CN106117356A - A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody - Google Patents

A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody Download PDF

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CN106117356A
CN106117356A CN201610507224.0A CN201610507224A CN106117356A CN 106117356 A CN106117356 A CN 106117356A CN 201610507224 A CN201610507224 A CN 201610507224A CN 106117356 A CN106117356 A CN 106117356A
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nanometer particle
golden nanometer
fetoprotein
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hrp
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常津
武玉东
宫晓群
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Tianjin University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

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Abstract

The present invention relates to a kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody；Utilize golden nanometer particle and alpha-fetoprotein traget antibody and the electrostatic adsorption of horseradish peroxidase, prepare the golden nanometer particle alpha-fetoprotein immunological probe of many enzyme labellings, to alpha-fetoprotein (alpha fetoprotein AFP), this protein marker being widely present primary hepatocarcinoma carries out the detection of qualitative, quantitative, setting up the new method of protein markers analyte detection, high degree improves detection sensitivity.Present invention is characterized in that whole preparation process is simple, be suitable for industrialization and produce；Golden nanometer particle coupling horseradish peroxidase and alpha-fetoprotein traget antibody, detection probe will have immunocompetence and enzymatic activity simultaneously, gained detection probe is many enzyme labelled probes, it is achieved that the nanometer enhancement effect of nm regime, can be effectively improved detection sensitivity.

