CN109142732A - Human breast carcinoma antigen electrochemiluminescdetection detection method and its kit - Google Patents
Human breast carcinoma antigen electrochemiluminescdetection detection method and its kit Download PDFInfo
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- CN109142732A CN109142732A CN201810894107.3A CN201810894107A CN109142732A CN 109142732 A CN109142732 A CN 109142732A CN 201810894107 A CN201810894107 A CN 201810894107A CN 109142732 A CN109142732 A CN 109142732A
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- 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
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
Abstract
The present invention discloses a kind of human breast carcinoma antigen electrochemiluminescdetection detection method and its kit.The present invention is using human breast carcinoma antigen as test object, high quantum production rate gold nano cluster electrogenerated chemiluminescence technology and immuno analytical method are organically combined, high quantum production rate gold nano cluster electrogenerated chemiluminescence probe is prepared using reduction method, using nano material of manganese dioxide as electrogenerated chemiluminescence quencher, the redox reaction of the ascorbic acid and manganese dioxide that are generated using enzyme linked immunoassay restores electrochemiluminescence signal, is a kind of high-performance electrogenerated chemiluminescence human breast carcinoma antigen detection method based on high quantum production rate gold nano cluster probe.The present invention is the U/mL of 0.03 U/mL ~ 1 to the range of linearity that human breast carcinoma antigens c A15-3 is detected, and detection is limited to 0.018 U/mL.Have the characteristics that quick, accurate, high sensitivity, selectivity and stability are good, amount of samples is few, there is preferable potential applicability in clinical practice.
Description
Technical field
The present invention relates to a kind of electrogenerated chemiluminescence human breast carcinoma antigens based on high quantum production rate gold nano cluster probe
Detection method and its detection kit belong to analytical chemistry and field of nanometer technology.
Background technique
Breast cancer is one of the malignant tumour of the most common threat to life occurred in global women.Human breast carcinoma antigen is
A kind of specific tumors marker of breast cancer is assessed and diagnosed, the level of human breast carcinoma antigen is early detection cancer in serum
Important indicator.Clinically human breast carcinoma antigen levels can be used as Computer-aided Diagnosis of Breast Cancer, the index of Follow-up After and transfer and relapse,
(80%) higher to the sensibility of advanced breast cancer, the positive rate of metastatic breast cancer are 80%.Currently used for people in detection serum
Many kinds have been developed in the analysis method of breast cancer antigen content, as enzyme-linked immunosorbent assay, microfluidic analysis method, electrochemistry are exempted from
Epidemic disease analytic approach, electrogenerated chemiluminescence immunoassay and fluoroimmunoassay etc..Wherein, electrogenerated chemiluminescence immunoassay is close
A kind of novel immune detection method quickly grown for several years, the technology have many excellent characteristics, such as zero background signal, controllably
Property it is strong, stability is good, low in cost etc., because due to be widely used in the fields such as clinical analysis and bioanalysis, analysis imaging.
In order to widen the application field of electrogenerated chemiluminescence, a large amount of research concentrates on developing the electricity constructed by nano material
Cause chemical luminous system, including quantum dot, carbon dots, two-dimentional transistion metal compound, metal nanometer cluster etc..What is studied at present
In electrogenerated chemiluminescence system based on nano material, gold nano cluster is due to its distinctive good biocompatibility, quantum size
The characteristics such as effect, special photoelectric property, high stability cause more and more concerns.Gold nano cluster is widely used to give birth to
The fields such as substance markers, cell imaging, chemical sensor, catalysis and life analysis.But due to its electrogenerated chemiluminescence intensity is small,
The limitation for the problems such as quantum yield is low, luminous mechanism is not known, research of the gold nano cluster in electrogenerated chemiluminescence field are also few
It has been reported that.In view of the above-mentioned problems, this seminar is electroluminescent by the gold nano cluster that high quantum production rate has been made in nanoscale regulation
Chemiluminescence probe new method, and the biomolecule such as detection and small molecule biological thiol for being applied to cell release dopamine
Highly sensitive, quick detection.For the urgent need of clinical tumor diagnosing and treating, design and prepare based on new functionization gold
The high quantum production rate electrochemiluminescimmunosensor immunosensor of nanocluster and the detection for being used for tumor markers have heavier
The Academic innovations and clinical value wanted.
It is anti-that the present invention provides a kind of electrogenerated chemiluminescence human breast carcinomas based on high quantum production rate gold nano cluster probe
Former detection method, and the detection kit based on this method preparation.
Summary of the invention
It is an object of the present invention to provide a kind of, and the electroluminescent chemistry based on high quantum production rate gold nano cluster probe is sent out
Light human breast carcinoma antigen detection method.
It is another object of the present invention to provide a kind of electroluminescent chemistry based on high quantum production rate gold nano cluster probe
Shine human breast carcinoma antigen detection kit.
