CN101776683A - Luminescent and magnetic nano material-based method for detecting carcinoembryonic antigen - Google Patents

Luminescent and magnetic nano material-based method for detecting carcinoembryonic antigen Download PDF

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CN101776683A
CN101776683A CN201010022455A CN201010022455A CN101776683A CN 101776683 A CN101776683 A CN 101776683A CN 201010022455 A CN201010022455 A CN 201010022455A CN 201010022455 A CN201010022455 A CN 201010022455A CN 101776683 A CN101776683 A CN 101776683A
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antibody
cea
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fluorescence
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周兴平
李靖
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Donghua University
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Abstract

The invention relates to a luminescent and magnetic nano material-based method for detecting carcinoembryonic antigen (CEA), which comprises the following steps: (1) preparing a FE3O4/SiO2 nuclear shell-first antibody compound; (2) preparing a CdTe-second antibody compound; (3) drawing a 'fluorescence intensity-CEA concentration' standard working curve and calculating a working equation; and (4) detecting a fluorescence intensity value, and obtaining the content of the CEA of the patient through the working equation. The detection method has the advantages of simpleness and high sensitivity, and can easily detect the illness state of patients in the early stage of cancer.

Description

The detection method of the carcinomebryonic antigen of and magnetic Nano material luminous based on fluorescence nano
Technical field
The invention belongs to the detection method field of carcinomebryonic antigen (CEA), particularly relate to a kind of detection method of carcinomebryonic antigen of and magnetic Nano material luminous based on fluorescence nano.
Background technology
Carcinomebryonic antigen (CEA) is a kind of glycoprotein in the tissues such as fetus intestines, liver, pancreas, and molecular weight 150～2000kb is very low in the normal human serum, takes place to measure rising after the canceration.At present known other malignant tumours in colon cancer, the carcinoma of the rectum and entoderm source as oesophagus, stomach, liver, pancreas etc., have also raise when canceration.The index of normal person CEA is less than 5ng/ml, but smoker or groups of people's CEA is detected and is higher than 5ng/ml and belongs to normal level.So CEA is not the specific antigen of cancer, and should think a kind of related antigen of cancer.The CEA level in the blood measured can be used as cancer patient monitoring, determine the result of treatment quality.After tumor post-operation or the medication, the CEA level will descend in the blood, perform the operation successfully, and CEA will continue to descend until normal level.If operative failure, or cancer return CEA level will go back up to undesired level.The method of traditional detection CEA has: chemiluminescence immunoassay quantitative test, radio-immunity and immune radiating method, enzyme linked immunosorbent assay etc.These detection techniques are with fluorescein isothiocyanate (FITC), rhodamine (RB 200) mark specific antibody (Ab), do solid phase fluorescent dyeing then, determine antigenic content thereby survey its fluorescence intensity with microplate reader at last.But there are a lot of limitation in they aspect optical property and the photochemical stability, and are short as fluorescent lifetime, light stability is bad, excitation spectrum is short and emission spectrum is long.
Fe 3O 4Being a kind of oxide of important iron, is one of soft magnetic material that is most widely used.The Fe that exists with the magnetic fluid form 3O 4Nano particle has general liquid attribute and paramagnetism in liquid, under the situation of externally-applied magnetic field, directed moving can be taken place.And Fe 3O 4Nano particle can be connected with various biomolecules through finishing, under the effect of externally-applied magnetic field, can realize the immobilization or the purifying of these biomolecule.
The CdTe quantum dot is that a kind of fluorescence semiconductor is nanocrystalline, quantum dot have intensity height, good stability, can enough a kind of wavelength light source activation multi-wavelength, emission wavelength along with the increase of particle diameter fluorescent characteristic such as red shift regularly.The CdTe quantum dot good hydrophilic property that water is synthetic through after the finishing, can interact with various biomolecules.
Summary of the invention
Technical matters to be solved by this invention provides a kind of detection method of carcinomebryonic antigen of and magnetic Nano material luminous based on fluorescence nano, and this detection method is simple, and is highly sensitive, can allow the patient at a lot of cancer initial stages detect the state of an illness more simply.
