CN103995103B - Method based on Prussian blue bionical marker detection micromolecular compound - Google Patents
Method based on Prussian blue bionical marker detection micromolecular compound Download PDFInfo
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- CN103995103B CN103995103B CN201410258017.7A CN201410258017A CN103995103B CN 103995103 B CN103995103 B CN 103995103B CN 201410258017 A CN201410258017 A CN 201410258017A CN 103995103 B CN103995103 B CN 103995103B
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The present invention relates to electro-chemistry immunity technical field, be a kind of method based on Prussian blue bionical marker detection micromolecular compound.Comprise the following steps: there is the analog of the active Prussian blue micropartical labelling determinand of catalase-like catalysis as labelled antigen using a kind of；By magnetic ferroferric oxide microgranule sessile antibody, and it is dispersed in reactant liquor；Form the oxidation current signal in competitive immunization reaction, Electrochemical Detection system and then determinand is realized quantitative measurement.This detection method uses Prussian blue replacement enzyme molecular marker small molecule antigens, and combine magnetic particle being at war with property immunoreation, by Electrochemical Detection labelled antigen for hydrogen peroxide and hydroquinone catalytic oxidation produce the signal of telecommunication, owing to its signal of telecommunication size is the most relevant to little molecular concentration to be measured, the detection of the high sensitivity quantitation to micromolecular compound can be realized.
Description
Technical field
The present invention relates to electro-chemistry immunity technical field, be a kind of based on Prussian blue bionical mark
The method of note detection micromolecular compound.
Background technology
At current biological medicine detection field, for environmental contaminants, pesticide, hormone,
The high sensitivity quantitation immune detection of the little molecule such as antibiotic has become vast research worker and has paid close attention to
One of focal issue.In order to realize the high sensitivity of the micromolecular compound of trace or trace
Detection, various based on biologic specificity reaction, such as antigen-antibody techniques, protein ligands technology
Have been set up etc. different methods and system.Wherein, have typicality is that competitiveness is exempted from
Epidemic disease detection technique, it utilizes labelled antigen and the competition of test analyte antagonist, by inspection
Survey the labelled antigen that adsorbed by antibody and can be inferred that the dense of the test analyte that contains in system
Degree.But conventional labelling technique has radioactive label, horseradish peroxidase (HRP) etc.
Enzyme labelling, and the optical markings such as fluorescence chemical probe.
Electrochemical immunoanalytical technology is immuno analytical method be combined with electrochemical techniques
Plant immune analysis method, by some are prone to measure and have the material mark of high susceptibility
Remember on antigen or antibody molecule, and the specific binding reaction between conjugated antigen and antibody,
The signal of telecommunication producing or amplifying by detection label, it is possible to achieve for test substance
High sensitivity specific detection.The material of electro-chemistry immunity labelling is usually biological enzyme, as
Horseradish peroxidase, alkaline phosphatase, glucoseoxidase etc., but these enzymes pair
Require strict in the external condition such as temperature, solvent, it is difficult to prepare and expensive.
Prussian blue microgranule has the catalytic capability of similar peroxidase, can be catalyzed peroxide
Change the decomposition of hydrogen, detect its hydrogen peroxide decomposed to reproducibility thing by electrochemical workstation
The electric current that matter hydroquinone oxidation produces.By electrostatic interaction, can be by Prussian blue labelling
The equimolecular surface of chitosan at band amino, then by chemical conjugation methods by little point to be measured
Son and the chitosan molecule coupling with Prussian blue labelling, can prepare Prussian blue labelling
Small molecule tags antigen.
Magnetic nano-particle has features such as big, the easy enrichment of specific surface area, modified after permissible
At its surface marker antibody, prepare Magnetic antibody.By being dispersed in reaction system and additional
The effect in magnetic field, can separate the micromolecular compound with antibody specificity identification with fast enriching.