Description

A kind of golden nanometer particle coupling horseradish peroxidase and alpha-fetoprotein traget antibody Preparation method
Technical field
The present invention relates to technical field of nanometer material preparation, more particularly to a kind of golden nanometer particle coupling Radix Cochleariae officinalis mistake Oxide enzyme and the preparation method of alpha-fetoprotein traget antibody.
Background technology
In recent years, drastically changing along with the life style of people there occurs, stress the most constantly increases, China's tumor Sickness rate rises year by year.In China urban and rural area, malignant tumor has become " the first killer " causing death.Generation Boundary's health organization has been made by up-to-date authoritative conclusion, if malignant tumor patient can be at morbidity early discovery, cure rate Can reach more than 80%, the early diagnosis of tumor has become as the focus of Recent study with treatment.
Serum protein markers detection tumor the most increasingly causes the concern of people, and protein markers analyte detection tumor is easy Noinvasive, testing result is directly perceived and can be quantitative, can have the highest using value clinically with dynamic monitoring tumor patient. Protein marker can be to occur and in breeding in malignant tumor, the synthesis secretion by the gene expression of tumor cell, It is present in cell, tissue or body fluid, it is possible to come quantitatively by certain method and the material that tumor exists can be confirmed；Can also be by In body, tumor is produced reaction, cause internal abnormal producing or raising, can reflect that tumor exists and growth, Neng Goujian Surveying oncotherapy and a class material of prognosis, these materials do not exist the internal of normal person, or occur in normal human Level be substantially less than the level in tumor patient body.
Alpha-fetoprotein is a kind of glycoprotein, and under normal circumstances, this albumen is essentially from the hepatocyte of embryo, fetal birth Within latter about two weeks, alpha-fetoprotein disappears from blood, therefore in normal human serum the content of alpha-fetoprotein still less than 20 micrograms per litre.But When hepatocyte generation canceration, but recovered the function of this protein of generation, and along with sb.'s illness took a turn for the worse it in serum Content can sharply increase, alpha-fetoprotein has just become a specific clinical index of diagnosing primary hepatocarcinoma.Radix Cochleariae officinalis peroxidating Thing enzyme is one of a kind of the most frequently used enzyme in a kind of clinical assay reagent, is widely used in multiple biochemistry detecting item, the most extensively transports Chromogenic reaction for immunity class (ELISA) test kit.So intending herein developing a kind of golden nanometer particle coupling Radix Cochleariae officinalis peroxidating Thing enzyme and the preparation method of alpha-fetoprotein traget antibody, be used for detecting alpha-fetoprotein tumor markers.
Summary of the invention
In view of protein marker critical role, the colour developing character of HRP detection uniqueness and enzyme connection in lesion detection is exempted from The advantage of epidemic disease detection technique.We will utilize the quiet of golden nanometer particle and alpha-fetoprotein traget antibody and horseradish peroxidase Electro-adsorption effect, prepares the golden nanometer particle alpha-fetoprotein immunological probe of many enzyme labellings, to alpha-fetoprotein (alpha Fetoprotein AFP), this protein marker being widely present primary hepatocarcinoma carries out the detection of qualitative, quantitative, sets up egg The new method of white marker detection, high degree improves detection sensitivity.
Technical scheme is as follows:
A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody；Its step is such as Under:
(1) gold chloride is joined reactor, inject sodium citrate after heated and boiled, be cooled to room temperature, obtain gold nano Particle (AuNPs)；
(2) by above-mentioned golden nanometer particle add reactor, by wet chemical regulation golden nanometer particle pH be 8.3～ 8.5, by alpha-fetoprotein traget antibody (Ab1) and horseradish peroxidase (HRP) mixed liquor mix with golden nanometer particle, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, is centrifuged and obtains Many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Described step 1) gold chloride is four hydration gold chlorides, the quality proportioning of gold chloride and sodium citrate is 1:0.8～3.
Described step 2) be by wet chemical regulation golden nanometer particle pH be 8.3～8.5；Alpha-fetoprotein labelling resists Body (Ab1): horseradish peroxidase (HRP) quality proportioning is 1:1～9；Golden nanometer particle AuNPs:(HRP and Ab1) mixed liquor matter Amount proportioning is 100～500:1.
Described step 3) golden nanometer particle: protein amount proportioning 10～20:1.
Described step 3) AuNPs-HRP-Ab1Detection probe is many enzyme labelled probes, can be effectively improved detection sensitivity.
As shown in Figure 4, can simultaneously coupling multiple HRP molecule and alpha-fetoprotein labelling above a golden nanometer particle molecule Antibody molecule, is effectively increased the coupling efficiency of enzyme molecule and antibody molecule；The golden nanometer particle that as shown in Figure 1 prepared by the present invention Particle diameter is 10～60nm；Particle diameter after golden nanometer particle coupling horseradish peroxidase and alpha-fetoprotein traget antibody as shown in Figure 2 Increase about 20nm, show that horseradish peroxidase and alpha-fetoprotein traget antibody pass flag are to golden nanometer particle.
As shown in Fig. 3 golden nanometer particle ultra-violet absorption spectrum, golden nanometer particle absorbing wavelength, can between 520～550nm Effectively coupling protein molecule.As it is shown in figure 5, multi-HRP-AuNPs-Ab1Detection probe immunoadsorption assay is substantially better than HRP-Ab1Detection probe immunoadsorption assay, illustrates multi-HRP-AuNFs-Ab1Detection probe is than traditional HRP-Ab1Detection Probe in detecting sensitivity is higher.