The detection method of the carcinomebryonic antigen of a kind of and magnetic Nano material luminous based on fluorescence nano of the present invention comprises:
(1) Fe 3O 4/ SiO 2The preparation of nucleocapsid-first antibody compound
(a) coprecipitation method prepares Fe 3O 4Particle
Get ultrapure water, vacuumize inflated with nitrogen 20min behind the 20min, Fe 3+Salt and Fe 2+Salt is dissolved in the ultrapure water, obtains the precipitation of black to wherein adding excessive strong aqua again, separates with deionized water wash precipitation 3-5 time in conjunction with magnetic;
(b) sol-gel process prepares Fe 3O 4/ SiO 2Nucleocapsid structure
2.3ml absolute ethyl alcohol, 2.5mL deionized water and 0.7mg sodium oleate are fully mixed, add the 7mg ferroferric oxide nano granules, stir the back and add 10 μ L crosslinking chemical γ-glycidyl ether oxygen base oxypropyl trimethyl silane (GMPS), add 100 μ L ethyl orthosilicates (TEOS) behind the ultrasonic 15min, dropwise add strong aqua after stirring 1h, stir 20h and promptly obtain Fe 3O 4/ SiO 2The nano particle of nucleocapsid structure;
(c) Fe 3O 4/ SiO 2The core-shell nano particle combines with first antibody
With above-mentioned Fe 3O 4/ SiO 2Be dissolved in the toluene solution that 10ml contains 5% γ-glycidyl ether oxygen base oxypropyl trimethyl silane (GPMS), magnetic agitation reaction 12-24h, γ-glycidyl ether oxygen base oxypropyl trimethyl the silane (GPMS) that does not have coupling then with the potpourri washing of toluene and ethanol, be dissolved in afterwards in the 10ml PBS damping fluid, add anti-people's cancer embryo antibody then, under 4 ℃ of conditions, reaction 12-24h, separate in conjunction with magnetic with the PBS damping fluid again and wash 3 times, promptly make Fe 3O 4/ SiO 2-antibody complex;
(2) preparation of CdTe-second antibody compound
(a) Hydrothermal Preparation CdTe quantum dot
Be full of N 2The buffer solution of boric acid-sodium citrate in, add 0.04M CdCl respectively 22.5H 2O, 0.01MNa 2TeO 3, 0.12M mercaptoacetic acid (TGA), 20mgNaBH 4, under 95～100 ℃, boil 1h;
(b) preparation of CdTe-second antibody compound
In above-mentioned CdTe quantum dot, add 0.05M N-maloyl imines (NHS), activate 10min down at 37 ℃, add anti-people's cancer embryo antibody and 0.05M carbodiimide (EDC) again, under 37 ℃ of conditions, react 0.5h, add 1M glycocoll cessation reaction, use PBS damping fluid constant volume to 1ml at last, place 12-24h for 4 ℃; The molecule interception of packing into afterwards is in the bag filter of 10-12kD, the 12h that in concentration is the PBS damping fluid of 0.02% mercaptoacetic acid (TGA), dialyses, and every 4h changes damping fluid one time, will not have CdTe, NHS, glycocoll and the responseless EDC of combination to dialyse;
(3) " fluorescence intensity-CEA concentration " standard working curve and evaluation work equation
(a) get the above-mentioned " Fe of 100 μ l respectively 3O 4/ SiO 2-first antibody " mix with the antigen of 1.0ng/ml, 2.ng/ml, 4.0ng/ml, 10.0ng/ml, 15.0ng/ml, 20.0ng/ml, 30.0ng/ml, 40.0ng/ml, at room temperature react 30min;
(b) separate in conjunction with magnetic, material in the step (a) is washed 2 times with the PBS buffer solution of pH 7.4 respectively, constant volume adds above-mentioned " CdTe-second antibody " compound 200 μ l respectively then to 1.0ml, reacts 30min under the room temperature;
(c) separate in conjunction with magnetic, with the PBS buffer solution washing of pH 7.4 2 times, then with same buffer solution constant volume to 1ml, survey fluorescence;
(d) according to antigen concentration and fluorescence intensity relation, draw " fluorescence intensity-CEA concentration " standard working curve;
(e) according to above-mentioned working curve, calculate work equation: Y=A (X-B), Y is the concentration of CEA in the formula, and unit is ng/ml, and X is the relative intensity of fluorescence of sandwich compound, and unit is a.u.;
(4) measure gained fluorescence intensity numerical value obtains patient CEA by the work equation content.
The Fe of described step (1) in (a) 3+Salt is hydration FeCl 3, Fe 2+Salt is hydration FeSO 4Or FeCl 2, Fe 3+Salt and Fe 2+The salt mol ratio is 2: 1, and ammonia concn is 28%.