Therefore, foundation combines the reaction of magnetic particle competitive immunization and electrification based on biomimetic material labelling
The micromolecular compound detection system learned a skill, for environmental contaminants, pesticide, hormone,
The high sensitivity quantitation immune detection of the little molecule such as antibiotic is significant.
Summary of the invention
In view of this, the invention provides one based on little point of Prussian blue bionical marker detection
The method detection method of sub-compound, with realize for environmental contaminants, pesticide, hormone,
The purpose of the high sensitivity quantitation immune detection of the little molecule such as antibiotic.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme: based on general Shandong
The method of the blue bionical marker detection micromolecular compound of scholar, it is characterised in that include following step
Rapid:
A) treat with a kind of Prussian blue micropartical labelling with catalase-like catalysis activity
Survey the analog of thing as labelled antigen；
B) by magnetic ferroferric oxide microgranule sessile antibody, and it is dispersed in reactant liquor；
C) after adding the labelled antigen prepared in step a and determinand in reactant liquor, labelling
Antigen competes antibody with determinand, forms competitive immunization reaction；
D) separated by Magnet, the antigen of Magnetic antibody and absorption thereof separated to test tube,
Draw supernatant, add containing hydroquinone and the solution of hydrogen peroxide wherein, pass through difference
Pulse voltammetry, oxidation current signal in Electrochemical Detection system and then determinand is realized
Quantitative measurement.
Optionally, described micromolecular compound refers to that molecular weight, and can not be straight below 1000
Practice midwifery and give birth to immunogenic organic compound molecule.
Optionally, described ferroferric oxide particle and Prussian blue atomic size are μm
Level or nm level.
Optionally, the material described in catalase-like catalysis activity is Prussian blue.
Optionally, described determinand is organic micromolecule compound.
Optionally, in the step that described electrochemistry is examined, electrochemical electrode be glass-carbon electrode and
Electrode after it is modified.
Optionally, described Prussian blue micropartical fixes the little molecule of determinand by chemical method
Analog, formed labelled antigen.
Optionally, the reaction of described competitive immunization occurs in phosphate buffer.
Optionally, described competitive immunization reacts by by magnetic particle and externally-applied magnetic field
Effect formed.
Oxidation current letter optionally, in step d), in described Electrochemical Detection system
Number so determinand is realized quantitative measurement, specifically include:
By the labelled antigen of residual in detection supernatant for containing hydrogen peroxide and hydroquinone
The signal of telecommunication that produces of the catalytic oxidation of solution, it is achieved quantitative for little molecule to be measured
Electrochemical Detection.
This detection method uses Prussian blue replacement enzyme molecular marker small molecule antigens, and
In conjunction with magnetic particle being at war with property immunoreation, by Electrochemical Detection labelled antigen for
The signal of telecommunication that the catalytic oxidation of hydrogen peroxide and hydroquinone produces, due to its signal of telecommunication
Size is the most relevant to little molecular concentration to be measured, can realize the Gao Ling to micromolecular compound
Sensitivity detection by quantitative.
Accompanying drawing explanation
Fig. 1 is in the method for the embodiment of the present invention, Prussian blue catalysis immunity electrochemistry difference
Pulse Voltammetry figure；
Fig. 2 utilizes the linear relationship of concentration that the method for the embodiment of the present invention surveyed and electric current
Figure；
Fig. 3 utilizes the logarithm of the concentration that the method for the embodiment of the present invention surveyed and the linear of electric current
Graph of a relation.
Detailed description of the invention
The present invention proposes and a kind of utilizes Prussian blue catalysis activity as the competition of antigenic mark
Immunoreation electrochemical detection method.This method first by the chitosan with multiple amino,
Prussian blue particle label constitutes on the analog of micromolecular compound to be measured labelling resist
Former.By by antibody and magnetic nano-particle coupling, by labelled antigen and object to be measured
Competition and externally-applied magnetic field to Magnetic antibody resist for Magnetic antibody combining target thing and labelling
Separation after former, constructs competitive immunoreaction system.Then by Electrochemical Detection supernatant
The remaining labelled antigen catalytic decomposition to hydrogen peroxide and the oxidation to hydroquinone thereof in liquid
Electric current, establishes the highly sensitive detection system of micromolecular compound.
