CN107238707A - A kind of homogeneous detection SCCA1 immunofluorescence technique - Google Patents
A kind of homogeneous detection SCCA1 immunofluorescence technique Download PDFInfo
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- CN107238707A CN107238707A CN201710392300.2A CN201710392300A CN107238707A CN 107238707 A CN107238707 A CN 107238707A CN 201710392300 A CN201710392300 A CN 201710392300A CN 107238707 A CN107238707 A CN 107238707A
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Abstract
The invention discloses a kind of homogeneous detection SCCA1 immunofluorescence technique, belong to nano immune analysis technical field.The main FRET detection SCCA1 using between quantum dot and dyestuff, comprises the following steps：CdS quantum dot, rhodamine B or red fluorescent microspheres mark the preparation of SCCA1 pairing antibody, i.e. probe 1 and probe 2 respectively；It is prepared by the FRET system of 1 SCCA1 probes of probe 2；SCCA1 detection.The features such as this method has simple, quick, sensitive, high selectivity, can be applied to the detection of tumor marker in clinical medicine.
Description
Technical field
The invention discloses a kind of homogeneous detection SCCA1 immunofluorescence technique, belong to nano immune analysis technical field..
Background technology
FRET（FRET）Refer in two different fluorophors, if a fluorophor（Donor
Donor）Emission spectrum and another group（Acceptor Acceptor）Absorption spectrum have certain overlapping, two fluorophors
Between distance it is suitable when（Generally less than 10 nm）, you can it was observed that the phenomenon that fluorescent energy is shifted from donor to acceptor.
Quantum dot（QDs）It is that diameter is about 2 ~ 20nm, the semiconductor nanoparticle that exciting light produces fluorescence can be received.Pass
What the FRET of system was studied focuses primarily upon organic fluorescence molecular number, and as a kind of new FRET reagents, QDs has many organic glimmering
The advantage that optical molecule does not possess, if any tunable excitation wave, bleach-resistant, multiple dye molecules while connection, narrow hair
Ejected wave length and the excellent optical property such as excite away from emission peak.Therefore, QDs can turn into more more preferable than organic fluorescence molecule
FRET donor or acceptor.
Fluorescence energy transfer system luminous intensity is big, and sensitivity is high, and method is simple.
The content of the invention
It is an object of the invention to provide a kind of homogeneous detection SCCA1 immunofluorescence technique, using fluorescence match dyestuff it
Between the quick homogeneous detection broad-spectrum tumor label SCCA1 of FRET.This method can the realization pair in homogeneous
SCCA1 detection, and have sensitivity high SCCA1 detection and the characteristics of selectivity is good.
The present invention uses following technical scheme：
A kind of SCCA1 detection methods based on FRET, comprise the following steps：
First, the preparation of probe 1 and probe 2
A, CdS preparation
It is kept stirring in building-up process in the system of state, by 0.1 mol/L CdCl2The mL of solution 2 ~ 8 is injected into 100 mL and gone
In ionized water, 0.2 ~ 2.0 mmol TGA is added as dressing agent, with 1 mol/L sodium hydroxide solution after 2 ~ 5min
Regulation system pH value is to 9 ~ 11, and solution system is in water white transparency, is subsequently added the Na that 0.5 ~ 4 mL concentration is 0.1 mol/L2S water
Solution, produces CdS quantum dot.
B, CdS quantum dot is taken with after the dilution of 0.05M MES cushioning liquid, to add EDC and NHS respectively（QDs:EDC's rubs
You are than being 1:50~1:200；EDC:NHS mol ratio is 1:1~1:5）, uniformly, room temperature activates 15~60 to vortex oscillation
min；The quantum dot of activation is centrifuged after 10 min, ultrafiltration purification with 30 KD super filter tubes under the conditions of 8000 r/min, 4 DEG C, plus
Enter PBS7.4 dilutions, add corresponding monoclonal SCCA1 antibody-solutions（The mol ratio of QDs and antibody is 1:1~10:1）, 4 DEG C
Reaction is stayed overnight, and produces probe 1.
