al is used2O3Suspension is by glassy carbon electrode surface sanding and polishing to minute surface, and the electrode after polishing is successively pure
Magnetic agitation washing in water, dehydrated alcohol, pure water；
(2) electrode is placed in piraha solution immersion, with pure water rinsing clean after again in pure water magnetic agitation wash
Wash；
(3) electrode is placed in H2SO4In be circulated voltammetric scan activated electrode surface after activated after glass
Carbon electrode is clean by pure water rinsing；
Step 2: the modification of electrode；
(1) the glass-carbon electrode immersion after activation is circulated volt-ampere containing near amino thiophenols and perchloric acid solution
Scanning, carries out magnetic agitation washing with pure water after having scanned；
(2) HCl, KCl, HAuCl are put the electrodes into4Liquid at the bottom of the deposition of composition carries out potentiostatic electrodeposition, deposition
Carry out magnetic agitation washing with pure water after completing, obtain having modified the glass-carbon electrode of Jenner's grain of rice.
Step 3: the structure at bio-sensing interface
(1) apoB-100 antibody drops to modify the glassy carbon electrode surface of Jenner's grain of rice, hatches 0.5-2 hour,
With with pure water, loose antibody is washed away after having hatched, carry out with glycine-NaOH buffer
Magnetic agitation is washed；
(2) BSA solution capping is dripped 0.5-2 hour at electrode surface；
(3) magnetic agitation washing is carried out with the glycine-NaOH buffer solution containing BSA；
The Specification Curve of Increasing of step 4:LDL-C
(1) the electrode surface dropping LDL solution securing ApoB-100 antibody obtained in step 3 is incubated
Educate, carry out magnetic agitation washing with glycine-NaOH buffer and LDL at large is washed away；
(2) at electrode surface dropping cholesteryl esterase (CHER), cholesterol oxidase (CHOD) and AgNO3
Solution, lucifuge hatches the working electrode obtaining depositing elemental silver, this electrode glycine-NaOH
Buffer carries out magnetic agitation washing；
(3) KNO is put the electrodes into3In solution, carry out linear scanning, the Stripping Voltammetry peak of record Ag；
(4) according to the Stripping Voltammetry current value size of elemental silver, LDL-C standard curve, meter sensitivity are drawn
With detection limit.
Step 5: the detection of testing sample
(1) electrode surface securing ApoB-100 antibody obtained in step 3 drips 15 μ L testing samples
Hatch, carry out magnetic agitation washing with glycine-NaOH buffer；
(2) in electrode surface dropping containing CHER enzyme, CHOD enzyme and AgNO3Solution, lucifuge is hatched
Have the working electrode of elemental silver to deposition, this electrode glycine-NaOH buffer carries out magnetic force and stirs
Mix washing；
(3) working electrode is put into KNO3In solution, carry out linear scanning, sweep limits 0.0～+0.8V,
Sweep speed is 50-200mV/s, the Stripping Voltammetry current value of record Ag；
(4) according to standard curve described in step 4, the concentration of LDL-C in described testing sample solution is obtained.
Further, Al in described step 12O3Suspension granular size is 0.3 μm and 0.05 μm.
Further, in described step 1, piraha solution is the 30%H of volume ratio 3:72O2And 98%H2SO4
Mixed configuration.
Further, electrode is placed in H by described step 12SO4In be circulated voltammetric scan after, rush with pure water
After wash clean, then electrode is placed in the potassium ferricyanide/potassium ferrocyanide solution be circulated respectively voltammetric scan and
AC impedance scans, and finally dries standby by pure water rinsing.
Further, in described step 2, near amino thiophenols concentration is 5mmol/L, and perchloric acid concentration is 1
mol/L。
Further, described step 2 deposits end liquid respectively by 0.01mol/L HCl, 0.1mol/L of same volume
KCl、10μmol/L HAuCl4Composition.
Further, described incubation temperature is 37 DEG C.
Further, described glycine-NaOH is the glycine 0.1mol/L-NaOH buffer of pH8.6.
Further, the linear scanning scope 0.0～+0.8V in described step 4 and step 5, sweep speed is
50-200mV/s。
Wherein, step 1 provides a fresh electrode surface for step 2, complete, stable, firm for formation,
The high-molecular polymerization membrane of good conductivity provides condition.In step 2, high-molecular polymerization membrane is formed as raw in step 3
The fixing offer site of thing identification molecule apoB-100 antibody, thus constitute the biological biography of specific recognition LDL
Sense interface, and the transmission of beneficially electronics.In step 3, bio-sensing interface is configured to LDL-C in step 4
Electrochemical Detection in requisite committed step.Visible step 1-4 mutually supports, and jointly acts on, ability
Double enzyme concerted catalysis deposition of silver reaction is utilized to realize Electrochemical Detection LDL-C.
