CN106970212A - A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared - Google Patents
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared Download PDFInfo
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- CN106970212A CN106970212A CN201710231535.3A CN201710231535A CN106970212A CN 106970212 A CN106970212 A CN 106970212A CN 201710231535 A CN201710231535 A CN 201710231535A CN 106970212 A CN106970212 A CN 106970212A
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Abstract
The invention discloses a kind of electrogenerated chemiluminescence immunologic detection method of near-infrared, comprise the following steps：S1：Prepare reaction electrode；S2：Draw standard curve；S3：Prepare test serum；S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence signal intensity is determined using light-emitting appearance, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.Immunologic detection method proposed by the present invention rationally utilizes electrogenerated chemiluminescence technology, secondary antibody is marked the fluorescence probe that being chosen near infrared region has identification function, strengthen the Detection capability of light intensity signal, the detection speed of antigen concentration is fast, and human error is small, the degree of accuracy and sensitivity are high, it is simple to operate, it is easy to use, it is applied widely, can draw the chemiluminescence signal strength criterion curve of not synantigen as needed, and the standard curve drawn can in Valid concentration Reusability.
Description
Technical field
The present invention relates to the electrogenerated chemiluminescence immune detection side of technical field of biological, more particularly to a kind of near-infrared
Method.
Background technology
Electrogenerated chemiluminescence refers to produce some special materials in electrode surface by electrochemical method, these materials it
Between or between the other compositions in system by electron transmission formation excitation state, then by excitation state return to ground state produce it is luminous
Phenomenon.Electrochemiluminescprocess process technology without excitation source, and the characteristics of signal to noise ratio and high sensitivity because being widely used
In bioassay technique field.Common electrogenerated chemiluminescence system has Luminol, acridine ester type compound, polycyclic fragrance
Hydrocarbon compound etc., is concentrated mainly on visible region, and trivial operations of these systems in practical application, and processing procedure is superfluous
Long, efficiency is low.
The content of the invention
The invention aims to solve shortcoming present in prior art, and a kind of electroluminescentization of the near-infrared proposed
Learn electrochemiluminescent immunoassay detection method.
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared, comprises the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is carried out at normal temperatures anti-
Should, it is to be precipitated to be cleaned not in generation with dilution and Tween-20, then add into reactor containing fluorescence labeling two
It is anti-, continue to react, until precipitation is not regenerated, then cleaned with dilution and Tween-20, that is, reaction electrode is made；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and
Reaction electrode is tested using light-emitting appearance, chemiluminescence signal intensity is recorded, the antigen and chemistry for drawing various concentrations are sent out
The curve of light signal strength, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 35~45 DEG C of water-bath to preheat 10 minutes, and by the blood of preheating
Liquid fast transfer carries out high speed centrifugation into centrifuge 5~10 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence is determined using light-emitting appearance
Signal intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
It is preferred that, the volume ratio of antigen and dilution is 1 in the S1 steps：100~300.
It is preferred that, the addition of the Tween-20 is the 1~5% of dilution.
It is preferred that, the dilution is one kind in TBST and PBST.
It is preferred that, pH buffer is added in the dilution, and the pH value of dilution is 9.0~9.5.
It is preferred that, the secondary antibody of fluorescence labeling is that fluorescence probe is tagged in secondary antibody group in the S1 steps, and fluorescence
Probe is any one of silver sulfide quantum dot and copper sulfide quantum dot.
The immunologic detection method that the present invention is provided rationally utilizes electrogenerated chemiluminescence technology, and being chosen near infrared region has knowledge
Secondary antibody is marked the fluorescence probe of other function, strengthens the Detection capability of light intensity signal, makes faint light intensity signal equal
It can be gone out by effective detection, improve the degree of accuracy and sensitivity of detection method；The detection method is simple to operate, easy to use, is applicable
Scope is wide, can draw the chemiluminescence signal strength criterion curve of not synantigen as needed, and the standard curve drawn can be with
A kind of Reusability in Valid concentration, antigen draws a standard curve, when in use by testing sample concentration
It is diluted in the effective range of standard curve, the chemiluminescence signal intensity detected according to light-emitting appearance can quickly calculate to be measured
The concentration of antigen in sample, human error is small, and accuracy rate is high；And using dilution and told during prepared by reaction electrode
Temperature -20 is cleaned, and can effectively reduce influence of the unreacted antibody to testing result, improves the accuracy rate of detection.