Golden nanometer particle coupling horseradish peroxidase and alpha-fetoprotein traget antibody advantage prepared by the present invention are:
1. use golden nanometer particle AuNPs and alpha-fetoprotein traget antibody Ab under the conditions of alkalescence1And horseradish peroxidase (HRP) Electrostatic Absorption interacts and obtains detecting probe.
2. golden nanometer particle coupling horseradish peroxidase and alpha-fetoprotein traget antibody, detection probe is exempted from having simultaneously Epidemic disease activity and enzymatic activity.
3. golden nanometer particle is because of its abundant specific surface area, and gained detection probe is many enzyme labelled probes, it is achieved that nanometer The nanometer enhancement effect in field, can be effectively improved detection sensitivity.
Accompanying drawing explanation
Golden nanometer particle AuNPs transmission electron microscope photo (a) 14.2nm (b) 25.7nm (c) 25.4nm prepared by Fig. 1 present invention (d)48.1nm(e)58.2nm(f)67nm。
Golden nanometer particle prepared by Fig. 2 present invention and many enzyme labellings AuNPs-HRP-Ab1Detection probe granularity picture.
Golden nanometer particle UV-Vis spectrum visible light curve prepared by Fig. 3 present invention.
Golden nanometer particle coupling horseradish peroxidase prepared by Fig. 4 present invention and alpha-fetoprotein traget antibody figure.
Detection probe multi-HRP-AuNPs-Ab prepared by Fig. 5 present invention1Immunization experiment and traditional detection probe HRP- Ab1Immunization experiment matched curve.
Detailed description of the invention
In following case study on implementation, the invention will be further elaborated, but the invention is not restricted to this.
Case study on implementation 1:
(1) 0.01g gold chloride is joined reactor, injects 0.018g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.3, by 0.025mg alpha-fetoprotein traget antibody (Ab1) and 0.025mg horseradish peroxidase (HRP) mixed liquor and gold nano Mix particles, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.5mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.multi-HRP-AuNPs-Ab1Detection probe is complete in 96 hole ELISA Plate Become immunoadsorption assay ELISA, choose AFP antigen concentration respectively 0.25ng/mL, 0.5ng/mL, 1ng/mL, 1ng/mL, 1.5ng/mL, 2ng/mL, 2.5ng/mL, 3ng/mL, 3.5ng/mL and 4ng/mL.
Gained gold nanometer particle grain size shown in Fig. 1 (a) is 14.2nm；Fig. 2 granularity data shows, golden nanometer particle coupling Before and after HRP enzyme and alpha-fetoprotein traget antibody molecule, granularity is increased to about 35nm by 15nm, illustrate horseradish peroxidase and Alpha-fetoprotein traget antibody pass flag is on golden nanometer particle；Fig. 3 shows that gained golden nanometer particle has by force at 520nm Strong uv absorption.As it is shown in figure 5, multi-HRP-AuNPs-Ab1Detection probe immunoadsorption assay is substantially better than HRP-Ab1Inspection Probing pin immunoadsorption assay, illustrates multi-HRP-AuNFs-Ab1Detection probe is than traditional HRP-Ab1Detection probe in detecting Sensitivity is higher.
Case study on implementation 2:
(1) 0.01g gold chloride is joined reactor, injects 0.02g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.3, by 0.01mg alpha-fetoprotein traget antibody (Ab1) and 0.04mg horseradish peroxidase (HRP) mixed liquor and Jenner's grain of rice Son mixing, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.5mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Gained gold nanometer particle grain size shown in Fig. 1 (b) is 25.7nm；Fig. 3 shows that gained golden nanometer particle has at 526nm Intense UV absorbs.
Case study on implementation 3:
(1) 0.01g gold chloride is joined reactor, injects 0.03g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.4, by 0.005mg alpha-fetoprotein traget antibody (Ab1) and 0.045mg horseradish peroxidase (HRP) mixed liquor and gold nano Mix particles, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.5mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Gained gold nanometer particle grain size shown in Fig. 1 (c) is 35.4nm；Fig. 3 shows that gained golden nanometer particle has at 530nm Intense UV absorbs.
Case study on implementation 4:
(1) 0.01g gold chloride is joined reactor, injects 0.008g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.4, by 0.01mg alpha-fetoprotein traget antibody (Ab1) and 0.01mg horseradish peroxidase (HRP) mixed liquor and Jenner's grain of rice Son mixing, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.4mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Gained gold nanometer particle grain size shown in Fig. 1 (d) is 48.1nm；Fig. 3 shows that gained golden nanometer particle has at 534nm Intense UV absorbs.
Case study on implementation 5:
(1) 0.01g gold chloride is joined reactor, injects 0.01g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.5, by 0.005mg alpha-fetoprotein traget antibody (Ab1) and 0.005mg horseradish peroxidase (HRP) mixed liquor and gold nano Mix particles, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.25mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Gained gold nanometer particle grain size shown in Fig. 1 (e) is 58.2nm；Fig. 3 shows that gained golden nanometer particle has at 540nm Intense UV absorbs.
Case study on implementation 6:
(1) 0.01g gold chloride is joined reactor, injects 0.018g sodium citrate after heated and boiled, be cooled to room temperature, Obtain golden nanometer particle (AuNPs)；
(2) above-mentioned for 5mg golden nanometer particle is added reactor, by wet chemical regulation golden nanometer particle pH be 8.5, by 0.01mg alpha-fetoprotein traget antibody (Ab1) and 0.04mg horseradish peroxidase (HRP) mixed liquor and Jenner's grain of rice Son mixing, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) 0.25mg bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, from Gains in depth of comprehension are to many enzyme labellings AuNPs-HRP-Ab1Detection probe.
Gained gold nanometer particle grain size shown in Fig. 1 (f) is 67.0nm；Fig. 3 shows that gained golden nanometer particle has at 550nm Intense UV absorbs.