C, take fluorescent material（Rhodamine B or red fluorescent microspheres）After being diluted respectively with 0.05 M MES cushioning liquid, plus
Enter EDC and NHS（Fluorescent material:EDC mass ratio is 1:50~1:200；EDC:NHS mol ratio is 1:1~1:5）, it is vortexed
Shaken well, room temperature activates 15~60 min；By the fluorescent material of activation with 30 KD super filter tubes in 8000 r/min, 4 DEG C of conditions
After 10 min of lower centrifugation, ultrafiltration purification, PBS7.4 dilutions are added, corresponding monoclonal SCCA1 antibody-solutions are added（Fluorescent material
Mass ratio with antibody is 100:1~10:1）, 4 DEG C of reactions stay overnight, and produce probe 2.
Wherein, rhodamine excitation wavelength 525-550nm, the nm of launch wavelength 580；Red fluorescent microspheres excitation wavelength 520~
535 nm, the nm of launch wavelength 600.Rhodamine B is purchased from sigma；It is biological for degree that red fluorescent microspheres are purchased from Suzhou.
2nd, prepared by the FRET system of probe 1-SCCA1- probes 2
The principle of the FRET system of probe 1-SCCA1- probes 2 as described in Figure 1, by probe 1, probe 2 with
SCCA1 formation sandwich structures, make the mark fluorescent material of probe 1,2 occur energy fluorescence and shift, and this change in fluorescence amount
Size and SCCA1 content be in certain linear relationship.
The FRET system of probe 1-SCCA1- probes 2 is：
Sample（Standard items or sample to be tested） 2 μL
The μ L of 0.05mg/mL probes 1 20
The μ L of 0.05mg/mL probes 2 10
The μ L of PBS7.4 buffer solutions 50
ddH2O complements to 100 μ L.
3rd, SCCA1 working curves
Concentration is first contacted mixed with probe 1, PBS respectively for the standard items sample of 0.05 ~ 40 ng/mL various concentrations
After even, probe 2, ultra-pure water constant volume to 100 μ L are separately added into.Then normal temperature is rotated after 30 ~ 60 min, the system is placed in many
Fluorescence intensity on function ELIASA.Mixed liquor is recorded in the maximum emission wavelength of probe 2（At 580 nm or 600 nm）It is glimmering
Luminous intensity, F when there is SCCA1, F during in the absence of SCCA10, with (F-F0) standard curve is drawn to SCCA1 concentration.
The fluorescence signal of sample to be tested is detected by the FRET system of probe 1-SCCA1- probes 2, will be treated
The fluorescence signal value of test sample product substitutes into SCCA1 standard curves, draws the content of SCCA1 in testing sample.
This method is simple to operate, it is not necessary to which the antibody being not connected with hybridization system is separated, and realizes homogeneous detection；By
Fluorescence signal will not be directly produced in the case where 364 nm light are excited in rhodamine B, red fluorescent microspheres, therefore ambient interferences are small, letter
It is number strong.This method also has special, quick, high selectivity and high-throughout feature, the wide spectrum available for tumour in clinical medicine
Examination.
Brief description of the drawings
Fig. 1 is FRET（FRET）Detect SCCA1 principle schematic.
Fig. 2 is the SCCA1 of different systems（0,0.05,0.1,0.5,1,2,5,10,20,40）It is strong to FRET system fluorescence
The influence (quantum dot-rhodamine B pairing fluorescent dye) of degree.
Fig. 3 is CEA canonical plotting, with（F-F0）Standard curve is set up with CEA concentration.
Embodiment
Embodiment is given below so that the present invention will be described in more detail, it is necessary to be pointed out that following examples can not be managed
Solve as limiting the scope of the invention, the person skilled in the art in the field makes according to the invention described above content to the present invention
Some nonessential modifications and adaptations still belong to protection scope of the present invention.
Embodiment
The probe 1 of embodiment 1, the preparation of probe 2
Using CdS and rhodamine B as pairing dyestuff, SCCA1 pairing antibody is marked respectively, probe 1 and probe 2 are prepared, for SCCA1
Detection.