Embodiment
The present invention is made with reference to specific embodiment further to explain.
Embodiment one
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared proposed by the present invention, comprises the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is carried out at normal temperatures anti-
Should, it is to be precipitated not in generation, cleaned with pH value for 9.0 TBST and Tween-20, then added into reactor containing vulcanization
The quantum dot-labeled secondary antibody of silver, continues to react, until precipitation is not regenerated, then is carried out with pH value for 9.0 TBST and Tween-20
Cleaning, that is, be made reaction electrode；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and
Reaction electrode is tested using light-emitting appearance, chemiluminescence signal intensity is recorded, the antigen and chemistry for drawing various concentrations are sent out
The curve of light signal strength, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 35 DEG C of water-bath to preheat 10 minutes, and the blood of preheating is fast
Speed, which is transferred in centrifuge, carries out high speed centrifugation 10 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence is determined using light-emitting appearance
Signal intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
In the present invention, using hepatitis B surface antigen as detection object, the volume ratio of antigen and dilution is in the S1 steps
1：100；The addition of the Tween-20 is the 2% of dilution.
Embodiment two
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared proposed by the present invention, comprises the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is carried out at normal temperatures anti-
Should, it is to be precipitated not in generation, cleaned with pH value for 9.5 PBST and Tween-20, then added into reactor containing vulcanization
The quantum dot-labeled secondary antibody of silver, continues to react, until precipitation is not regenerated, then is carried out with pH value for 9.5 PBST and Tween-20
Cleaning, that is, be made reaction electrode；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and
Reaction electrode is tested using light-emitting appearance, chemiluminescence signal intensity is recorded, the antigen and chemistry for drawing various concentrations are sent out
The curve of light signal strength, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 40 DEG C of water-bath to preheat 10 minutes, and the blood of preheating is fast
Speed, which is transferred in centrifuge, carries out high speed centrifugation 5 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence is determined using light-emitting appearance
Signal intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
In the present invention, the volume ratio of antigen and dilution is 1 in the S1 steps：300；The addition of the Tween-20
For the 5% of dilution
Embodiment three
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared proposed by the present invention, comprises the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is carried out at normal temperatures anti-
Should, it is to be precipitated not in generation, cleaned with pH value for 9.0 PBST and Tween-20, then added into reactor containing vulcanization
The quantum dot-labeled secondary antibody of copper, continues to react, until precipitation is not regenerated, then is carried out with pH value for 9.0 PBST and Tween-20
Cleaning, that is, be made reaction electrode；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and
Reaction electrode is tested using light-emitting appearance, chemiluminescence signal intensity is recorded, the antigen and chemistry for drawing various concentrations are sent out
The curve of light signal strength, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 45 DEG C of water-bath to preheat 10 minutes, and the blood of preheating is fast
Speed, which is transferred in centrifuge, carries out high speed centrifugation 7 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence is determined using light-emitting appearance
Signal intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
In the present invention, the volume ratio of antigen and dilution is 1 in the S1 steps：200；The addition of the Tween-20
For the 2% of dilution.
Example IV
A kind of electrogenerated chemiluminescence immunologic detection method of near-infrared proposed by the present invention, comprises the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is carried out at normal temperatures anti-
Should, it is to be precipitated not in generation, cleaned with pH value for 9.5 TBST and Tween-20, then added into reactor containing vulcanization
The quantum dot-labeled secondary antibody of copper, continues to react, until precipitation is not regenerated, then is carried out with pH value for 9.5 TBST and Tween-20
Cleaning, that is, be made reaction electrode；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and
Reaction electrode is tested using light-emitting appearance, chemiluminescence signal intensity is recorded, the antigen and chemistry for drawing various concentrations are sent out
The curve of light signal strength, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 40 DEG C of water-bath to preheat 10 minutes, and the blood of preheating is fast
Speed, which is transferred in centrifuge, carries out high speed centrifugation 5 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence is determined using light-emitting appearance
Signal intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
In the present invention, the volume ratio of antigen and dilution is 1 in the S1 steps：200；The addition of the Tween-20
For the 5% of dilution.