To achieve the goals above, the invention adopts the following technical scheme: a kind of be based on high quantum production rate gold nano cluster
The electrogenerated chemiluminescence human breast carcinoma antigen detection method of probe, it is characterised in that: first in electrode face finish gold nano group
Cluster prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe by reduction method, and further in electrode face finish
Nano material of manganese dioxide, as electrochemiluminescence signal quencher；It is anti-that human breast carcinoma is fixed on blank ELISA Plate
Human breast carcinoma antigen is added in original antibody, adds the human breast carcinoma antigen-antibody of biotin labeling, and it is compound to form sandwich immunoassay
Object；Streptavidin-alkaline phosphatase is added, adds 2- phosphoric acid-L-AA trisodium salt, is selected by alkaline phosphatase
Property hydrolysis 2- phosphoric acid-L-AA trisodium salt generate ascorbic acid；Reaction solution is taken out, electrode is immersed in reaction solution, electrode
Redox reaction occurs for the ascorbic acid in the manganese dioxide and reaction solution on surface；Acquire the electrogenerated chemiluminescence of modified electrode
Signal realizes the detection of human breast carcinoma antigen according to the change of electrochemiluminescence signal.
The reduction method prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe and uses following electrochemistry also
Former method or chemical reduction method preparation:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode be to electrode, Ag/AgCl is reference electrode, by above-mentioned electrode insertion 0.1 mol/L, pH 7.4 phosphate delay
It rushes in solution, applies the negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electricity
Chemiluminescence gold nano cluster probe；
(2) gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the gold nano cluster probe modification electrode of chemical reduction method preparation.
It is described to be prepared in electrode face finish nano material of manganese dioxide using following electrochemical deposition methods or drop-coating:
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in the KMnO of 1:1 V/V4-H2SO4In solution, electricity
Manganese dioxide is deposited, current potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, and cleaning is dried with nitrogen；
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to 1.25
In the buffer solution of 0.1 mol/L pH 6.0 of mL, the volume that addition deionized water keeps solution last is at ultrasound 30 after 5 mL
Min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters again, obtain dioxy
Change manganese nanometer sheet solution；Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
The method that human breast carcinoma antigen-antibody is fixed on blank ELISA Plate is self-assembly method: blank ELISA Plate is taken,
50 ~ 100 μ L of dopamine solution of 1 ~ 5 mg/mL, pH 8.5 is added in blank ELISA Plate sample well, reacts 1 ~ 5 in 4 DEG C
H is gently rinsed with water after reaction, is patted dry；It is added dropwise 0.1 ~ 0.5 mg/mL human breast carcinoma antigens c A15-3 antibody-solutions again, 4
DEG C be incubated for 8 ~ 12 h, rinsing；Be added 50 ~ 100 μ L, the wt of 1wt% ~ 5 % bovine serum albumin solution, in 4 DEG C react 0.5 ~
2 h, washing, pat dry.
The addition human breast carcinoma antigen adds the human breast carcinoma antigen-antibody of biotin labeling, forms sandwich immunoassay
The method of compound are as follows: human breast carcinoma is added in the example reaction hole for being fixed with human breast carcinoma antigens c A15-3 abzyme target
Antigen sample covers sealing plate film, and gently oscillation mixes, 37 DEG C of incubation min of 30 min ~ 60, and washing pats dry；It is added 0.1 ~ 10
Human breast carcinoma antigens c A15-3 antibody 50 μ L, the 37 DEG C of incubation min of 30 min ~ 60 of μ g/mL biotin labeling are washed, and are clapped
It is dry.
Addition Streptavidin-the alkaline phosphatase adds 2- phosphoric acid-L-AA trisodium salt method are as follows:
Every hole is separately added into 0.1 ~ 1 U/mL Streptavidin-alkalinity of 50 μ L in the ELISA Plate hole for forming sandwich immunoassay compound
Phosphatase and 5 ~ 10 mmol/L 2- phosphoric acid-L-AA trisodium-salt solution, gently oscillation mixes, and is protected from light, 37 DEG C of temperature
Educate the min of 30 min ~ 60.
The electrochemiluminescence signal method of the acquisition modified electrode is as follows: it is tested using three-electrode system, with
Modified electrode is working electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, and buffer solution is phosphate buffer
Or Tris-HCl buffer solution, electrolyte used are KCl or KNO3；The insertion of above-mentioned electrode is contained into over cure acid ion coreaction
In the buffer solution of agent, apply certain scanning voltage, working electrode surface generates electrogenerated chemiluminescence radiation, photomultiplier tube
High pressure is set as 600 ~ 800 V, detects electrogenerated chemiluminescence radiation signal.