First, the preparation of probe 1
It is kept stirring in building-up process in the system of state, by 0.1mol/L CdCl2The mL of solution 2 ~ 8 is injected into 100 mL and gone
In ionized water, 0.2 ~ 2.0 mmol TGA is added as dressing agent, with 1 mol/L sodium hydroxide solution after 2 ~ 5min
Regulation system pH value is to 9 ~ 11, and solution system is in water white transparency, is subsequently added the Na that 0.5 ~ 4 mL concentration is 0.1 mol/L2S water
Solution, produces CdS quantum dot（Its excitation wavelength 364 nm, the nm of launch wavelength 530）.
CdS quantum dot is taken with after 0.05 M MES cushioning liquid dilution, to add EDC and NHS respectively（QDs:EDC's rubs
You are than being 1:50；EDC:NHS mol ratio is 1:2.5）, uniformly, room temperature activates 20 min to vortex oscillation；By the quantum of activation
Point is centrifuged after 10 min, ultrafiltration purification with 30 KD super filter tubes under the conditions of 8000 r/min, 4 DEG C, adds PBS7.4 dilutions, plus
Enter corresponding monoclonal SCCA1 antibody-solutions（The mol ratio of QDs and antibody is 4:1）, 4 DEG C of reactions stay overnight, and produce probe 1.
2nd, the preparation of probe 2
Rhodamine B is taken with after 0.05 M MES cushioning liquid dilution, to add EDC and NHS respectively（Rhodamine B:EDC quality
Than for 1:50；EDC:NHS mol ratio is 1:2.5）, uniformly, room temperature activates 15 min to vortex oscillation；By the rhodamine B of activation
Centrifuged with 30 KD super filter tubes under the conditions of 8000 r/min, 4 DEG C after 10 min, ultrafiltration purification, add PBS7.4 dilutions, add
Corresponding monoclonal SCCA1 antibody-solutions（The mass ratio of rhodamine B and antibody is 10:1）, 4 DEG C of reactions stay overnight, and produce probe 2.
Wherein, the nm of rhodamine excitation wavelength 525 ~ 550, the nm of launch wavelength 580.
Embodiment 2 homogeneously detects SCCA1 immunofluorescence technique
Specification Curve of Increasing：First by concentration for 0.05 ~ 40 ng/mL various concentrations each 2 μ L of standard items sample respectively with 20
μ L concentration is after 0.05 mg/mL probes 1,50 μ LPBS7.4 Buffer fluid contacts are mixed, to be separately added into 10 μ L concentration for 0.05
The system then after 30 ~ 60 min of normal temperature rotation, is finally placed in multi-functional by mg/mL probes 2, ultra-pure water constant volume to 100 μ L
Fluorescence intensity on ELIASA.Mixed liquor is recorded in the maximum emission wavelength of probe 2（At 580 nm or 600 nm）Fluorescence it is strong
Degree, F when there is SCCA1, F during in the absence of SCCA10, with（F-F0）Standard curve is drawn to SCCA1 concentration（See Fig. 3）.
Pattern detection：The fluorescence of sample to be tested is detected by the FRET system of probe 1-SCCA1- probes 2
Signal, substitutes into SCCA1 standard curves by the fluorescence signal value of testing sample, draws the content of SCCA1 in testing sample.
Claims (8)
1. a kind of homogeneous detection SCCA1 immunofluorescence technique, it is characterised in that utilize fluorescence resonance energy between fluorescence pairing dyestuff
The quick homogeneous detection broad-spectrum tumor label SCCA1 of amount transfer, comprises the following steps：The preparation of probe 1 and probe 2；Probe 1-
It is prepared by the FRET system of SCCA1- probes 2；SCCA1 working curve；Test sample.
2. the immunofluorescence technique of the homogeneous detection SCCA1 described in claim 1, it is characterised in that fluorescence pairing dyestuff is CdS
QDs- rhodamine Bs pairing dyestuff, CdS QDs- red fluorescent microspheres pairing dyestuff.