The Fe of described step (1) in (a) 3+Salt is FeCl 36H 2O, Fe 2+Salt is FeSO 47H 2O.
Described step (1) (c) or the anti-people cancer embryo antibody of step (2) in (b) be mouse-anti people cancer embryo antibody, the anti-people's cancer of rabbit embryo antibody, its amount is 100 μ L, the pH of PBS damping fluid is 7.4.
Described (2) CdCl in (a) 2With Na 2TeO 3Mol ratio be 6.0: 1.
The concentration of the boric acid of described step (2) in (a) is 15mM, and the concentration of sodium citrate is 15mM.
The present invention replaces FITC, RB by using the semiconductor-quantum-point of handling 200Mark specific antibody and the separation and purification of use magnetic, solve a series of problems of this immunoassay, and realize combining of fluorescence and magnetic Nano material and antibody with covalently bound mode, compare with the static combination, covalent bond is more stable, more help the enrichment object, thereby significantly simplify the testing process of carcinomebryonic antigen (CEA).
Beneficial effect
The present invention replaces with FITC, RB by the semiconductor-quantum-point that uses magnetic separation and purification and processing 200Mark specific antibody method, problems such as the bias light that solves this immunoassay disturbs, selectivity is bad, thereby enrichment object, can significantly simplify the detection means of carcinomebryonic antigen (CEA), this detection means is simple, and highly sensitive, can allow the patient at a lot of cancer initial stages detect the state of an illness more simply, and in time take treatment means, for the clinical detection of gastrointestinal cancer has been opened up a brand-new road.
Description of drawings
Fig. 1 is embodiment 1 prepared Fe 3O 4/ SiO 2The transmission electron microscope picture of core-shell nano particle (TEM);
Fig. 2 is embodiment 3 gained Fe 3O 4/ SiO 2With the infared spectrum situation of change before and after the antibodies;
Fig. 3 is that embodiment 4 prepared antibody combine front and back fluorescence spectrum variation diagram with quantum dot;
" fluorescence intensity-CEA concentration " graph of relation that Fig. 4 is drawn for embodiment 5.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
(1) Fe 3O 4/ SiO 2The preparation of core-shell nano particle:
1. take by weighing the FeCl of 1.08g 36H 2The FeSO of O and 0.555g 47H 2O is dissolved in the 20ml ultrapure water, vacuumizes 20min, N 2Saturated 20min; Add excessive ammonia at it and obtain black precipitate, separate, clean for several times with ultrapure water in conjunction with magnetic.
2. go step 1 product 7mg fully to mix with 2.3mL absolute ethyl alcohol, 2.5mL deionized water and 0.7mg sodium oleate, magneton stirs and adds the GMPS of 10 μ L.
3. will add TEOS 100 μ L behind the ultrasonic 15min of step 2 potpourri, dropwise add 28% ammoniacal liquor 10.2mL after the stirring 1h, and stir 20h and promptly obtain Fe 3O 4/ SiO 2The core-shell nano particle separates in conjunction with magnetic, cleans for several times with ultrapure water.
(2) CdTe quantum dot preparation:
1. 40ml is comprised and vacuumize 20min earlier in the buffer solution of 15mM boric acid and 15mM sodium citrate and charge into nitrogen 20min again.
2. under magnetic agitation, add 0.04M CdCl successively 22.5H 2O solution, 0.01M Na 2TeO 3Solution, 1.2M TGA and 20mgNaBH 4
3. magnetic agitation reaction 5min forms limpid green solution.The 1h that refluxes in 95～100 ℃ of boiling water baths promptly prepares the CdTe quantum dot.
(3) " Fe 3O 4/ SiO 2-first antibody " preparation of compound:
1. with the Fe that makes 3O 4/ SiO 2Be dissolved in the toluene solution that 10ml contains 5%GPMS, the magnetic agitation reaction overnight does not have the GPMS of coupling then with the potpourri washing of toluene and ethanol.
2. with the Fe of above-mentioned preparation 3O 4/ SiO 2-GPMS is dissolved in the 10ml PBS damping fluid, adds 100 μ L antibody, and under 4 ℃ of conditions, reaction overnight is separated in conjunction with magnetic with the PBS damping fluid and washed 3 times, promptly makes Fe 3O 4/ SiO 2-GPMS-IgG compound.
