CN102636649B - Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material - Google Patents
Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material Download PDFInfo
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- CN102636649B CN102636649B CN201210062667.5A CN201210062667A CN102636649B CN 102636649 B CN102636649 B CN 102636649B CN 201210062667 A CN201210062667 A CN 201210062667A CN 102636649 B CN102636649 B CN 102636649B
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Abstract
The invention relates to a kit for detecting a carcinoembryonic antigen (CEA) based on an antibody functionalized magnetic nanometer material and an up-conversion fluorescence nanometer material. The kit provided by the invention is characterized in that an anti-CEA first antibody (capturing antibody) functionalized magnetic nanometer material is used to enrich and separate CEAs in a sample; moreover, the up-conversion fluorescence nanometer material NaY0.78F4:Yb0.20 is marked with an anti-CEA second antibody (detecting antibody) based on the characteristics of the high laser-induced fluorescence sensitivity of the up-conversion fluorescence nanometer material and the capability of effectively avoiding biological background fluorescence interference of the sample, and Ho0.02 is used as a detecting probe; and the high-sensitively, stable and rapid CEA detecting method is established by combining the two nanometer materials and an antigen-antibody specificity reaction principle. The kit can be used for detecting the content of CEA in clinic or in scientific researches and is used for auxiliary tumor diagnosis, guide therapy and prompt prognosis.
Description
Technical field
The invention belongs to nano material and biological technical field, the kit based on antibody function magnetic Nano material and up-conversion fluorescence nano material conjugated antigen-antibody specific reaction, carcinomebryonic antigen (CEA) being detected for one.Feature is that the magnetic Nano material of anti-CEA first antibody (capture antibody) functionalization carries out separation and concentration to antigen in sample, and detection sensitivity is improved; The up-conversion fluorescence nano material NaY that simultaneously utilizes anti-CEA second antibody (detection antibody) to modify
0.78f
4: Yb
0.20, Ho
0.02the thing that serves as a mark induces up-conversion fluorescence signal further to improve the sensitivity of detection by detection laser.
Background technology
Antigen, refers to and can stimulate body to produce specific immune response, and can with immune response product---antibody and sensitized lymphocyte are combined in vivo and in vitro, there is the material of specific reaction.Tumor associated antigen refers in kinds of tumor cells to express to be increased, and in normal cell the antigen molecule of microsynthesis only.Carcinomebryonic antigen (CEA) is the most frequently used tumor associated antigen, and it is a kind of acidoglycoprotein, and synthesize embryonic period, embryonic phase at small intestine, liver, pancreas, and in normal human serum, only trace exists.CEA is considered to the mark (60%-90% patient's rising) of colorectal cancer the earliest, but finds also have higher CEA to express in cancer of pancreas (～80%), cancer of the stomach (～60%), lung cancer (～75%) and breast cancer (～60%) patients serum later.CEA is as a broad spectrum activity tumor markers, and curative effect judgement, PD, monitoring and the prognosis to tumor in digestive tract and lung cancer estimated according to all having important value.Change of serum C EA detects has become the most widely used tumour auxiliary diagnosis and prognosis judging means at present.
Utilizing the detection method of Ag-Ab specific reaction principle exploitation antigen, antibody is the basic ideas of current immunology detection, is also the basis that the various tumor associated antigens including CEA detect.Traditional CEA detection method mainly contains the methods such as enzyme-linked immuno assay (ELISA), electrochemiluminescence immunoassay, radiommunoassay.These methods all exist not enough separately at aspects such as optical property, stability, susceptibility, security, detection time or instrument requirements.As all not good enough in elisa assay sensitivity, stability and repeatability; Although electrochemiluminescence immunoassay has improved detection sensitivity, exist functional electrode to prepare the deficiency such as difficulty and running time length; Radiommunoassay because need radiation proof drawbacks limit its large-scale promotion.So be necessary that development is a kind of quick and convenient, good stability, highly sensitive, high specificity, the novel detection method that use cost is low.