The detection that human breast carcinoma antigen is realized according to the change of electrochemiluminescence signal are as follows: electrode surface is acquired
Electrochemiluminescence signal electrogenerated chemiluminescence intensity value and human breast carcinoma antigens c A15-3 concentration logarithm 0.03
In a linear relationship in the range of the U/mL of U/mL ~ 1, detection is limited to 0.018 U/mL.
Electrogenerated chemiluminescence human breast carcinoma antigen inspection of the present invention based on high quantum production rate gold nano cluster probe
The detection kit of survey method, which is characterized in that anti-including gold nano cluster solution, nano material of manganese dioxide, human breast carcinoma
Original antibody fixes human breast carcinoma antigen-antibody, the Streptavidin-of ELISA Plate, human breast carcinoma antigen standard, biotin labeling
Alkaline phosphatase, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence test electrolyte solution.
The fixed ELISA Plate of the human breast carcinoma antigen-antibody are as follows: blank ELISA Plate is taken, in blank ELISA Plate sample well
50 ~ 100 μ L of dopamine solution of 1 ~ 5 mg/mL pH 8.5 is added, in 4 DEG C of 1 ~ 5 h of reaction, after reaction gently with water
Rinsing, pats dry；0.1 ~ 0.5 mg/mL human breast carcinoma antigen-antibody solution, 4 DEG C of 8 ~ 12 h of incubation, rinsing are added dropwise again；Addition 50 ~
The bovine serum albumin solution of 100 μ L 1wt% ~ 5wt%, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry；The cleaning solution is
Phosphate buffer or Tris-HCl buffer solution；The electrogenerated chemiluminescence test electrolyte solution is to contain potassium peroxydisulfate
Buffer solution.
Specifically, that the present invention uses the specific technical proposal is: to prepare high quantum production rate gold nano cluster first electroluminescent
Chemiluminescence probe using manganese dioxide as electrogenerated chemiluminescence quencher, and then utilizes enzyme linked immunoassay, anti-in sandwich immunoassay
On the basis of answering, generated based on Streptavidin-alkaline phosphatase enzyme solutions and 2- phosphoric acid-L-AA trisodium reactant salt anti-
Thus the oxygen chemical original reacting recovery electrochemiluminescence signal of bad hematic acid and manganese dioxide develops a kind of based on high quantum production rate
The electrogenerated chemiluminescence immune analysis method of gold nano cluster probe, the efficient inspection for tumor markers human breast carcinoma antigen
It surveys.The present invention the following steps are included:
(1) prepared by high quantum production rate gold nano cluster probe
High quantum production rate gold nano cluster probe preparation method is as follows:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode are to electrode, and Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into buffer solution, apply negative potential voltage
(within the scope of -1.4 V of V ~ -2) carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrochemical luminescence gold nano cluster probe.
(2) it is anti-that gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution
5 ~ 30 min(preferably 0.1 mol/L sodium borohydride solution is answered to react 5 min), obtain the gold nano cluster of chemical reduction method preparation
Probe modification electrode.
The gold nano cluster material is functional modification gold nano cluster, such as: N- acetylation-L-cysteine-gold
Nanocluster, bovine serum albumin(BSA)-gold nano cluster etc..
(2) electrochemiluminescence signal acquisition method
By polishing electrode, then it is sequentially placed into HNO3It is cleaned by ultrasonic in solution, dehydrated alcohol and deionized water, N2Drying.Using three
Electrode system is tested, and using modified glassy carbon electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode,
Above-mentioned electrode is inserted into the buffer solution containing coreagent.Using step pulse method, initial potential is 0 V, burst length
For 10 s, termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as the V of 600 V ~ 800, detects work
Make the electrochemiluminescence signal of electrode surface generation.
The electrode is glass-carbon electrode, screen printing electrode or ITO electrode etc..Above-mentioned coreagent be over cure acid group from
Son, concentration range are 0.01 ~ 1 mol/L, preferably 0.1 mol/L.The buffer solution is phosphate buffer or Tris-HCl
Buffer solution, added electrolyte is KCl or KNO in buffer solution3, concentration is 0.01 ~ 1 mol/L, preferably 0. 1 mol/
L。
(3) nano material of manganese dioxide method of modifying
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in KMnO4-H2SO4In (1:1 V/V) solution, electrification
Deposition manganese dioxide is learned, sedimentation potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, preferably -0.2 V, 300
S is rinsed well with distilled water later, is dried with nitrogen spare.