3. the immunofluorescence technique of the homogeneous detection SCCA1 described in claim 2, it is characterised in that CdS QDs emission maximum light
Spectrum has preferably overlapping with the maximum absorption spectrum of rhodamine B, red fluorescent microspheres.
4. the immunofluorescence technique of the homogeneous detection SCCA1 described in claim 1, it is characterised in that probe 1 marks for CdS QDs
SCCA1 antibody, probe 2 is the SCCA1 antibody that rhodamine B or red fluorescent microspheres are marked.
5. the immunofluorescence technique of the homogeneous detection SCCA1 described in claim 4, it is characterised in that probe 1 and probe 2 are marked
SCCA1 antibody for pairing antibody.
6. the SCCA1 detection methods based on FRET described in claim 1, it is characterised in that probe 1-
The FRET system of SCCA1- probes 2 is：
Sample（Standard items or sample to be tested） 2 μL
The μ L of 0.05mg/mL probes 1 20
The μ L of 0.05mg/mL probes 2 10
The μ L of PBS 50
ddH2O complements to 100 μ L.
7. the SCCA1 detection methods according to claim 6 based on FRET, it is characterised in that fluorescence is common
Energy system of shaking detects that SCCA1 working curve step is：First various concentrations standard items sample is buffered with probe 1, PBS respectively
After liquid contact is mixed, probe 2, ultra-pure water constant volume to 100 μ L, then after 30 ~ 60 min of normal temperature rotation, by the body are separately added into
System is placed in fluorescence intensity on multi-function microplate reader, and record mixed liquor is in the maximum emission wavelength of probe 2（580 nm or 600 nm
Place）Fluorescence intensity, F when there is SCCA1, F during in the absence of SCCA10, with（F-F0）Standard curve is drawn to SCCA1 concentration.
8. the SCCA1 detection methods based on FRET described in claim 7, it is characterised in that testing sample is examined
Survey method is that the fluorescence signal of sample to be tested is detected by the FRET system of probe 1-SCCA1- probes 2, will be treated
The fluorescence signal changing value of test sample product substitutes into SCCA1 standard curves, draws the content of SCCA1 in testing sample.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051421A (en) * | 2018-01-18 | 2018-05-18 | 上海交通大学 | The method of recombined milk is mixed based on two-dimentional external source fluorescence spectrum combination principal component analysis detection fresh milk |
| CN112391158A (en) * | 2019-11-08 | 2021-02-23 | 重庆科技学院 | Multicolor fluorescent probe for detecting early markers of cardiac injury and preparation and use methods thereof |
| CN112763463A (en) * | 2020-11-05 | 2021-05-07 | 北京工业大学 | Method for improving sensitivity of whispering gallery mode microcavity sensing |
Citations (1)
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|---|---|---|---|---|
| CN101519696A (en) * | 2009-02-19 | 2009-09-02 | 中国人民解放军第三军医大学第一附属医院 | Nucleic acid sensor based on quantum dots and preparation method and detection method thereof |
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2017
- 2017-05-27 CN CN201710392300.2A patent/CN107238707A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519696A (en) * | 2009-02-19 | 2009-09-02 | 中国人民解放军第三军医大学第一附属医院 | Nucleic acid sensor based on quantum dots and preparation method and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| SULE CATALTEPE ET AL: "Development of specific monoclonal antibodies and a sensitive discriminatory immunoassay for the circulating tumor markers SCCA1 and SCCA2", 《CLINA CHIMICA ACTA》 * |
| 陈玫君等: "基于量子点的新型匀相荧光免疫分析技术", 《中国科技论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051421A (en) * | 2018-01-18 | 2018-05-18 | 上海交通大学 | The method of recombined milk is mixed based on two-dimentional external source fluorescence spectrum combination principal component analysis detection fresh milk |
| CN112391158A (en) * | 2019-11-08 | 2021-02-23 | 重庆科技学院 | Multicolor fluorescent probe for detecting early markers of cardiac injury and preparation and use methods thereof |
| CN112763463A (en) * | 2020-11-05 | 2021-05-07 | 北京工业大学 | Method for improving sensitivity of whispering gallery mode microcavity sensing |
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