Add 200 μ l, 5% BSA again, spend the night under 4 ℃ of conditions, the group of participating in reaction just has been closed;
(4) preparation of " CdTe-second antibody " compound:
1.500 add N-maloyl imines (NHS) among the μ l CdTe, activate 10min down at 37 ℃, add 100 μ l antibody and carbodiimide (EDC) again, under 37 ℃ of conditions, react 0.5h, add the glycocoll cessation reaction, use PBS damping fluid constant volume to 1ml at last.
2. above-mentioned sample is put 4 ℃ of refrigerator overnight.The molecule interception of packing into then is in the bag filter of 10-12kD, the 12h that in the PBS damping fluid of mercaptoacetic acid (TGA), dialyses, and every 4h changes damping fluid one time.So just can will there be CdTe, NHS, glycocoll and the responseless EDC of combination to dialyse.
(5) preparation of " fluorescence intensity-CEA concentration " curve:
1. get " the Fe of 100 μ l respectively 3O 4/ SiO 2-first antibody " with 1.0,2.0,4.0,10.0,15.0,20.0,30.0, the antigen of 40.0ng/ml mixes, and at room temperature reacts 30min;
2. separate in conjunction with magnetic, the PBS buffer solution of each the miniature centrifuge tube sample in the step 1 with pH 7.4 is washed 2 times, fixed molten then to 1.0ml, add " CdTe-second antibody " compound 200 μ l then respectively, react 30min under the room temperature;
3. separate in conjunction with magnetic, with the careful washing of the PBS buffer solution of pH 7.4 2 times, then with same buffer solution constant volume to 1ml, survey fluorescence;
4. according to antigen concentration and fluorescence intensity relation, draw " fluorescence intensity-CEA concentration " standard working curve;
5. according to the working curve of step 4, calculate work equation: Y=A (X-B), Y is the concentration of CEA in the formula, and unit is ng/ml, and X is the relative intensity of fluorescence of sandwich compound.
Embodiment 2
Detect the content of CEA among the patients serum:
1. get serum 1ml, with " the Fe of 100 μ l 3O 4/ SiO 2-first antibody " mix, at room temperature react 30min;
2. separate in conjunction with magnetic, the PBS buffer solution of each the miniature centrifuge tube sample in the step 1 with pH 7.4 is washed 2 times, fixed molten then to 1.0ml, add " CdTe-second antibody " compound 200 μ l then respectively, react 30min under the room temperature;
3. separate in conjunction with magnetic,, use this buffer solution constant volume, survey fluorescence to 0.5ml with the careful washing of the PBS buffer solution of pH 7.4 2 times;
4. resulting result is brought among the work equation Y=A (X-B), calculate the content of CEA in the serum, and, see Table 1 result and the contrast of immunofluorescence testing result.
Table 1 detection method of the present invention and the contrast of immunofluorescence testing result
Figure G2010100224555D00061

Claims (6)

1. the detection method of the carcinomebryonic antigen CEA of an and magnetic Nano material luminous based on fluorescence nano comprises:
(1) Fe 3O 4/ SiO 2The preparation of nucleocapsid one first antibody compound
(a) get ultrapure water, vacuumize inflated with nitrogen 20min behind the 20min, Fe 3+Salt and Fe 2+Salt is dissolved in the ultrapure water, obtains the precipitation of black to wherein adding excessive strong aqua again, separates with deionized water wash precipitation 3-5 time in conjunction with magnetic, must Fe 3O 4Particle;
(b) 2.3ml absolute ethyl alcohol, 2.5mL deionized water and 0.7mg sodium oleate are fully mixed, add the 7mg ferroferric oxide nano granules, stir the back and add 10 μ L crosslinking chemical γ-glycidyl ether oxygen base oxypropyl trimethyl silane GMPS, add 100 μ L ethyl orthosilicate TEOS behind the ultrasonic 15min, dropwise add strong aqua after stirring 1h, stir 20h, promptly get Fe 3O 4/ SiO 2The nano particle of nucleocapsid structure;