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to
In the buffer solution of 1.25 mL, 0.1 mol/L pH 6.0, the volume that addition deionized water keeps solution last is at ultrasonic after 5 mL
30 min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters, obtain again
Manganese dioxide nano-plates solution.Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
(4) sandwich immunoassay reaction system constructs
Specific step is as follows for the building of sandwich immunoassay reaction system: (1) blank ELISA Plate being set blank well, (blank control wells are not loaded
Product, remaining each step operation are identical), standard sample wells, sample to be tested hole.Blank ELISA Plate is taken, in blank ELISA Plate sample well
The dopamine solution (the Tris-HCl buffer solution of pH 8.5 dissolves) of 50 μ L, 1 ~ 5 mg/mL is added, reacts 1 ~ 5 in 4 DEG C
H is gently rinsed with water after reaction, is patted dry.0.01 ~ 0.5 mg/mL human breast carcinoma antigen-antibody solution is added dropwise again, 4 DEG C incubate
8 ~ 12 h are educated, are rinsed.The bovine serum albumin solution that 50 ~ 200 μ L 1% ~ 5% are added has been reacted in 4 DEG C of 0.5 ~ 2 h of reaction
It (carefully takes sealing plate film off at rear washing, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so repeatedly 4
It is secondary, pat dry).Then 50 μ L of human breast carcinoma antigen standard solution is added in standard sample wells, 50 μ are added in example reaction hole
L sample covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 ~ 60 min of incubation, and washing pats dry, adds 0.1 ~ 10 μ g/mL
Human breast carcinoma antigen-antibody 50 μ L, 37 DEG C of 30 ~ 60 min of incubation of biotin labeling, washing, pat dry.Every hole is added 0.1 ~ 1
50 μ L of U/mL Streptavidin-alkaline phosphatase enzyme solutions, gently oscillation mixes, 37 DEG C of 30 ~ 60 min of incubation, with 10 mM pH
The washing of=7.3 Tris-HCl buffers, pats dry.
(5) measurement of human breast carcinoma antigen
Human breast carcinoma antigen-measuring step is as follows: being added into the ELISA Plate of the sandwich immunoassay reaction system of above-mentioned building
5 ~ 10 mmol/L 2- phosphoric acid -50 μ L of L-AA trisodium-salt solution add 10 Tris-HCl of mM pH=8.0 buffering
50 μ L of solution, gently oscillation mixes, and 37 DEG C are protected from light 25 ~ 30 min.By above-mentioned manganese dioxide/gold nano cluster modification
Electrode, which immerses, impregnates 4 ~ 10 min in above-mentioned reaction solution, rinsed well after taking-up with distilled water, N2Drying.Collecting work electrode table
The electrochemiluminescence signal that face generates, it is bent to human breast carcinoma antigen concentration mapping drafting standard with electrochemiluminescence signal
Line.
(6) kit
A kind of electrogenerated chemiluminescence human breast carcinoma antigen detection kit based on high quantum production rate gold nano cluster probe, wherein
Including gold nano cluster solution, nano material of manganese dioxide, human breast carcinoma antigen-antibody fixed ELISA Plate, human breast carcinoma antigen mark
Quasi- product, the human breast carcinoma antigen-antibody of biotin labeling, Streptavidin-alkaline phosphatase, 2- phosphoric acid-L-AA trisodium
Salt, cleaning solution, electrogenerated chemiluminescence test electrolyte solution.
Compared with prior art, the invention has the benefit that
The present invention is using high quantum production rate gold nano cluster probe as electrogenerated chemiluminescence material, with nano-manganese dioxide for electroluminescentization
Luminescence quenchers are learned, restores its electrochemiluminescence signal using enzyme linked immunoassay generation ascorbic acid and realizes that human breast carcinoma is anti-
Former detection.The present invention is high to the detection sensitivity of human breast carcinoma antigen, easy to operate, can effectively avoid practical in detection process
The interference of sample complexity composition, thus specificity is good, accuracy is high.Also, the present invention is at low cost, production is simple, stability is good,
Sensitivity is good, and the range of linearity is wide (U/mL of 0.03 U/mL ~ 1), and detection limits low (0.018 U/mL), has preferable market price
Value.
Detailed description of the invention
Fig. 1 is electrogenerated chemiluminescence-time plot of gold nano cluster probe modification glass-carbon electrode.
Fig. 2 is manganese dioxide/gold nano cluster probe modification glass-carbon electrode electrogenerated chemiluminescence-time plot.
Fig. 3 is electrogenerated chemiluminescence-time plot (human breast carcinoma antigen concentration of present invention detection human breast carcinoma antigen
For 1 U/mL).
Linear relationship chart of the Fig. 4 between electrogenerated chemiluminescence Strength Changes and human breast carcinoma antigen concentration logarithm.
Specific embodiment
The present invention is further elaborated in the following with reference to the drawings and specific embodiments, and the present invention is not limited thereto.
Human breast carcinoma antigen-antibody used in the present invention (write from memory picogram Biotechnology Co., Ltd in Wuhan), human breast carcinoma antigen
(write from memory picogram Biotechnology Co., Ltd in Wuhan), the human breast carcinoma antigen-antibody of biotin labeling (write from memory picogram biotechnology by Wuhan
Co., Ltd), Streptavidin-alkaline phosphatase (Wuhan Boster Biological Technology Co., Ltd.), 2- phosphoric acid-L-AA
Trisodium salt (Sigma-Aldrich company) is prior art products.
