CN109164091A - 4 detection method of people's epididymal proteins and its kit based on gold nano cluster probe - Google Patents
4 detection method of people's epididymal proteins and its kit based on gold nano cluster probe Download PDFInfo
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Abstract
The present invention discloses a kind of 4 detection method of people's epididymal proteins and its kit based on gold nano cluster probe.The present invention is test object with people's epididymal proteins 4, 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 4 detection method of high-performance electrogenerated chemiluminescence people epididymal proteins based on high quantum production rate gold nano cluster probe.It is the pmol/L of 0.01 pmol/L ~ 6 that it, which detects the range of linearity to people's epididymal proteins 4, and detection is limited to 0.004 pmol/L.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, and the electrogenerated chemiluminescence people epididymal proteins 4 based on high quantum production rate gold nano cluster probe are examined
Survey method and its detection kit belong to analytical chemistry and field of nanometer technology.
Background technique
Malignant tumour is a kind of important diseases for influencing people's life health, becomes the maximum public health problem in the whole world.
In female reproductive system, disease incidence of the oophoroma in gynecologic malignant tumor is number three, and the death rate makes number one.Closely
70% ovarian cancer patients are diagnosed as advanced stage, and 5 years survival rates of ovarian cancer patients rise to 90% by early stage effectively treatment.
Therefore, it early diagnoses most important for the prognosis for improving oophoroma.Currently, the serum for being most commonly used to oophoroma early diagnosis is raw
Object marker is CA125.CA125 but for Patients with Pelvic Inflammatory Disease, such as endometriosis, tubercular peritonitis, in serum
It may also increase.Therefore, application of the CA125 in ovarian cancer diagnosis shows higher false positive rate, and sometimes resulting in need not
The operation wanted.People epididymal proteins 4(Human epididymis protein 4, HE4) it is considered as oophoroma early diagnosis
Neoformation marker.It is reported that HE4 is highly sensitive to early ovarian cancer, can be used in combination with CA125, be oophoroma and other
The antidiastole of pelvic mass provides the best approach.Immunoassay side currently used for 4 content of people's epididymal proteins in detection serum
Many kinds have been developed in method, such as enzyme-linked immunosorbent assay, chemiluminescence immunoassay, electrogenerated chemiluminescence immunoassay and glimmering
Light immunoassay etc..Enzyme-linked immunosorbent assay has always been considered as being most mature method and gold mark interior over the past thirty years
Standard, but enzyme-linked immunosorbent assay still has certain defect, it is such as sensitive low, it can not detect the biological marker of denier in serum
Object.There are also disadvantage, including the biggish analysis instrument of figure, high cost and special markings to require for chemiluminescence immunoassay
Deng.These factors limit the clinical application of these technologies.Electrogenerated chemiluminescence immunoassay is one quickly grown in recent years
Kind novel immune detection method, the technology have diversification, better sensitivity and selectivity, and background signal is low, Optical devices
Simply, the performance characteristics such as time/space controlling is good, and the range of linearity is wide, oneself is extensive in clinical analysis and the fields such as medicine, immune
Using such as the diagnosis of chronic obstructive pulmonary disease and asthma.
Novel light-emitting body is found, the electrogenerated chemiluminescence system for developing function admirable is the important of Electrochemiluminescprocess process
Research direction.The ECL phenomenon for reporting silicon nano for 2002 for the first time has then emerged in large numbers a large amount of nano materials as new
ECL illuminator, especially quantum dot (CdS, ZnS, CdTe) have been widely used in ECL system.Gold nano cluster by tens to
Several hundred a gold atom compositions, the characteristics such as good biocompatibility, quantum size effect, special photoelectric property for having in catalysis, pass
The fields such as sense and photoelectronics are widely used.But since its electrogenerated chemiluminescence intensity is small, quantum yield is low, luminous mechanism
The limitation for the problems such as not knowing, research of the gold nano cluster in electrogenerated chemiluminescence field are also considerably less.In view of the above-mentioned problems, this
The gold nano cluster probe of high quantum production rate has been prepared by the method for controlling golden cluster valence state in seminar, and is applied to
Cell discharges highly sensitive, the quick detection of the biomolecule such as detection and the small molecule biological thiol of dopamine.In face of clinical tumor
The urgent need of diagnosis and treatment designs and develops a kind of being immunized for novel high quantum production rate gold nano cluster electrogenerated chemiluminescence probe
Sensor is simultaneously used for the detection of tumor markers with more important Academic innovations and clinical value.