In addition, it is necessary to it is noted that in all embodiments of the invention, it is preferred that can
To possess one or more qualifications following；Optionally, described micromolecular compound is
Refer to that molecular weight, below 1000, and can not directly produce immunogenic organic compound and divides
Son.Optionally, described ferroferric oxide particle and Prussian blue atomic size are μm
Level or nm level.Optionally, the material described in catalase-like catalysis activity is
Prussian blue.Optionally, described determinand is organic micromolecule compound.Optionally,
In the step that described electrochemistry is examined, electrochemical electrode be glass-carbon electrode and modified after
Electrode.Optionally, described Prussian blue micropartical fixes little point of determinand by chemical method
The analog of son, forms labelled antigen.Optionally, described competitive immunization reacts at phosphoric acid
Buffer occurs.Optionally, described competitive immunization reaction by by magnetic particle with
And the effect of externally-applied magnetic field is formed.Optionally, in step d), described Electrochemical Detection
Oxidation current signal in system and then determinand is realized quantitative measurement, specifically includes: logical
Cross the labelled antigen remained in detection supernatant for the solution containing hydrogen peroxide and hydroquinone
Catalytic oxidation produce the signal of telecommunication, it is achieved for the quantitative electrochemical of little molecule to be measured
Detection.
Embodiment one
The detection method that the present embodiment provides comprises the following steps:
1, the preparation of Prussian blue labelling molecular antigen:
Amidation process will be passed through with the micromolecular compound of amino or carboxyl isoreactivity group
(EDC/NHS reaction), diazotising method, glutaraldehyde method etc. are coupled in chitosan molecule,
By regulation pH value, make marked the chitosan positively charged of micromolecular compound, then exist
Adding ferrous sulfate and the potassium ferricyanide in its dispersion liquid, its surface synthesizes electronegative Prussia
Indigo plant, realizes the Prussian blue labelling to small molecule antigens by electrostatic interaction.
2, the preparation of Magnetic antibody:
By ferrous chloride and ferric chloride ratio 1:2, it is separately added in water dissolving and is heated to
70 DEG C, add the sodium hydroxide solution of ferrous chloride molal quantity 8 times, mix rapidly, it is seen that
Generate the magnetic nano-particle of brownish black, be then slowly added dropwise and ferrous chloride equimolar number
Oleic acid, adds potassium permanganate solution, aoxidizes 24h, make magnetic nano particle after ripening 20min
The oleic acid of sub-surface is oxidized to carboxyl, with milli-Q water for several times, it is thus achieved that with carboxyl
Magnetic nano-particle.The magnetic nano-particle of band carboxyl is dispersed in phosphate buffer,
And add a certain amount of antibody, even with antibody by amidation process (EDC/NHS reaction)
Connection 2h.Afterwards, with 1% skimmed milk powder at 37 DEG C, hatch 1h, close unreacted
Carboxyl, washed once, and i.e. obtains Magnetic antibody.
3, electrochemical detection method:
The method is method based on Prussian blue bionical marker detection micromolecular compound, tool
Body comprises the following steps:
Step 1: with a kind of Prussian blue micropartical with catalase-like catalysis activity
The analog of labelling determinand is as labelled antigen:
Concrete, weigh a certain amount of hapten being modified with amino and be dissolved in the dd containing methanol
H2O constitutes I liquid；Weigh the chitosan that a certain amount of deacetylation is 90% and be dissolved in pH value 6.5
Phosphate buffer in, constitute II liquid；Measure the glutaraldehyde of 100 μ l25%, add distillation
Water is diluted to 5ml, constitutes III liquid.Under stirring at room temperature, I liquid and III liquid are dropwise added
Enter in II liquid and stir, load bag filter after 8h, dialyse 3d with distilled water, change 2 every day
To 3 dialysis solution to dialysis completely, the antigen (Ag-CS) of coupled chitosan is i.e. obtained.?