The present invention set up detection LDL-C method have the beneficial effects that simple to operate, the detection time is short, it is easy to
Miniaturization.
Accompanying drawing explanation
Fig. 1 electrochemical method schematic diagram based on double enzyme concerted catalysis deposition of silver reaction principles detection LDL-C；
The cyclic voltammetric phenogram of Fig. 2 electrode surface different modifying process；
The AC impedance phenogram of Fig. 3 electrode surface different modifying process；
Fig. 4 (a) sensor response current under variable concentrations LDL-C；
The working curve of Fig. 4 (b) sensor.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in detail.Fig. 1 is based on double enzyme concerted catalysis silver
The electrochemical method schematic diagram of deposition reaction principle detection LDL-C.Fig. 2 is electrode surface different modifying process
Cyclic voltammetric phenogram；Fig. 3 is the AC impedance phenogram of electrode surface different modifying process.Implement step
As follows:
1. the pretreatment of electrode: be 0.3 μm and the Al of 0.05 μm by granular size2O3Suspension is by glass carbon
Electrode surface sanding and polishing is to minute surface, and the electrode after polishing distinguishes magnetic agitation successively in pure water, dehydrated alcohol
Washing 5min, by pure water rinsing, in pure water, magnetic agitation washs 5min again；Electrode is placed in volume ratio 3:7
30%H2O2And 98%H2SO4The piraha solution of preparation soaks 1min, with pure water rinsing clean after again
Magnetic agitation washing 5min；Electrode is placed in 0.5M H2SO4In be circulated under-0.3～+1.5V current potential
Voltammetric scan 10min activated electrode surface, sweep limits-0.3～+1.5V, sweep speed is 50-100mV/s,
Clean by pure water rinsing；Electrode is placed in the potassium ferricyanide/potassium ferrocyanide solution and is circulated volt-ampere respectively and sweeps
Retouch (-0.5～+1.0V, 50mV/s) and AC impedance scanning (0.24V, 1 × 105Hz, 0.1Hz), finally
Dry standby by pure water rinsing.
2. the modification of electrode: the glass-carbon electrode after activation is dipped vertically into (oATP) containing near amino thiophenols (dense
Degree is for 5mmol/L) and perchloric acid (concentration is 1mol/L) solution in be circulated voltammetric scan and (scan model
Enclosing 0.0～+0.8V, sweep speed is 10-100mV/s, and the scanning number of turns is 40), use pure water after having scanned
Carry out magnetic agitation to wash 5 minutes；Put the electrodes into 1mL deposition end liquid and (consist of same volume: 0.01
mol/L HCl、0.1mol/L KCl、10μmol/L HAuCl4Potentiostatic electrodeposition (sedimentation potential is carried out in)
For-0.5V, sedimentation time is the 100-150 second), carry out magnetic agitation with pure water after having deposited and wash 5 minutes,
I.e. obtain having modified the glass-carbon electrode of Jenner's grain of rice.
3. fix ApoB-100 antibody: drip 10 μ l20 μ at the electrode surface having modified nano Au particle
The ApoB-100 antibody of g/ml, hatches 1h in 37 DEG C, is washed by loose antibody with pure water after having hatched
Go, carry out magnetic agitation washing 5min with the glycine-NaOH buffer of pH8.6；Drip at electrode surface
The BSA solution of 10 μ l1%, with enclosed-electrode surface non-specific adsorption sites, closes at 37 DEG C
Reaction 1h, delays with glycine (the 0.1mol/L)-NaOH of the pH8.6 containing BSA (0.1%) after having reacted
Rush liquid to rinse well.
4. detect the standard curve of LDL-C: drip 10 μ at the electrode surface securing ApoB-100 antibody
LLDL solution, hatches 30min at 37 DEG C, after immunoreation completes, with the glycine (0.1 of pH8.6
Mol/L) LDL at large is washed away by-NaOH buffer；5 μ l 100 μ g/ml are dripped at electrode surface
CHER enzyme liquid, 5 μ l100 μ g/ml CHOD enzyme liquid, 10 μ l5mMAgNO3Solution, in 37 DEG C
Under hatch 30min, obtain depositing the working electrode of elemental silver, enzymic catalytic reaction complete after sweet with pH8.6
Propylhomoserin-NaOH buffer carries out magnetic agitation washing 5min；At KNO3(1mol/L) in solution, with washing
After electrode be working electrode, platinum electrode for being reference electrode to electrode, saturated calomel electrode, in electrochemistry work
Making the enterprising line linearity surface sweeping LSV that stands, sweep limits is-0.5～0.6V, and sweep speed 50mV/s obtains list
The Stripping Voltammetry current value of matter silver.Stripping Voltammetry current value (Y) size of elemental silver is at 10ng/mL-1000
In the range of ng/mL linear with the concentration of LDL-C (X), its linear equation is:
Y=0.9459+0.4413X, linearly dependent coefficient R2=0.9982.Fig. 4 is based on double enzyme concerted catalysis deposition of silver
The electrochemical method standard curve of reaction principle detection LDL-C, wherein Fig. 4 (a) sensor is at variable concentrations
Response current under LDL-C；The working curve of Fig. 4 (b) sensor.