The present invention provides a kind of electrogenerated chemiluminescence people epididymal proteins 4 based on high quantum production rate gold nano cluster probe
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
4 detection method of light people epididymal proteins.
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 4 detection kit of people's epididymal proteins.
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
4 detection method of electrogenerated chemiluminescence people epididymal proteins 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-in the fixed people's epididymal proteins 4 of blank ELISA Plate
People's epididymal proteins 4 are added in body, add 4 antibody of people's epididymal proteins of biotin labeling, form sandwich immunoassay compound；It is added
Streptavidin-alkaline phosphatase adds 2- phosphoric acid-L-AA trisodium salt, passes through alkaline phosphatase selective hydrolysis
2- phosphoric acid-L-AA trisodium salt generates ascorbic acid；Reaction solution is taken out, electrode is immersed in reaction solution, electrode surface
Redox reaction occurs for the ascorbic acid in manganese dioxide and reaction solution；The electrochemiluminescence signal of modified electrode is acquired,
The detection of people's epididymal proteins 4 is realized 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 negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrification
Learn the gold nano cluster probe that shines；
(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.
The electrode face finish nano material of manganese dioxide is prepared 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, sedimentation 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
ML, 0.1 mol/L, pH 6.0 buffer solution in, the deionized water volume that keeps solution last is added into 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 in fixed 4 antibody of people's epididymal proteins of blank ELISA Plate is self-assembly method: blank ELISA Plate is taken, in sky
50 ~ 100 μ L of dopamine solution of 1 ~ 5 mg/mL pH 8.5 is added in white ELISA Plate sample well, in 4 DEG C of 1 ~ 5 h of reaction, instead
It is gently rinsed, is patted dry with water after answering；0.1 ~ 0.5 mg/mL people's epididymal proteins, 4 antibody-solutions are added dropwise again, 4 DEG C are incubated for 8 ~ 12
H, rinsing；The bovine serum albumin solution of 50 ~ 100 μ L 1wt% ~ 5wt% is added, in 4 DEG C of 0.5 ~ 2 h of reaction, washs, claps
It is dry.
The addition people epididymal proteins 4 add 4 antibody of people's epididymal proteins of biotin labeling, and it is multiple to form sandwich immunoassay
The method for closing object are as follows: 4 sample of people's epididymal proteins is added in the example reaction hole of fixed 4 abzyme target of someone's epididymal proteins,
Sealing plate film is covered, gently oscillation mixes, 37 DEG C of incubation min of 30 min ~ 60, and washing pats dry；0.1 ~ 10 μ g/mL biology is added
4 antibody of people's epididymal proteins 50 μ L, the 37 DEG C of incubation min of 30 min ~ 60 of element label, washing pat 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 people's epididymal proteins 4 are realized according to the change of electrochemiluminescence signal are as follows: electrode surface is acquired
Electrochemiluminescence signal electrogenerated chemiluminescence intensity value and 4 concentration of people's epididymal proteins logarithm 0.01 pmol/L ~
In a linear relationship in the range of 6 pmol/L, detection is limited to 0.004 pmol/L.
Electrogenerated chemiluminescence people epididymal proteins 4 of the present invention based on high quantum production rate gold nano cluster probe detect
The detection kit of method, which is characterized in that anti-including gold nano cluster solution, nano material of manganese dioxide, people's epididymal proteins 4
Body fixes ELISA Plate, 4 standard items of people's epididymal proteins, 4 antibody of people's epididymal proteins of biotin labeling, Streptavidin-alkalinity phosphorus
Sour enzyme, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence test electrolyte solution.