Relation between nano material and immunological technique is more and more tightr in recent years, and the development prospect of novel nano-material in biomedicine detects is also more and more wide.Wherein upload and change fluorescent nano material and its unique characteristic of magnetic Nano material enjoys people to pay close attention to.In rear-earth-doped inorganic nano material, there is the special luminescent material of a class, it can convert shortwave radiation to long-wave radiation (near infrared light) by multi-photon mechanism, launches the fluorescence (ultraviolet-visible) than excitation light wave length, i.e. up-conversion fluorescence.Up-conversion has following plurality of advantages: photochemical stable, detection background is low, and penetrability is strong, and can be by selecting different host materials and doping ion to regulate nano particle to launch different up-conversion fluorescences, really realize single excitation multi and penetrate, thereby be conducive to detect polycomponent simultaneously.In view of above-mentioned advantage, up-conversion nano material has become a kind of outstanding biological labled material at present.Meanwhile, in the detection of some biological sample, because measured target content of material is very low, the separating step complexity in mark and detection, the separation of mark sample and bioaccumulation efficiency will directly affect sensitivity and the accuracy of detection.Fe
3o
4the magnetic material being most widely used, the Fe existing with magnetic fluid form in liquid
3o
4can, under the effect of exterior magnetic field, there is displacement in magnetic-particle.And Fe
3o
4the stable performance of magnetic magnetic nano-particle, more easily preparation, can be connected with various biomolecules through finishing, can be under the effect of externally-applied magnetic field, the immobilization or the enrichment that realize these molecules are concentrated.Therefore, utilize magnetic Nano material can fast and effeciently separate and enrichment target substance with up-conversion nano material, have specificity high, separate fast, high repeatability and other advantages.
The first synthetic Fe that has obtained surface biological functionalization of the present invention
3o
4magnetic Nano material and NaY
0.78f
4: Yb
0.20, Ho
0.02up-conversion fluorescence nano material, utilize antigen-antibody reaction specificity to catch the CEA in sample to be tested by the anti-CEA first antibody (capture antibody) of magnetic Nano material combination again, and separate in addition concentration and separation by magnetic, then be combined with the up-conversion fluorescence nano particle of anti-CEA second antibody (detection antibody) mark and form CEA capture antibody/CEA/CEA and detect the nano-particle complex of antibody sandwich structure sample.Taking 980nm laser excitation induction up-conversion fluorescence as detection signal, with the Criterion curve of variable concentrations CEA standard items.Under 980nm induced with laser, NaY
0.78f
4: Yb
0.20, Ho
0.02the luminous signal (wavelength is 542nm) that up-conversion fluorescence nano material produces and the amount positive correlation of CEA, to reach the object to carrying out containing CEA sample quantitatively detecting.This invention can be used for the mensuration of CEA content in human serum sample.
Summary of the invention
The present invention is a kind of kit that detects CEA based on antibody function magnetic Nano material magnetic separation-up-conversion fluorescence nano material mark, and described kit comprises:
As the magnetic Nano material of anti-CEA first antibody (capture antibody) functionalization of antigen capture and magnetic separation agent; Finishing anti-CEA second antibody (detection antibody) up-conversion fluorescence nano material form fluorescence probe; Positive control; Negative control; Antigen standard items.
CEA standard items are the CEA antigen freeze-dried powder of known quality, the human serum of positive control for containing finite concentration (1-10ng/mL) CEA, and negative control is PBS damping fluid (pH=7.4).
Prepare respectively NaY
0.78f
4: Yb
0.20, Ho
0.02up-conversion fluorescence nano material and amidized Fe
3o
4magnetic nano-balls, carries out surface-functionalized modification to nano particle, subsequently by amidized Fe
3o
4magnetic nano-balls is combined with anti-CEA first antibody, as catching and magnetic separation agent, and by NaY
0.78f
4: Yb
0.20, Ho
0.02up-conversion fluorescence nano material and anti-CEA second antibody (detection antibody) combination, the detector probe of formation mark.When add CEA in CEA capture antibody functional magnetic nano material system time, both are by antigen-antibody reaction generation specific binding, through washing and magnetic separating step removing after supernatant, add CEA to detect the up-conversion fluorescence nano material of antibody labeling, the fluorescence probe that the magnetic Nano material that CEA detects antibody labeling detects antibody labeling by antigen-antibody reaction and CEA is combined and is formed the nano-particle complex of CEA capture antibody/CEA/CEA detection antibody sandwich structure sample, after washing separates with magnetic, then with 980nm induced with laser NaY
0.78f
4: Yb
0.20, Ho
0.02up-conversion fluorescence nano material is luminous.Luminous signal intensity is relevant to CEA content within the specific limits, based on this, CEA standard items is detected, and Criterion curve, to reach the object to carrying out containing CEA sample quantitatively detecting.
Beneficial effect:
(1) to utilize 980nm induced with laser up-conversion fluorescence be detection signal in the present invention, disturb without bias light and autofluorescence, the bias light that has solved the immune analysis technology of FITC, RB200 labelled antibody disturbs and selectivity not good shortcoming, greatly lower signal to noise ratio (S/N ratio), improved detection stability and specificity.
(2) it is concentrated that the magnetic nano-balls that the present invention utilizes CEA antibody function carries out separation and concentration to target antigen in sample, and detection sensitivity is high, sense cycle is short, and simple to operate.
Brief description of the drawings
Fig. 1: the experimental principle figure that detects CEA based on antibody function magnetic Nano material magnetic separation-up-conversion fluorescence nano material mark.