5. the detection of LDL-C in actual sample: securing 10 μ l20 μ g/ml ApoB-100 antibody
Electrode surface, add 10 μ L LDL-C solution to be measured, at 37 DEG C, hatch 30min, immunoreation is complete
Cheng Hou, washes away LDL at large with the glycine-NaOH buffer of pH8.6；Drip at electrode surface
Add 5 μ l100 μ g/ml CHER enzyme liquid, 5 μ l 100 μ g/ml CHOD enzyme liquid, 10 μ l5mM
AgNO3Solution, hatches 30min at 37 DEG C and obtains depositing the working electrode of elemental silver, enzymic catalytic reaction
Magnetic agitation washing 5min is carried out with the glycine-NaOH buffer of pH8.6 after completing；At 1mol/L KNO3
In solution, with the electrode after washing as working electrode, platinum electrode be to be that reference is electric to electrode, saturated calomel electrode
Pole, scans at the enterprising line linearity of electrochemical workstation, and sweep limits is-0.5～0.6V, sweep speed 50mV/s,
The Stripping Voltammetry current-responsive value obtaining elemental silver is respectively (23.6 μ A, 217.3 μ A, 395.7 μ A).
Standard curve Y=0.9459+0.4413X according to step 4, LDL-C in available corresponding actual sample solution
Concentration is respectively (51.3ng/mL, 490.3ng/mL, 894.5ng/mL).
The present invention compared with prior art has the advantage that
1. using double enzyme concerted catalysis deposition of silver principle to realize Electrochemical Detection LDL-C, the detection time is short, background
Disturbing little, detection sensitivity is high.
2. high-molecular polymerization membrane can be the fixing offer site of biological identification molecule apoB-100 antibody, and is conducive to electricity
The transmission of son.
3. the immunoreation carried out on gold nano-material surface is a kind of interfacial reaction system, can improve ApoB-100
Antibody and the reaction efficiency of LDL
The above is only the better embodiment to the present invention, not makees the present invention any pro forma
Limiting, every any simple modification done embodiment of above according to the technical spirit of the present invention, equivalent becomes
Change and modify, belonging in the range of technical solution of the present invention.
Claims (9)
1. the method for double enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol, it is characterised in that press
Carry out according to following steps:
Step 1: pretreatment of glassy carbon electrode；
(1) Al is used2O3Suspension is by glassy carbon electrode surface sanding and polishing to minute surface, and the electrode after polishing is successively
Magnetic agitation washing in pure water, dehydrated alcohol, pure water；
(2) electrode is placed in piraha solution immersion, with pure water rinsing clean after again in pure water magnetic force stir
Mix washing；
(3) electrode is placed in H2SO4In be circulated voltammetric scan activated electrode surface after activated after
Glass-carbon electrode is clean by pure water rinsing；
Step 2: the modification of electrode；
(1) the glass-carbon electrode immersion after activation is circulated volt containing near amino thiophenols and perchloric acid solution
Peace scanning, carries out magnetic agitation washing with pure water after having scanned；
(2) HCl, KCl, HAuCl are put the electrodes into4Liquid at the bottom of the deposition of composition carries out potentiostatic electrodeposition,
Carry out magnetic agitation washing with pure water after having deposited, obtain having modified the glass-carbon electrode of Jenner's grain of rice.
Step 3: the structure at bio-sensing interface
(1) apoB-100 antibody drops to modify the glassy carbon electrode surface of Jenner's grain of rice, hatch 0.5-2
Hour, with pure water, loose antibody is washed away after having hatched, carry out with glycine-NaOH buffer
Magnetic agitation is washed；
(2) BSA solution capping is dripped 0.5-2 hour at electrode surface；
(3) magnetic agitation washing is carried out with the glycine-NaOH buffer solution containing BSA；
The Specification Curve of Increasing of step 4:LDL-C
(1) the electrode surface dropping LDL solution securing ApoB-100 antibody obtained in step 3 is carried out
Hatch, carry out magnetic agitation washing with glycine-NaOH buffer and LDL at large is washed away；
(2) cholesteryl esterase, cholesterol oxidase and AgNO are dripped at electrode surface3Solution, lucifuge is incubated
Educating the working electrode obtaining depositing elemental silver, this electrode glycine-NaOH buffer carries out magnetic agitation
Washing；
(3) KNO is put the electrodes into3In solution, carry out linear scanning, the Stripping Voltammetry peak of record Ag；
(4) according to the Stripping Voltammetry current value size of elemental silver, draw LDL-C standard curve, calculate sensitive
Degree and detection limit.