Wherein add 1ml1mol/l ferrous chloride and the hydrochloric acid of 2 ‰, add 1mol/l ferrum cyaniding
The formation of the Prussian blue particle of the i.e. visible blue of potassium, lyophilization, can obtain and marked
Prussian blue chitosan determinand molecular marker antigen (PB/CS/Ag).
Step 2: the magnetic nano-particle of anamorphic zone carboxyl, by magnetic ferroferric oxide microgranule
Sessile antibody, and be dispersed in reactant liquor: by 8.1g FeCl3·6H2O is at 142.5mL
After deionized water dissolves, transfer in there-necked flask, be heated with stirring to 70 DEG C.Weigh
4.4gFeCl2·4H2O is dissolved in 10mL deionized water, filters, takes 7.5mL and join three mouthfuls
In flask.With vigorous stirring, the NaOH solution of 24mL20%, 1min are rapidly joined
After be added dropwise over 4.66g oleic acid, continue quickly to stir 20min at 70 DEG C.Reaction terminates
After, obtain black sol shape material, utilize externally-applied magnetic field by the precipitation of gained from reaction system
In separate.
Remove unnecessary oleic acid 2 times by washing with alcohol, then be washed with deionized to pH=7.
It is subsequently adding the KMnO that 160mL concentration is 10mg/mL4Solution, in ultrasonic waves for cleaning
Sonic oscillation 24h in instrument, is washed with deionized after Magneto separate 3 times, obtains magnetic fluid.
Or vacuum lyophilization 40h after washing, must arrive surface and be modified with the magnetic Nano of carboxyl
Particle.The magnetic nano-particle of 25mg band carboxyl is dispersed in the PBS of 1mL PH=6.5
In phosphate buffer, it is separately added into 25mgEDC and NHS wherein, lives in 37 DEG C
Change 30min, and add a certain amount of antibody, by amidation process (EDC/NHS reaction)
With antibody coupling 2h.Afterwards, with 1% skimmed milk powder at 37 DEG C, hatch 1h, closing is fallen
Unreacted carboxyl, washed once, and i.e. obtains Magnetic antibody.
Step 3: after adding the labelled antigen prepared in step 1 and determinand in reactant liquor,
Labelled antigen competes antibody with determinand, forms competitive immunization reaction: contained by 100 μ L
25mg magnetic ferroferric oxide microgranule sessile antibody is dispersed in 5mL reactant liquor, in reaction
Liquid adds the labelled antigen and one group of variable concentrations determinand prepared in 100 μ L steps a
After (sulfamethazine), hatching 1h at 37 DEG C, labelled antigen is competed with determinand
Antibody, forms competitive immunization reaction.
Step 4: separated by Magnet, is separated the antigen of Magnetic antibody and absorption thereof to examination
Bottom pipe, draw 4mL supernatant, add 1mL and contain 77mg hydroquinone and 11 μ L30%
In the solution of hydrogen peroxide, with glass-carbon electrode as working electrode, platinum plate electrode is to electrode,
Saturated calomel electrode is that reference electrode leads to, and between-0.6～2.0V, passes through differential pulse voltammetry
Oxidation current signal in (DPV method) Electrochemical Detection system so determinand is realized fixed
Measurement (refer to Fig. 1).Found that between 20ng/mL～10 μ g/mL, electric current
Signal magnitude and target concentration to be measured present good linear relationship.
The reagent that the inventive method uses in implementation process includes following several composition:
Antibody and antigen molecule: antibody generally refer to can specific recognition environmental contaminants, pesticide,
The antibody of the little molecule such as hormone, antibiotic.Chitosan, the shell of deacetylation 70%～90% gathers
Sugar containing a large amount of amino, magnetic nano-particle (surface is with carboxyl), Prussian blue particle,
Glass-carbon electrode, platinum plate electrode, saturated calomel electrode, Prussian blue, determinand antigen molecule
And the like, hydrogen peroxide, hydroquinone, sodium nitrite, glutaraldehyde, potassium permanganate,
Ferric chloride hexahydrate, Iron dichloride tetrahydrate.