Above-described embodiment one~tri- is detected using with a collection of testing sample using the detection method of embodiment one~tri-
The concentration of hepatitis B surface antigen is as follows：
It is above-mentioned test result indicates that, using the detection method of embodiment one~tetra-, detect the content of hepatitis B surface antigen
Unanimously, and embodiment one~tetra- draw standard curve match, show the favorable reproducibility of the detection method, the mark once drawn
Directrix curve can be with Reusability, and the result precision determined is high, and error is small.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (6)
1. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared, it is characterised in that comprise the following steps：
S1：Prepare reaction electrode：Antigen and primary antibody are added into the reactor equipped with electrode, is reacted, treated at normal temperatures
Precipitation in generation, is not cleaned with dilution and Tween-20, then adds the secondary antibody containing fluorescence labeling into reactor, after
Continuous reaction, until precipitation is not regenerated, then is cleaned with dilution and Tween-20, that is, reaction electrode is made；
S2：Draw standard curve：Series reaction electrode is prepared by matrix of the antigen of various concentrations according to S1 steps, and is utilized
Light-emitting appearance is tested reaction electrode, records chemiluminescence signal intensity, and the antigen and chemiluminescence for drawing various concentrations are believed
The curve of number intensity, produces standard curve；
S3：Prepare test serum：Take blood to be measured to be placed in 35~45 DEG C of water-bath to preheat 10 minutes, and the blood of preheating is fast
Speed, which is transferred in centrifuge, carries out high speed centrifugation 5~10 minutes, produces test serum；
S4：Above-mentioned serum is taken, according to S1 step operations, electrode to be measured is prepared, and chemiluminescence signal is determined using light-emitting appearance
Intensity, the concentration of antigen in serum can be calculated according to the standard curve that S2 steps are drawn.
2. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared according to claim 1, it is characterised in that described
The volume ratio of antigen and dilution is 1 in S1 steps：100~300.
3. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared according to claim 1, it is characterised in that described
The addition of Tween-20 is the 1~5% of dilution.
4. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared according to claim 1, it is characterised in that described
Dilution is one kind in TBST and PBST.
5. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared according to claim 1, it is characterised in that described
PH buffer is added in dilution, and the pH value of dilution is 9.0~9.5.
6. the electrogenerated chemiluminescence immunologic detection method of a kind of near-infrared according to claim 1, it is characterised in that described
The secondary antibody of fluorescence labeling is that fluorescence probe is tagged in secondary antibody group in S1 steps, and fluorescence probe be silver sulfide quantum dot and
Any one of copper sulfide quantum dot.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108088816A (en) * | 2018-01-23 | 2018-05-29 | 深圳市国赛生物技术有限公司 | Small-sized specific protein analyzer |
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| CN102277157A (en) * | 2011-05-30 | 2011-12-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared silver sulphide quantum dot as well as preparation method and application thereof |
| CN102749452A (en) * | 2012-07-26 | 2012-10-24 | 山东大学 | Near-infrared electro-generated chemiluminescence immunodetection method |
| CN104830322A (en) * | 2015-04-12 | 2015-08-12 | 桂林理工大学 | High-stability near-infrared water-soluble CuS fluorescent quantum dots preparation method |
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2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102277157A (en) * | 2011-05-30 | 2011-12-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared silver sulphide quantum dot as well as preparation method and application thereof |
| CN102749452A (en) * | 2012-07-26 | 2012-10-24 | 山东大学 | Near-infrared electro-generated chemiluminescence immunodetection method |
| CN104830322A (en) * | 2015-04-12 | 2015-08-12 | 桂林理工大学 | High-stability near-infrared water-soluble CuS fluorescent quantum dots preparation method |
Non-Patent Citations (1)
| Title |
|---|
| RAN CUI 等: "Near-Infrared Electrogenerated Chemiluminescence of Ultrasmall Ag2Se Quantum Dots for the Detection of Dopamine", 《ANALYTICAL CHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108088816A (en) * | 2018-01-23 | 2018-05-29 | 深圳市国赛生物技术有限公司 | Small-sized specific protein analyzer |
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