Step 5: the detection of testing sample
(1) electrode surface securing ApoB-100 antibody obtained in step 3 drips 15 μ L and treats test sample
Product are hatched, and carry out magnetic agitation washing with glycine-NaOH buffer；
(2) in electrode surface dropping containing CHER enzyme, CHOD enzyme and AgNO3Solution, lucifuge is incubated
Educating the working electrode obtaining depositing elemental silver, this electrode glycine-NaOH buffer carries out magnetic agitation
Washing；
(3) working electrode is put into KNO3In solution, carry out linear scanning, sweep limits 0.0～+0.8V,
Sweep speed is 50-200mV/s, the Stripping Voltammetry current value of record Ag；
(4) according to standard curve described in step 4, the concentration of LDL-C in described testing sample solution is obtained.
2. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: Al in described step 12O3Suspension granular size is 0.3 μm and 0.05 μm.
3. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: in described step 1, piraha solution is the 30%H of volume ratio 3:72O2With 98%
H2SO4Mixed configuration.
4. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: electrode is placed in H by described step 12SO4In be circulated voltammetric scan after,
With pure water rinsing clean after, then electrode is placed in the potassium ferricyanide/potassium ferrocyanide solution and is circulated volt respectively
Peace scanning and AC impedance scan, and finally dry standby by pure water rinsing.
5. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: in described step 2, near amino thiophenols concentration is 5mmol/L, and perchloric acid is dense
Degree is 1mol/L.
6. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: in described step 2 deposit end liquid respectively by the 0.01mol/L HCl of same volume, 0.1
mol/L KCl、10μmol/L HAuCl4Composition.
7. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: described incubation temperature is 37 DEG C.
8. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: described glycine-NaOH is the glycine 0.1mol/L-NaOH buffering of pH8.6
Liquid.
9. according to a kind of pair of enzyme concerted catalysis deposition of silver detection low-density lipoprotein cholesterol described in claim 1
Method, it is characterised in that: the linear scanning scope 0.0～+0.8V in described step 4 and step 5, scanning
Speed is 50-200mV/s.
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| CN107664659A (en) * | 2017-09-07 | 2018-02-06 | 桂林电子科技大学 | A kind of method of enzyme and graphene concerted catalysis deposition of silver cholesterol detection |
| CN110146578A (en) * | 2019-06-03 | 2019-08-20 | 桂林电子科技大学 | A method of based on RGO-CS-Fc/Pt NPs nanocomposite cholesterol detection |
| CN110455883A (en) * | 2019-08-26 | 2019-11-15 | 浙江大学山东工业技术研究院 | A kind of stepwise reaction formula electrochemical detection method and device |
| CN111721822A (en) * | 2020-06-29 | 2020-09-29 | 山东理工大学 | Preparation method and application of electrochemical sensor based on AuNPs @ rGO @ PS NSs |
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Cited By (7)
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|---|---|---|---|---|
| CN107664659A (en) * | 2017-09-07 | 2018-02-06 | 桂林电子科技大学 | A kind of method of enzyme and graphene concerted catalysis deposition of silver cholesterol detection |
| CN107664659B (en) * | 2017-09-07 | 2020-02-07 | 桂林电子科技大学 | Method for detecting cholesterol by cooperatively catalyzing silver deposition with enzyme and graphene |
| CN110146578A (en) * | 2019-06-03 | 2019-08-20 | 桂林电子科技大学 | A method of based on RGO-CS-Fc/Pt NPs nanocomposite cholesterol detection |
| CN110146578B (en) * | 2019-06-03 | 2021-09-17 | 桂林电子科技大学 | Method for detecting cholesterol based on RGO-CS-Fc/Pt NPs nano composite material |
| CN110455883A (en) * | 2019-08-26 | 2019-11-15 | 浙江大学山东工业技术研究院 | A kind of stepwise reaction formula electrochemical detection method and device |
| CN111721822A (en) * | 2020-06-29 | 2020-09-29 | 山东理工大学 | Preparation method and application of electrochemical sensor based on AuNPs @ rGO @ PS NSs |
| CN111721822B (en) * | 2020-06-29 | 2022-08-19 | 山东理工大学 | Preparation method and application of electrochemical sensor based on AuNPs @ rGO @ PS NSs |
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