Fig. 2: (a): NaY
0.78f
4: Yb
0.20, Ho
0.02up-conversion fluorescence nano material Electronic Speculum figure; (b) NaY
0.78f
4: Yb
0.20, Ho
0.02, SiO
2up-conversion fluorescence nano material Electronic Speculum figure.
Fig. 3: (a): the ultra-violet absorption spectrum of magnetic Nano material after anti-CEA primary antibodie (0.1mg/mL) and modification thereof, wherein A is anti-CEA primary antibodie, B is magnetic Nano material after anti-CEA primary antibodie is modified; (b): the ultra-violet absorption spectrum of up-conversion fluorescence nano material after anti-CEA bis-anti-(0.15mg/mL) and modification thereof, wherein A is that anti-CEA bis-is anti-, B is up-conversion fluorescence nano material after the anti-modification of anti-CEA bis-.
Fig. 4: the ultra-violet absorption spectrum after antibody is combined with nano particle.A: anti-CEA primary antibodie and magnetic nanoparticle (2mgmL
-1) combination, b: anti-CEA bis-resists and up-conversion fluorescence nano material (2mgmL
-1) combination.
Fig. 5: CEA examination criteria curve map, concentration range is 2.5 × 10
-12-1 × 10
-8gmL
-1
Embodiment
Embodiment 1: the preparation of the magnetic Nano material of finishing and upper conversion nano particle
(1) use hydro-thermal-solvent heat technology to prepare NaY
0.78f
4: Yb
0.20, Ho
0.02upper conversion nano particle, and it is done to finishing.Take 0.18g Y
2o
3(28%), 0.788g Yb
2o
3and 0.022g Ho (70%)
2o
3(2%) potpourri adds heating for dissolving in nitric acid, vapor away the nitrate powder that obtains rare earth element after redundant nitric acid, be dissolved in 8mL deionized water, add again 1.0635g bis-ethylenediamine hydrate Sequestrene AAs (EDTA), and regulate pH to alkalescent, form the EDTA-Ln solution of clear.Get 25mL ethylene glycol, add 3mL HF and 0.4g cetyl trimethyl ammonium bromide (CTAB), add while stirring the above-mentioned EDTA-Ln solution of 8mL, obtain white liliquoid.Finally add 5.5mL red fuming nitric acid (RFNA), after stirring, transfer in 50mL band teflon-lined reactor 195 DEG C of reaction 24h.Reaction finish after, be allowed to condition in air and naturally cool to room temperature, abandon supernatant liquid, the solid at the bottom of still with hot water injection in beaker, ultrasonic 10 minutes, then leave standstill several minutes, discard supernatant liquid, then add hot water ultrasonic, repeat 3 times.Then add the ultrasonic dispersion of ethanol, last centrifugal gained solid is placed in 70 DEG C of oven drying 10h, and pressed powder stores for future use.
(2) utilize improved
method is carried out surface amination modification to up-conversion nanoparticles.
Up-conversion fluorescence nano material after treatment 60mg is added in the 250mL conical flask that fills 60mL isopropyl alcohol, ultrasonic 40min reaches completely and disperses.Under quick magnetic agitation, add successively the ammoniacal liquor of 20mL distilled water, 2.5mL 25%, sealing.At 35 DEG C, after magnetic agitation 10min, add the mixed solution that dropwise adds 20mL isopropyl alcohol and 50 μ L ethyl orthosilicates.React after 3h, more dropwise add the mixed solution of the 3-aminopropyl triethoxysilane of 30mL isopropyl alcohol and 200 μ L, continue to stop stirring after reaction 1h ageing 2h under room temperature.Finally, by product centrifuging, washing repeatedly, is dried 12h for 60 DEG C, and obtaining surface has amido modified up-conversion fluorescence nano material NaY
0.78f
4: Yb
0.20, Ho
0.02.
(3) adopt single stage method to prepare surperficial amidized magnetic nanoparticle.
In 30mL ethylene glycol, add 6.5g1,6-hexane diamine, 2.0g anhydrous sodium acetate (CH
3and 1.0g Iron(III) chloride hexahydrate (FeCl COONa)
3.6H
2o).Then be heated to 50 DEG C, stir and form uniform colloidal solution.Gained solution is transferred in 50mL band teflon-lined reactor, under 198 DEG C of parts, reacted 6h.Take out after reactor, at room temperature naturally cooling, abandon supernatant liquid in still, lower black solid to beaker, then utilizes magnetic separated and collected with deionized water rinsing.Under ultrasound condition, then use aqueous dispersion, then magnetic separated and collected, by method is again with ethanol washing 2 times above, gained black solid is dry 5-10h under 50 DEG C of conditions.
(4) combination of magnetic nanoparticle and anti-CEA first antibody (capture antibody).