The fixed ELISA Plate of 4 antibody of people's epididymal proteins are as follows: take blank ELISA Plate, add in blank ELISA Plate sample well
50 ~ 100 μ L of dopamine solution for entering 1 ~ 5 mg/mL pH 8.5 is gently floated with water after reaction in 4 DEG C of 1 ~ 5 h of reaction
It washes, pats dry；0.1 ~ 0.5 mg/mL people's epididymal proteins, 4 antibody-solutions, 4 DEG C of 8 ~ 12 h of incubation, rinsing are added dropwise again；It is added 50 ~ 100
The bovine serum albumin solution of μ L 1wt% ~ 5wt%, in 4 DEG C of 0.5 ~ 2 h of reaction, washing is patted dry；The cleaning solution is phosphoric acid
Salt buffer or Tris-HCl buffer solution；The electrogenerated chemiluminescence test electrolyte solution is slow containing potassium peroxydisulfate
Rush solution.
Specifically, to achieve the goals above, the present invention is using technical solution in detail below: preparing high quantum first and produce
Rate gold nano cluster electrogenerated chemiluminescence probe using manganese dioxide as electrogenerated chemiluminescence quencher, and then utilizes enzyme linked immunological
Reaction is based on Streptavidin-alkaline phosphatase enzyme solutions and 2- phosphoric acid-L-AA on the basis of sandwich immunoassay reaction
The oxygen chemical original reacting recovery electrochemiluminescence signal of ascorbic acid and manganese dioxide that trisodium reactant salt generates, thus develops
A kind of electrogenerated chemiluminescence immune analysis method based on high quantum production rate gold nano cluster probe, it is attached for tumor markers people
The efficient detection of testis albumen 4.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.0.1 ~ 0.5 mg/mL people's epididymal proteins, 4 antibody-solutions, 4 DEG C of incubations are added dropwise again
8 ~ 12 h, rinsing.The bovine serum albumin solution of 50 ~ 200 μ L 1% ~ 5% is added, in 4 DEG C of 0.5 ~ 2 h of reaction, reaction is completed
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 repeatedly 4 afterwards
It is secondary, pat dry).Then 4 standard solution of people's epididymal proteins, 50 μ L is added in standard sample wells, 50 μ L are added in example reaction hole
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
4 antibody of people's epididymal proteins 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 people's epididymal proteins 4
4 detecting step of people's epididymal proteins is as follows: being added 5 into the ELISA Plate of the sandwich immunoassay reaction system of above-mentioned building
It is molten to add 10 Tris-HCl of mM pH=8.0 buffering for ~ 10 mmol/L 2- phosphoric acid -50 μ L of L-AA trisodium-salt solution
50 μ L of liquid, gently oscillation mixes, and 37 DEG C are protected from light 25 ~ 30 min.By above-mentioned manganese dioxide/gold nano cluster modification electricity
Pole, which is immersed, impregnates 4 ~ 10 min in above-mentioned reaction solution, rinsed well after taking-up with distilled water, N2Drying.Collecting work electrode surface
The electrochemiluminescence signal of generation maps to 4 concentration of people's epididymal proteins with electrochemiluminescence signal and draws standard curve.
(6) kit
A kind of 4 detection kit of electrogenerated chemiluminescence people epididymal proteins based on high quantum production rate gold nano cluster probe, wherein
Including gold nano cluster solution, nano material of manganese dioxide, 4 antibody of people's epididymal proteins fixed ELISA Plate, 4 standard of people's epididymal proteins
Product, 4 antibody of people's epididymal proteins 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, ascorbic acid is generated using enzyme linked immunoassay and restores its electrochemiluminescence signal realization people's epididymal proteins
4 detection.The present invention is high to the detection sensitivity of people's epididymal proteins 4, easy to operate, can effectively avoid practical sample in detection process
The interference of product complexity composition, thus specificity is good, accuracy is high.Also, the present invention is at low cost, production is simple, stability is good, clever
Sensitivity is good, and the range of linearity is wide (pmol/L of 0.01 pmol/L ~ 6), and detection limits low (0.004 pmol/L), has good market
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 that (4 concentration of people's epididymal proteins is for electrogenerated chemiluminescence-time plots of present invention detection people's epididymal proteins 4
6 pmol/L).
Linear relationship chart of the Fig. 4 between 4 log concentration value of electrogenerated chemiluminescence Strength Changes and people's epididymal proteins.