Claims (6)

1. a golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody；It is characterized in that step Rapid as follows:
(1) gold chloride is joined reactor, inject sodium citrate after heated and boiled, be cooled to room temperature, obtain golden nanometer particle (AuNPs)；
(2) above-mentioned golden nanometer particle is added reactor, is 8.3～8.5 by wet chemical regulation golden nanometer particle pH, By alpha-fetoprotein traget antibody (Ab1) and horseradish peroxidase (HRP) mixed liquor mix with golden nanometer particle, gained AuNPs, HRP and Ab1Mixed liquor is rotating standing process after rotation mixing on hybrid frame；
(3) bovine serum albumin (BSA) is joined above-mentioned AuNPs, HRP and Ab1Mixed liquor sealing treatment, is centrifuged and obtains multienzyme Labelling AuNPs-HRP-Ab1Detection probe.
2. the method for claim 1, is characterized in that gold chloride is four hydration gold chlorides, gold chloride and the matter of sodium citrate Amount proportioning is 1:0.8～3.
3. the method for claim 1, it is characterized in that by wet chemical regulation golden nanometer particle pH be 8.3～ 8.5。
4. the method for claim 1, is characterized in that alpha-fetoprotein traget antibody (Ab1): horseradish peroxidase (HRP) Quality proportioning is 1:1～9；Golden nanometer particle AuNPs:(HRP and Ab1) mixed liquor quality proportioning is 100～500:1.
5. the method for claim 1, is characterized in that golden nanometer particle: protein amount proportioning 10～20:1.
6. the method for claim 1, is characterized in that AuNPs-HRP-Ab1Detection probe is many enzyme labelled probes.
CN201610507224.0A 2016-06-30 2016-06-30 A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody Pending CN106117356A (en)

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Cited By (3)

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CN107824800A (en) * 2017-11-01 2018-03-23 石河子大学 A kind of preparation method of sea urchin shape nano Au particle and the method for labelled protein
CN109254152A (en) * 2018-06-28 2019-01-22 广西医科大学 A kind of preparation and application of liver cancer marker GP73 detection probe
CN111686826A (en) * 2019-03-15 2020-09-22 国家纳米科学中心 Micro-fluidic chip with layered structure and application thereof

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Publication number Priority date Publication date Assignee Title
CN107824800A (en) * 2017-11-01 2018-03-23 石河子大学 A kind of preparation method of sea urchin shape nano Au particle and the method for labelled protein
CN107824800B (en) * 2017-11-01 2020-06-09 石河子大学 Preparation method of sea urchin-shaped gold nanoparticles and method for labeling protein
CN109254152A (en) * 2018-06-28 2019-01-22 广西医科大学 A kind of preparation and application of liver cancer marker GP73 detection probe
CN111686826A (en) * 2019-03-15 2020-09-22 国家纳米科学中心 Micro-fluidic chip with layered structure and application thereof
CN111686826B (en) * 2019-03-15 2023-05-23 国家纳米科学中心 Microfluidic chip with layered structure and application thereof

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