The device used includes: water-bath, electrochemical workstation, ultra-pure water water-making machine.
With glass-carbon electrode as working electrode, platinum plate electrode is to electrode, and saturated calomel electrode is
Reference electrode, uses differential pulse voltammetry, the oxidation current letter in Electrochemical Detection system
Number so determinand is realized quantitative measurement.
Concrete, with Prussian blue for labelling catalysis material, with ferriferrous oxide nano-particle
For magnetic particle, with sulfonamides antibody for detection antibody, sulfamethazine is to be measured point
Analysis thing, as a example by sulfanilamide is to the analog that dimethoxypyridin is test analyte, illustrates we
Method be embodied as flow process.
1, the test analyte of Prussian blue labelling, analog are as the synthesis side of labelled antigen
Method:
Weigh 28mg sulfanilamide and dimethoxypyridin is dissolved in the 1ml dd containing 50 μ l methanol
H2O constitutes I liquid；Weigh the chitosan that 20mg deacetylation is 90% and be dissolved in 0.35ml PH
In the phosphate buffer of value 6.5, constitute II liquid；Measure the glutaraldehyde of 100 μ l25%,
Add distilled water diluting to 5ml, composition III liquid.Under stirring at room temperature, by I liquid and III liquid
It is added dropwise in II liquid stirring, loads bag filter after 8h, dialyse 3d with distilled water, every day
Change 2 to 3 dialysis solution to dialysis completely, i.e. obtain the sulfanilamide dimethyl of coupled chitosan
Pyrimidine (SD-CS).Add 1ml1mol/l ferrous chloride and the hydrochloric acid of 2 ‰ wherein, then
Add the formation of the Prussian blue particle of the i.e. visible blue of the 1mol/l potassium ferricyanide, freezing dry
Dry, can obtain and marked Prussian blue chitosan determinand molecular marker antigen
(PB/CS/SD)。
2, the synthesis of Fe 3 O 4 magnetic antibody:
By 8.1g FeCl3·6H2After O dissolves in 142.5mL deionized water, transfer to three
In mouth flask, it is heated with stirring to 70 DEG C.Weigh 4.4gFeCl2·4H2O be dissolved in 10mL go from
Sub-water, filters, takes 7.5mL and join in there-necked flask.With vigorous stirring, quickly add
Enter the NaOH solution of 24mL20%, after 1min, be added dropwise over 4.66g oleic acid, in 70 DEG C
20min is quickly stirred in lower continuation.After reaction terminates, obtain black sol shape material, utilize
The precipitation of gained is separated from reaction system by externally-applied magnetic field.Remove for 2 times by washing with alcohol
Remove unnecessary oleic acid, then be washed with deionized to pH=7.It is subsequently adding 160mL concentration
KMnO for 10mg/mL4Solution, sonic oscillation 24h in ultrasonic washing instrument,
It is washed with deionized after Magneto separate 3 times, obtains magnetic fluid.Or vacuum is cold after washing
The dry 40h of lyophilizing, must arrive surface and be modified with the magnetic nano-particle of carboxyl.By 25mg band carboxyl
Magnetic nano-particle be dispersed in the PBS phosphate buffer of 1mL PH=6.5, at it
In be separately added into 25mgEDC and NHS, 37 DEG C activate 30min, and add certain
The antibody of amount, by amidation process (EDC/NHS reaction) and antibody coupling 2h.It
After, with 1% skimmed milk powder at 37 DEG C, hatch 1h, close unreacted carboxyl, wash
Wash once, i.e. obtain Magnetic antibody.