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.
4 antibody of people's epididymal proteins used in the present invention (write from memory picogram Biotechnology Co., Ltd in Wuhan), people's epididymal proteins 4
(write from memory picogram Biotechnology Co., Ltd in Wuhan), (the silent picogram biotechnology in Wuhan has 4 antibody of people's epididymal proteins of biotin labeling
Limit company), Streptavidin-alkaline phosphatase (Wuhan Boster Biological Technology Co., Ltd.), 2- phosphoric acid-L-AA three
Sodium 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 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 protection of N-acetyl-L-cysteine made from 1 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 protection of N-acetyl-L-cysteine made from 1 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 -0.2 V, and the time is 300 s, and obtained electrode is that manganese dioxide/gold nano cluster modifies glass 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/L persulfuric acid
In 0.1 mol/L pH, 7.4 phosphate buffer solution of potassium and 0.1 mol/L KCl.Using step pulse method, initial potential
For 0 V, 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 working electrode surface generates is detected, electrochemical luminescence signals (see figure 2) is obtained.
Embodiment 4
Take blank ELISA Plate, be added in blank ELISA Plate sample well 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
4 antibody-solutions of mL people's epididymal proteins, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, it is non-to remove electrode surface
The antibody of 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, after the reaction was completed
Washing (it carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, and it discards after standing 30 seconds, is so repeated 4 times,
Pat dry), obtain the fixed ELISA Plate of 4 antibody of people's epididymal proteins.
Embodiment 5
People's epididymis of 6 pmol/L is added in the standard sample wells of the fixed ELISA Plate of 4 antibody of people's epididymal proteins made from embodiment 4
4 standard solution of albumen, 50 μ L, covers sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing is patted dry, added
4 antibody of people's epididymal proteins 50 μ L, 37 DEG C of 30 min of incubation of 0.5 μ g/mL biotin labeling, washing, pat dry.Every hole is added
0.2 U/mL Streptavidin -50 μ L(37 DEG C of alkaline phosphatase enzyme solutions preheats 30 min), gently oscillation mixes, 37 DEG C of temperature
45 min are educated, washs, pats dry.2- acid phosphorus-L-AA trisodium-salt solution of 8 mmol/L is added into above-mentioned ELISA Plate again
50 μ L(37 DEG C preheat 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
25 min.Liquid in each hole is taken out, is added in the EP pipe of 2 mL, the electrode handled well is put into each corresponding concentration
10 min are impregnated in 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 mistake
In 0.1 mol/L pH, 7.4 phosphate buffer solution of potassium sulfate and 0.1 mol/L KCl.Using step pulse method, initially
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 people's epididymal proteins
4 solution 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
4 antibody-solutions of mL people's epididymal proteins, 4 DEG C of 12 h of incubation.It is gently rinsed after incubation with water, it is non-to remove electrode surface
The antibody of 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, after the reaction was completed
Washing (it carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, and it discards after standing 30 seconds, is so repeated 4 times,
It pats dry).Then 4 standard solution of people's epididymal proteins, the 50 μ L of various concentration is added in standard sample wells, covers sealing plate film, gently
Oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, adds 4 antibody of people's epididymal proteins of 0.5 μ g/mL biotin labeling
50 μ L, 37 DEG C of 30 min of incubation, washing, pat dry.0.2 U/mL Streptavidin -50 μ of alkaline phosphatase enzyme solutions is added in every hole
L(37 DEG C of 30 min of preheating), gently oscillation mixes, 37 DEG C of 45 min of incubation, and washing pats dry.It is added again into above-mentioned ELISA Plate
The 2- acid phosphorus of 8 mmol/L -50 μ L(37 DEG C of L-AA trisodium-salt solution preheats 30 min), add the Tri- of pH=8.0
50 μ L of Hcl, gently oscillation mixes, and 37 DEG C are protected from light 25 min.Liquid in each hole is taken out, the EP of 2 mL is added
In pipe, 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.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, the logarithm and electricity of 4 concentration of people's epididymal proteins in the range of 4 concentration of people's epididymal proteins is 0.01 pmol/L ~ 6 pmol/L