Embodiment 1
The 20 mg/mL chlorauric acid solution of NaOH and 0.4 mL of 0.6 mL, 0.5 moL/L is taken to be added to 4 mL, 0.08 mol/L
N-acetyl-L-cysteine solution in, mixing is placed in 37 DEG C of thermostatic water baths and is incubated for 3 hours.To after reaction, use
The bag filter that molecular weight is 3000 obtains N-acetyl-L-cysteine protection after purification to 24 h of reaction solution dialysis purification
Gold nano cluster solution is kept in dark place in 4 DEG C of refrigerators.
Embodiment 2
By the glass-carbon electrode of 3 mm of diameter with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
The gold nano cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and room temperature is dry
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, obtain gold nano cluster probe
Modified electrode.Above-mentioned electrode is inserted into the 0.1 mol/L pH 7.4 containing 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl
In phosphate buffer solution.Using step pulse method, initial potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V,
Burst length is 1 s.Photomultiplier tube high pressure is set as 750 V, the electrogenerated chemiluminescence letter that detection working electrode surface generates
Number, obtain electrochemical luminescence signals (see figure 1).
Embodiment 3
By the glass-carbon electrode of 3 mm of diameter 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
The gold nano cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and room temperature is dry
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, obtain gold nano cluster probe
Modified glassy carbon electrode.Using three-electrode system, using gold nano cluster probe modification glass-carbon electrode as working electrode, platinum electrode is
To electrode, Ag/AgCl is reference electrode, using chronoamperometry, in KMnO4-H2SO4In (1:1 V/V) solution, electro-deposition two
Manganese oxide, sedimentation potential are -0.1 V of V ~ -0.5, and the time is 300 s, and obtained electrode is manganese dioxide/gold nano cluster
Modified glassy carbon electrode is rinsed well with distilled water later, N2Gas gently dries up spare.The insertion of above-mentioned electrode is contained into 0.1 mol/
In 0.1 mol/L pH, 7.4 phosphate buffer solution of L potassium peroxydisulfate and 0.1 mol/L KCl.Using step pulse method, just
Beginning current potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as
750 V, the electrochemiluminescence signal that detection working electrode surface generates, obtain electrochemical luminescence signals (see figure 2).
Embodiment 4
Take blank ELISA Plate, be added in the sample well of blank ELISA Plate 50 μ L, 5 mg/mL dopamine solution (pH's 8.5
The dissolution of Tris-HCl buffer solution), in 4 DEG C of 2 h of reaction, is gently rinsed, patted dry with water after reaction.100 μ g/ are added dropwise again
ML human breast carcinoma antigens c A15-3 antibody-solutions, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, to remove electrode
The antibody of surface non-specific adsorption.It is subsequently added into 1% 50 μ L of bovine serum albumin solution, in 4 DEG C of 1 h of reaction, reaction
Washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so weight after the completion
It is 4 times multiple, pat dry), obtain the fixed ELISA Plate of human breast carcinoma antigens c A15-3 antibody.
Embodiment 5
The human breast carcinoma of 1 U/mL is added in the standard sample wells of the fixed ELISA Plate of human breast carcinoma antigen-antibody prepared by embodiment 4
50 μ L of antigen standard solution, covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, adds 0.5
Human breast carcinoma antigens c A15-3 antibody 50 μ L, 37 DEG C of 30 min of incubation of μ g/mL biotin labeling, washing, pat dry.Every hole adds
Enter 0.2 U/mL Streptavidin -50 μ L(37 DEG C of alkaline phosphatase enzyme solutions and preheat 30 min), gently oscillation mixes, and 37 DEG C
45 min are incubated, washs, pats dry.2- acid phosphorus-L-AA trisodium salt that 8 mmol/L are added into above-mentioned ELISA Plate again is molten
50 μ L(37 DEG C of liquid preheats 30 min), 50 μ L of the Tri-Hcl of pH=8.0 is added, gently oscillation mixes, and 37 DEG C are protected from light instead
Answer 25 min.Liquid in each hole is taken out, is added in the EP pipe of 2 mL, it is dense that the electrode handled well is put into each correspondence
10 min are impregnated in degree EP pipe, are rinsed well after taking-up with distilled water, N2Drying.The insertion of above-mentioned electrode is contained into 0.1 mol/L
In 0.1 mol/L pH, 7.4 phosphate buffer solution of potassium peroxydisulfate and 0.1 mol/L KCl.Using step pulse method, just
Beginning current potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as
750 V, the electrochemiluminescence signal that detection working electrode surface generates, obtained electrochemical luminescence signals are not than adding human milk gland
The solution of cancer antigen significantly increases (Fig. 3).