(c) with above-mentioned Fe 3O 4/ SiO 2The nano particle of nucleocapsid structure is dissolved in the toluene solution that 10ml contains 5% γ-glycidyl ether oxygen base oxypropyl trimethyl silane GPMS, magnetic agitation reaction 12-24h, γ-glycidyl ether oxygen base oxypropyl trimethyl silane the GPMS that does not have coupling then with the potpourri washing of toluene and ethanol, be dissolved in afterwards in the 10ml PBS damping fluid, add anti-people's cancer embryo antibody then, under 4 ℃ of conditions, reaction 12-24h, separate in conjunction with magnetic with the PBS damping fluid again and wash 3 times, promptly get Fe 3O 4/ SiO 2-antibody complex;
(2) preparation of CdTe-second antibody compound
(a) be full of N 2The buffer solution of boric acid-sodium citrate in, add 0.04M CdCl respectively 22.5H 2O, 0.01MNa 2TeO 3, 0.12M mercaptoacetic acid TGA, 20mg NaBH 4, under 95～100 ℃, boil 1h, promptly get the CdTe quantum dot;
(b) in above-mentioned CdTe quantum dot, add 0.05M N-maloyl imines NHS, activate 10min down at 37 ℃, add anti-people's cancer embryo antibody and 0.05M carbodiimide EDC again, under 37 ℃ of conditions, react 0.5h, add 1M glycocoll cessation reaction, use PBS damping fluid constant volume to 1ml at last, place 12-24h for 4 ℃; The molecule interception of packing into afterwards is in the bag filter of 10-12kD, the 12h that in concentration is the PBS damping fluid of 0.02% mercaptoacetic acid TGA, dialyses, and every 4h changes damping fluid one time, promptly gets CdTe-second antibody compound;
(3) " fluorescence intensity-CEA concentration " standard working curve and evaluation work equation
(a) get the above-mentioned " Fe of 100 μ l respectively 3O 4/ SiO 2-first antibody " mix with the antigen of 1.0ng/ml, 2.ng/ml, 4.0ng/ml, 10.0ng/ml, 15.0ng/ml, 20.0ng/ml, 30.0ng/ml, 40.0ng/ml, at room temperature react 30min;
(b) separate in conjunction with magnetic, material in the step (a) is washed 2 times with the PBS buffer solution of pH 7.4 respectively, constant volume adds above-mentioned " CdTe-second antibody " compound 200 μ l respectively then to 1.0ml, reacts 30min under the room temperature;
(c) separate in conjunction with magnetic, with the PBS buffer solution washing of pH 7.4 2 times, then with same buffer solution constant volume to 1ml, survey fluorescence;
(d) according to antigen concentration and fluorescence intensity relation, draw " fluorescence intensity-CEA concentration " standard working curve;
(e) according to above-mentioned working curve, calculate work equation: Y=A (X-B), Y is the concentration of CEA in the formula, and unit is ng/ml, and X is the relative intensity of fluorescence of sandwich compound, and unit is a.u.;
(4) measure gained fluorescence intensity numerical value obtains patient CEA by the work equation content.
2. the detection method of the carcinomebryonic antigen CEA of a kind of and magnetic Nano material luminous based on fluorescence nano according to claim 1 is characterized in that: the Fe of described step (1) in (a) 3+Salt is hydration FeCl 3, Fe 2+Salt is hydration FeSO 4Or FeCl 2, Fe 3+Salt and Fe 2+The salt mol ratio is 2: 1, and ammonia concn is 28%.
3. the detection method of the carcinomebryonic antigen CEA of a kind of and magnetic Nano material luminous based on fluorescence nano according to claim 1 is characterized in that: the Fe of described step (1) in (a) 3+Salt is FeCl 36H 2O, Fe 2+Salt is FeSO 47H 2O.
4. the detection method of the carcinomebryonic antigen CEA of a kind of and magnetic Nano material luminous according to claim 1 based on fluorescence nano, it is characterized in that: described step (1) (c) or the anti-people cancer embryo antibody of step (2) in (b) be mouse-anti people cancer embryo antibody, the anti-people's cancer of rabbit embryo antibody, its amount is 100 μ L, and the pH of PBS damping fluid is 7.4.
5. the detection method of the carcinomebryonic antigen CEA of a kind of and magnetic Nano material luminous based on fluorescence nano according to claim 1 is characterized in that: described (2) CdCl in (a) 2With Na 2TeO 3Mol ratio be 6.0: 1.
6. the detection method of the carcinomebryonic antigen CEA of a kind of and magnetic Nano material luminous based on fluorescence nano according to claim 1 is characterized in that: the concentration of the boric acid of described step (2) in (a) is 15mM, and the concentration of sodium citrate is 15mM.
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Application publication date: 20100714