3, the carrying out of competitive immunoreaction and Electrochemical Detection:
100 μ L are contained 25mg magnetic ferroferric oxide microgranule sessile antibody and is dispersed in 5mL
In reactant liquor, the labelled antigen and the one group of variable concentrations that add 100 μ L in reactant liquor are to be measured
After thing (sulfamethazine), hatching 1h at 37 DEG C, labelled antigen is competing with determinand
Strive antibody, form competitive immunization reaction.Separated by Magnet, by Magnetic antibody and suction thereof
Attached antigen separates to test tube, draws 4mL supernatant, adds 1mL and contains 77mg
In the solution of hydroquinone and 11 μ L30% hydrogen peroxide. with glass-carbon electrode as working electrode,
Platinum plate electrode is to electrode, and saturated calomel electrode is reference electrode, between-0.6～2.0V, logical
The oxidation current signal crossed in differential pulse voltammetry (DPV method) Electrochemical Detection system enters
And determinand is realized quantitative measurement (refer to Fig. 2 and Fig. 3, wherein, in fig. 2,
Abscissa is electric current, and vertical coordinate is concentration；In figure 3, abscissa is electric current, vertical coordinate
The logarithm of concentration).Found that between 20ng/mL～10 μ g/mL, current signal is big
Little and target concentration to be measured presents good linear relationship.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention,
For a person skilled in the art, the present invention can have various modifications and variations.All
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made,
Should be included within the scope of the present invention.
Claims (8)
1. method based on Prussian blue bionical marker detection micromolecular compound, its feature exists
In, comprise the following steps:
A) treat with a kind of Prussian blue micropartical labelling with catalase-like catalysis activity
Survey the analog of thing as labelled antigen；
B) by magnetic ferroferric oxide microgranule sessile antibody, and it is dispersed in reactant liquor；
C) after adding the labelled antigen prepared in step a and determinand in reactant liquor, labelling
Antigen competes antibody with determinand, forms competitive immunization reaction；
D) separated by Magnet, the antigen of Magnetic antibody and absorption thereof separated to test tube,
Draw supernatant, add containing hydroquinone and the solution of hydrogen peroxide wherein, pass through difference
Pulse voltammetry, oxidation current signal in Electrochemical Detection system and then determinand is realized
Quantitative measurement；In the step that described electrochemistry is examined, electrochemical electrode be glass-carbon electrode and
Electrode after modified.
2. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that:
Described micromolecular compound refers to that molecular weight, below 1000, and can not directly produce and exempts from
The organic compound molecule of epidemic focus.
3. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that:
Described ferroferric oxide particle and Prussian blue atomic size are μm level or nm
Level.
4. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that: described determinand is organic micromolecule compound.
5. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that: described Prussian blue micropartical is treated by chemical method is fixing
Survey the analog of the little molecule of thing, form labelled antigen.
6. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that: the reaction of described competitive immunization occurs in phosphate buffer.
7. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that: described competitive immunization reaction by by magnetic particle with
And the effect of externally-applied magnetic field is formed.
8. as claimed in claim 1 based on Prussian blue bionical marker detection little molecule chemical combination
The method of thing, it is characterised in that: in step d), in described Electrochemical Detection system
Oxidation current signal and then determinand is realized quantitative measurement, specifically includes:
By the hydrogen peroxide in the labelled antigen added solution of catalysis of residual in detection supernatant and
Hydroquinone carries out the signal of telecommunication of oxidation reaction generation, it is achieved the quantitative electricity to little molecule to be measured
Chemical detection.
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| CN110261600A (en) * | 2019-07-02 | 2019-09-20 | 济南大学 | It is a kind of based on ferroso-ferric oxide/prussian blue nano enzyme marker preparation method and application |
| CN110927378A (en) * | 2019-11-18 | 2020-03-27 | 广东工业大学 | Nano enzyme-linked immunosorbent assay method for detecting glycocholic acid |
| CN111024634B (en) * | 2019-12-26 | 2021-02-02 | 湖南大学 | Prussian blue-based nano probe and preparation method and application thereof |
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