Causing chemiluminescence intensity value is in good linear relationship, and detection is limited to 0.004 pmol/L(and sees Fig. 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 6 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 4 antibody of people's epididymal proteins prepared by Example 4, is set to blank well (blank control
Sample is not added in hole, remaining each step operation is identical), standard sample wells, sample to be tested hole.50 μ are added in each hole of blank ELISA Plate
The dopamine solution (the Tris-HCl buffer solution of pH 8.5 dissolves) of 5 mg/mL of L, in 4 DEG C of 2 h of reaction, after reaction
It is gently rinsed, is patted dry with water.200 μ g/mL people epididymal proteins, 4 antibody-solutions, 4 DEG C of 12 h of incubation are added dropwise again.After incubation
It is gently rinsed with water, to remove the antibody of electrode surface non-specific adsorption.It is subsequently added into the BSA solution of 50 μ L 1%, in
4 DEG C of 1 h of reaction, washing (carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole, stands 30 after the reaction was completed
It is discarded after second, is so repeated 4 times, pats dry).Then 4 standard solution of people's epididymal proteins of various concentration is added in standard sample wells
50 μ L, cover sealing plate film, and gently oscillation mixes, 37 DEG C of 30 min of incubation, and washing pats dry, adds 0.5 μ g/mL biotin
4 antibody of people's epididymal proteins 50 μ L, 37 DEG C of 30 min of incubation of label, washing, pat dry.The strepto- parent of 0.2 U/mL is added in every hole
30 min are preheated with 50 μ L(37 DEG C of element-alkaline phosphatase enzyme solutions), gently oscillation mixes, 37 DEG C of 45 min of incubation, washing,
It pats dry.2- acid phosphorus -50 μ L(37 DEG C of L-AA trisodium-salt solution preheating of 8 mmol/L is added into above-mentioned ELISA Plate again
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 25 min.It (3) will be each
Liquid in hole 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
In 0.1 mol/L pH, 7.4 phosphate buffer solution of mol/L KCl.Using step pulse method, initial potential is 0 V, arteries and veins
Rushing the time 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, detects work
The electrochemiluminescence signal that electrode surface generates, in the range that 4 concentration of people's epididymal proteins is the pmol/L of 0.01 pmol/L ~ 6
The logarithm and electrogenerated chemiluminescence Strength Changes value of interior 4 concentration of people's epididymal proteins are in good linear relationship, and detection is limited to
0.004 pmol/L。
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 4 detection method of electrogenerated chemiluminescence people epididymal proteins 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；In fixed 4 antibody of people's epididymal proteins of blank ELISA Plate, people's epididymal proteins 4 are added, add people's epididymis of biotin labeling
4 antibody of albumen forms sandwich immunoassay compound；Streptavidin-alkaline phosphatase is added, adds 2- phosphoric acid-L- Vitamin C
Sour trisodium salt generates ascorbic acid by alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt；Take out reaction
Liquid immerses electrode in reaction solution, and redox reaction occurs for the ascorbic acid in the manganese dioxide and reaction solution of electrode surface；
The electrochemiluminescence signal for acquiring modified electrode realizes the inspection of people's epididymal proteins 4 according to the change of electrochemiluminescence signal
It surveys.
2. a kind of electrogenerated chemiluminescence people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, 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 negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtain electrification
Learn the gold nano cluster probe that shines；
(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 people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, it is characterized in that the electrode face finish nano material of manganese dioxide uses following electrochemical deposition methods
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, sedimentation 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
ML, 0.1 mol/L, pH 6.0 buffer solution in, the deionized water volume that keeps solution last is added into 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 people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, it is characterized in that the method in fixed 4 antibody of people's epididymal proteins of blank ELISA Plate is self-assembly method: taking
50 ~ 100 μ L of dopamine solution of 1 ~ 5 mg/mL pH 8.5 is added, in 4 in blank ELISA Plate in blank ELISA Plate sample well
DEG C reaction 1 ~ 5 h, gently rinsed, patted dry with water after reaction；It is molten that 0.1 ~ 0.5 mg/mL people's epididymal proteins, 4 antibody is added dropwise again
Liquid, 4 DEG C of 8 ~ 12 h of incubation, rinsing；The bovine serum albumin solution of 50 ~ 100 μ L 1wt% ~ 5wt% is added, is reacted in 4 DEG C
0.5 ~ 2 h, washing, pats dry.