Embodiment 6
Take blank ELISA Plate, by blank ELISA Plate set blank well (sample is not added in blank control wells, remaining each step operation is identical),
Standard sample wells, sample to be tested hole.Be added in each hole of blank ELISA Plate 50 μ L, 5 mg/mL dopamine solution (pH's 8.5
The dissolution of Tris-HCl buffer solution), in 4 DEG C of 2 h of reaction, is gently rinsed, patted dry with water after reaction.200 μ g/ are added dropwise again
ML human breast carcinoma antigens c A15-3 antibody-solutions, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, to remove electrode
The antibody of surface non-specific adsorption.It is subsequently added into 1% 50 μ L of bovine serum albumin solution, in 4 DEG C of 1 h of reaction, reaction
Washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, so weight after the completion
It is 4 times multiple, pat dry).Then the 50 μ L of human breast carcinoma antigens c A15-3 standard solution of various concentration, lid are added in standard sample wells
Upper sealing plate film, gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, adds the people of 0.5 μ g/mL biotin labeling
Breast cancer antigen CA15-3 antibody 50 μ L, 37 DEG C of 30 min of incubation, washing, pat dry.It is affine that 0.2 U/mL strepto- is added in every hole
50 μ L(37 DEG C of element-alkaline phosphatase enzyme solutions preheats 30 min), gently oscillation mixes, 37 DEG C of 45 min of incubation, washs, and claps
It is dry.2- acid phosphorus -50 μ L(37 DEG C of L-AA trisodium-salt solution preheating 30 of 8 mmol/L is added into above-mentioned ELISA Plate again
Min), 50 μ L of the Tri-Hcl of pH=8.0 is added, gently oscillation mixes, and 37 DEG C are protected from light 25 min.It will be in each hole
Liquid takes out, and is added in the EP pipe of 2 mL, the electrode handled well is put into each corresponding concentration EP pipe and impregnates 10 min.
It is rinsed well after taking-up with distilled water, N2Drying.The insertion of above-mentioned electrode is contained into 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L
In 0.1 mol/L pH, 7.4 phosphate buffer solution of KCl.Using step pulse method, initial potential is 0 V, burst length
For 10 s, termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as 750 V, detects working electrode table
The electrochemiluminescence signal that face generates, human breast carcinoma in the range of human breast carcinoma antigen concentration is 0.03 U/mL ~ 1 U/mL
The logarithm and electrogenerated chemiluminescence intensity value of antigen concentration are in good linear relationship, and detection is limited to 0.018 U/mL(and sees figure
4).
Embodiment 7
Take the KMnO of 500 μ L, 10 mmol/L4Solution is added to 2-(the N- morpholines of 1.25 mL, 0.1 mol/L pH 6.0
Generation) in ethanesulfonic acid (MES) buffer solution, volume that deionized water keeps solution last is added into 30 min of ultrasound after 5 mL, end
12000 r.p.m are centrifuged 10 min afterwards, are washed with water 3 times, are scattered in 2.5 mL deionized waters again, obtain manganese dioxide nano
Piece solution.
Embodiment 8
Kit application method: (1) the gold nano cluster drop for the N-acetyl-L-cysteine protection for taking 5 μ L embodiments 1 to prepare
It is added in the glassy carbon electrode surface handled well, drying at room temperature.And the electrode is further immersed in 0.1 mol/L sodium borohydride solution
Middle reaction 5 minutes, obtains gold nano cluster probe modification glass-carbon electrode.The MnO for taking 5 μ L embodiments 7 to prepare2Nanometer sheet solution
It is added dropwise in above-mentioned gold nano cluster probe modification glassy carbon electrode surface, drying at room temperature, obtains manganese dioxide/gold nano cluster modification
Glass-carbon electrode.(2) the fixed ELISA Plate of human breast carcinoma antigens c A15-3 antibody prepared by Example 4, it is (empty to be set to blank well
Sample is not added in white control wells, remaining each step operation is identical), standard sample wells, sample to be tested hole.In each Kong Zhongjia of blank ELISA Plate
The dopamine solution (the Tris-HCl buffer solution of pH 8.5 dissolves) for entering 50 μ L, 5 mg/mL, in 4 DEG C of 2 h of reaction, reaction
After gently rinsed with water, pat dry.200 μ g/mL human breast carcinoma antigen-antibody solution, 4 DEG C of 12 h of incubation are added dropwise again.It incubates
It is gently rinsed after educating with water, to remove the antibody of electrode surface non-specific adsorption.It is subsequently added into 50 μ L's 1%
BSA solution, in 4 DEG C of 1 h of reaction, washing (carefully takes sealing plate film off, discards liquid, dry, every hole, which is filled it up with, to be washed after the reaction was completed
Liquid is washed, is discarded after standing 30 seconds, is so repeated 4 times, pats dry).Then the human breast carcinoma of various concentration is added in standard sample wells
50 μ L of antigens c A15-3 standard solution, covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, then