5. a kind of electrogenerated chemiluminescence people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, it is characterized in that the addition people epididymal proteins 4, add 4 antibody of people's epididymal proteins of biotin labeling,
The method for forming sandwich immunoassay compound are as follows: people is added in the example reaction hole of fixed 4 abzyme target of someone's epididymal proteins
4 sample of epididymal proteins, 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
4 antibody of people's epididymal proteins 50 μ L, the 37 DEG C of incubation min of 30 min ~ 60 of 0.1 ~ 10 μ g/mL biotin labeling are washed, and are clapped
It is dry.
6. a kind of electrogenerated chemiluminescence people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, it is characterized in that the addition Streptavidin-alkaline phosphatase, adds 2- phosphoric acid-L-AA three
The method of sodium salt are as follows: every hole is separately added into 0.1 ~ 1 U/mL chain of 50 μ L in the ELISA Plate hole for forming sandwich immunoassay compound
Mould Avidin-alkaline phosphatase and 5 ~ 10 mmol/L 2- phosphoric acid-L-AA trisodium-salt solution, gently oscillation mixes, and keeps away
Light reaction, 37 DEG C of incubation 30 min ~ 60 min.
7. a kind of electrogenerated chemiluminescence people's epididymis based on high quantum production rate gold nano cluster probe according to claim 1
4 detection method of albumen, it is characterized in that the electrochemiluminescence signal method of the acquisition modified electrode is as follows: using three electrode bodies
System is tested, and using modified electrode as working electrode, 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 persulfuric acid
In the buffer solution of radical ion coreagent, apply certain scanning voltage, working electrode surface generates electrogenerated chemiluminescence spoke
It penetrates, photomultiplier tube high pressure is set as 600 ~ 800 V, detects 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
4 detection method of people's epididymal proteins, it is characterized in that the inspection for realizing people's epididymal proteins 4 according to the change of electrochemiluminescence signal
It surveys are as follows: the electrogenerated chemiluminescence intensity value of electrode surface electrochemiluminescence signal collected and 4 concentration of people's epididymal proteins
Logarithm is in a linear relationship in the range of 0.01 pmol/L ~ 6 pmol/L, and detection is limited to 0.004 pmol/L.
9. a kind of any electrogenerated chemiluminescence people based on high quantum production rate gold nano cluster probe of claim 1-8 is attached
The detection kit of 4 detection method of testis albumen, which is characterized in that including gold nano cluster solution, nano material of manganese dioxide, people
4 antibody of epididymal proteins fixes ELISA Plate, 4 standard items of people's epididymal proteins, 4 antibody of people's epididymal proteins of biotin labeling, strepto- parent
Electrolyte solution is tested with element-alkaline phosphatase, 2- phosphoric acid-L-AA trisodium salt, cleaning solution, electrogenerated chemiluminescence.
10. electrogenerated chemiluminescence people's epididymis egg according to claim 9 based on high quantum production rate gold nano cluster probe
The detection kit of white 4 detection method, it is characterized in that the fixed ELISA Plate of 4 antibody of people's epididymal proteins are as follows: take blank enzyme mark
Plate, in blank ELISA Plate sample well be added 1 ~ 5 mg/mL pH 8.5 50 ~ 100 μ L of dopamine solution, in 4 DEG C react 1 ~
5 h are gently rinsed with water after reaction, are patted dry；0.1 ~ 0.5 mg/mL people's epididymal proteins, 4 antibody-solutions are added dropwise again, 4 DEG C incubate
8 ~ 12 h are educated, are rinsed；The bovine serum albumin solution that 50 ~ 100 μ L 1wt% ~ 5wt% are added is washed in 4 DEG C of 0.5 ~ 2 h of reaction
It washs, pats dry；The cleaning solution is phosphate buffer or Tris-HCl buffer solution；The electrogenerated chemiluminescence tests electrolysis
Matter solution is the buffer solution containing potassium peroxydisulfate.
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