Human breast carcinoma antigen-antibody 50 μ L, 37 DEG C of 30 min of incubation of 0.5 μ g/mL biotin labeling are added, washs, pats dry.Every hole
The 50 μ L(37 DEG C of Streptavidin-alkaline phosphatase enzyme solutions that 0.2 U/mL is added preheats 30 min), gently oscillation mixes, and 37
DEG C incubate 45 min, washing, pat dry.2- acid phosphorus-L-AA trisodium salt of 8 mmol/L is added into above-mentioned ELISA Plate again
50 μ L(37 DEG C of solution preheats 30 min), 50 μ L of the Tri-Hcl of pH=8.0 is added, gently oscillation mixes, and 37 DEG C are protected from light
React 25 min.(3) liquid in each hole is taken out, is added in the EP pipe of 2 mL, the electrode handled well is put into each
10 min are impregnated in corresponding concentration EP pipe.It is rinsed well after taking-up with distilled water, N2Drying.The insertion of above-mentioned electrode is contained 0.1
In 0.1 mol/L pH, 7.4 phosphate buffer solution of mol/L potassium peroxydisulfate and 0.1 mol/L KCl.Using step pulse
Method, initial potential are 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set
750 V are set to, the electrochemiluminescence signal that detection working electrode surface generates is in human breast carcinoma antigens c A15-3 concentration
The logarithm of human breast carcinoma antigens c A15-3 concentration and electrogenerated chemiluminescence Strength Changes in the range of the U/mL of 0.03 U/mL ~ 1
Value is in good linear relationship, and detection is limited to 0.018 U/mL.
The foregoing is merely exemplary embodiments of the invention, are not intended to limit the invention, all in essence of the invention
Made any modification within mind and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of electrogenerated chemiluminescence human breast carcinoma antigen detection method based on high quantum production rate gold nano cluster probe, special
Sign is: first in electrode face finish gold nano cluster, preparing high quantum production rate gold nano cluster electroluminescentization by reduction method
Luminescence probe is learned, and further in electrode face finish nano material of manganese dioxide, it is sudden as electrochemiluminescence signal
It goes out agent；Human breast carcinoma antigen-antibody is fixed on blank ELISA Plate, and human breast carcinoma antigen is added, adds the people of biotin labeling
Breast cancer antigen antibody forms sandwich immunoassay compound；Streptavidin-alkaline phosphatase is added, it is anti-to add 2- phosphoric acid-L-
Bad hematic acid trisodium salt generates ascorbic acid by alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt；It takes out
Reaction solution immerses electrode in reaction solution, and redox occurs for the ascorbic acid in the manganese dioxide and reaction solution of electrode surface
Reaction；The electrochemiluminescence signal for acquiring modified electrode realizes that human breast carcinoma is anti-according to the change of electrochemiluminescence signal
Former detection.
2. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that the reduction method prepares the use of high quantum production rate gold nano cluster electrogenerated chemiluminescence probe
Following electrochemical reducings or chemical reduction method preparation:
(1) electrochemical reducing: being restored using three-electrode system, using gold nano cluster modified glassy carbon electrode as work electricity
Pole, platinum electrode be to electrode, Ag/AgCl is reference electrode, by above-mentioned electrode insertion 0.1 mol/L, pH 7.4 phosphate delay
It rushes in solution, applies the negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electricity
Chemiluminescence gold nano cluster probe；
(2) gold nano cluster modified glassy carbon electrode chemical reduction method: is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the gold nano cluster probe modification electrode of chemical reduction method preparation.
3. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that described use following electrochemical depositions in electrode face finish nano material of manganese dioxide
Method or drop-coating preparation:
(1) electrochemical deposition method: using three-electrode system, using reduction method processing gold nano cluster modified electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method, in the KMnO of 1:1 V/V4-H2SO4In solution, electricity
Manganese dioxide is deposited, current potential is -0.1 V of V ~ -0.5, and sedimentation time is the s of 100 s ~ 600, and cleaning is dried with nitrogen；
(2) drop-coating: preparing manganese dioxide nano-plates first, takes the KMnO of 500 μ L, 10 mmol/L4Solution is added to 1.25
In the buffer solution of 0.1 mol/L pH 6.0 of mL, the volume that addition deionized water keeps solution last is at ultrasound 30 after 5 mL
Min, after 12000 r.p.m be centrifuged 10 min, be washed with water 3 times, be scattered in 2.5 mL deionized waters again, obtain dioxy
Change manganese nanometer sheet solution；Take 5 μ L drop coatings in gold nano cluster modified electrode surface, naturally dry.
4. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that the method for fixing human breast carcinoma antigen-antibody on blank ELISA Plate is self assembly
Method: taking blank ELISA Plate, and 50 ~ 100 μ of dopamine solution of 1 ~ 5 mg/mL, pH 8.5 is added in blank ELISA Plate sample well
L is gently rinsed with water after reaction, is patted dry in 4 DEG C of 1 ~ 5 h of reaction；0.1 ~ 0.5 mg/mL human breast carcinoma antigen is added dropwise again
CA15-3 antibody-solutions, 4 DEG C of 8 ~ 12 h of incubation, rinsing；It is molten that 50 ~ 100 μ L, the bovine serum albumin(BSA) of the wt of 1wt% ~ 5 % is added
Liquid, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry.
5. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that the addition human breast carcinoma antigen, the human breast carcinoma antigen for adding biotin labeling is anti-
Body, the method for forming sandwich immunoassay compound are as follows: in the example reaction for being fixed with human breast carcinoma antigens c A15-3 abzyme target
Human breast carcinoma antigen sample is added in hole, covers sealing plate film, gently oscillation mixes, 37 DEG C of incubation min of 30 min ~ 60, washing,
It pats dry；Human breast carcinoma antigens c A15-3 antibody 50 the μ L, 37 DEG C of 30 min of incubation of 0.1 ~ 10 μ g/mL biotin labeling of addition ~
60 min, washing, pat dry.
6. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that the addition Streptavidin-alkaline phosphatase, adds 2- phosphoric acid-L-AA
The method of trisodium salt are as follows: every hole is separately added into 0.1 ~ 1 U/mL of 50 μ L in the ELISA Plate hole for forming sandwich immunoassay compound
Streptavidin-alkaline phosphatase and 5 ~ 10 mmol/L 2- phosphoric acid-L-AA trisodium-salt solution, gently oscillation mixes,
It is protected from light, 37 DEG C of incubation 30 min ~ 60 min.
7. a kind of electrogenerated chemiluminescence human milk gland based on high quantum production rate gold nano cluster probe according to claim 1
Cancer antigen detection method, it is characterized in that the electrochemiluminescence signal method of the acquisition modified electrode is as follows: using three electrodes
System is tested, and using modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, buffer solution
For phosphate buffer or Tris-HCl buffer solution, electrolyte used is KCl or KNO3；The insertion of above-mentioned electrode is contained into over cure
In the buffer solution of acid ion coreagent, apply certain scanning voltage, working electrode surface generates electrogenerated chemiluminescence
Radiation, photomultiplier tube high pressure are set as 600 ~ 800 V, detect electrogenerated chemiluminescence radiation signal.
8. any a kind of electrogenerated chemiluminescence based on high quantum production rate gold nano cluster probe according to claim 1 ~ 7
Human breast carcinoma antigen detection method, it is characterized in that described realize human breast carcinoma antigen according to the change of electrochemiluminescence signal
Detection are as follows: the electrogenerated chemiluminescence intensity value and human breast carcinoma antigen of electrode surface electrochemiluminescence signal collected
The logarithm of CA15-3 concentration is in a linear relationship in the range of 0.03 U/mL ~ 1 U/mL, and detection is limited to 0.018 U/mL.
9. a kind of any electrogenerated chemiluminescence human milk based on high quantum production rate gold nano cluster probe of claim 1-8
The detection kit of gland cancer antigen detection method, which is characterized in that including gold nano cluster solution, nano material of manganese dioxide,
Human breast carcinoma antigen-antibody fixes human breast carcinoma antigen-antibody, the chain of ELISA Plate, human breast carcinoma antigen standard, biotin labeling
Mould Avidin-alkaline phosphatase, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence test electrolyte are molten
Liquid.
10. the electrogenerated chemiluminescence human breast carcinoma according to claim 9 based on high quantum production rate gold nano cluster probe
The detection kit of antigen detection method, it is characterized in that the fixed ELISA Plate of the human breast carcinoma antigen-antibody are as follows: take blank enzyme
50 ~ 100 μ L of dopamine solution of 1 ~ 5 mg/mL pH 8.5 is added in target in blank ELISA Plate sample well, reacts in 4 DEG C
1 ~ 5 h is gently rinsed with water after reaction, is patted dry；It is added dropwise 0.1 ~ 0.5 mg/mL human breast carcinoma antigen-antibody solution again, 4
DEG C be incubated for 8 ~ 12 h, rinsing；The bovine serum albumin solution of 50 ~ 100 μ L 1wt% ~ 5wt% is added, reacts 0.5 ~ 2 in 4 DEG C
H, washing, pats dry；The cleaning solution is phosphate buffer or Tris-HCl buffer solution；The electrogenerated chemiluminescence test
Electrolyte solution is the buffer solution containing potassium peroxydisulfate.
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