10mg is shown to amidized Fe
3o
4magnetic nanoparticle joins 5mL 10mM PBS damping fluid (pH7.4), and ultrasonic dispersion, after 20 minutes, adds 1.25mL 25% glutaraldehyde and 100mg NaBH
4.Mixed solution is at room temperature slowly rocked to 1h, and reaction finishes rear magnetic Nano material and separates through magnetic, and the pressed powder obtaining is washed 3 times thoroughly to remove glutaraldehyde with 10mM PBS.Powder dissolves with 10mM PBS again, and ultrasonic scattering, adds 5mL 0.1mgmL
-1anti-CEA primary antibodie, this solution at room temperature slowly rocks 6h, reaction finish rear cleaning repeatedly, abandon supernatant.Gained sediment, i.e. magnetic nanoparticle/anti-CEA primary antibodie compound (capture probe), resuspended with 5mL0.01M PBS damping fluid, under 4 DEG C of conditions, save backup.
(5) combination of upper conversion nano particle and anti-CEA second antibody (detection antibody).
Up-conversion fluorescence nano material amido modified 10mg is joined to 5mL 10mM PBS damping fluid (pH7.4), and ultrasonic dispersion, after 20 minutes, adds 1.25mL 25% glutaraldehyde and 100mg NaBH
4.Mixed solution is at room temperature slowly rocked to 1h, and reaction finishes rear magnetic Nano material and separates through magnetic, and the pressed powder obtaining is washed 3 times thoroughly to remove glutaraldehyde with 10mM PBS.Powder dissolves with 10mM PBS again, and ultrasonic scattering, adds 5mL 0.15mgmL
-1anti-CEA bis-anti-, this solution at room temperature slowly rocks 6h, reaction finishes rear cleaning repeatedly, abandons supernatant.Gained sediment, goes up conversion nano particle/anti-compound of anti-CEA bis-(capture probe), resuspended with 5mL 10mM PBS damping fluid, under 4 DEG C of conditions, saves backup.
Embodiment 2: the foundation of typical curve
With PBS damping fluid preparation variable concentrations (1 × 10
-12, 5 × 10
-12, 1 × 10
-11, 5 × 10
-11, 1 × 10
-10, 5 × 10
-10, 1 × 10
-9, 5 × 10
-9, 1 × 10
-8gmL
-1) CEA standard items, get respectively 100 μ L variable concentrations CEA standard items and equal-volume 0.1mgmL
-1the magnetic nanoparticle of CEA antibody labeling fully mixes, and magnetic separates 5 minutes removes supernatant, and resuspended magnetic nanoparticle also separates 3 times thoroughly to remove unconjugated CEA molecule with the washing of PBS damping fluid, magnetic.Add again the anti-compound of conversion nano particle/anti-CEA bis-, fully mix and hatch after 30min and divide and abandon supernatant through magnetic again, clean 3 times to ensure thoroughly to remove unconjugated detector probe.By the resuspended precipitation of 100 μ L0.01M PBS damping fluid, under 980nm laser excitation, obtain the up-conversion fluorescence signal at 542nm place, fluorescence signal (I) progressively strengthens along with the increase of CEA concentration.According to fluorescence intensity and corresponding CEA standard items concentration Criterion curve.Experimental result shows, this detection method is 2.5 × 10
-12-1 × 10
-8gmL
-1cEA concentration obtains good linear relation in interval.
Embodiment 3:
The detection of CEA in blood serum sample
Get 7 routine clinical serum specimens and CEA antigen standard items, 10 times of PBS damping fluid dilutions for serum specimen, CEA standard items are made into variable concentrations (1 × 10 with PBS damping fluid
-12, 5 × 10
-12, 1 × 10
-11, 5 × 10
-11, 1 × 10
-10, 5 × 10
-10, 1 × 10
-9, 5 × 10
-9, 1 × 10
-8gmL
-1).Utilize this kit and electrochemiluminescence kit (Roche diagnostic companies product) to measure respectively the content of CEA in these samples, the results are shown in Table one, the data that obtain are carried out to correlativity comparison, and both are without significant difference, and result has very high correlation (R
2=0.9929, P < 0.0001).Illustrate that compared with the similar products of CEA detection kit of the present invention and external well-known manufacturer production, technical indicator is roughly suitable, but this kit detects fast and reliable, sensitivity is higher, and stability is better, is suitable for the detection of CEA in clinical blood serum sample.
Table one: clinical blood serum sample CEA detects, the inventive method and electrochemical luminescence method contrast
Note: NE is PBS damping fluid, and n.d. is not for detecting.
Embodiment 4: the detection of CEA and recovery of standard addition experiment in human serum sample
9 groups of CEA concentration datas that obtain from embodiment 1, select 2 groups as background values, then choose 0.5ngmL
-1, 1ngmL
-1, 5ngmL
-1the CEA standard items of three kinds of variable concentrations add to respectively in determinand, utilize equally this kit again to detect the wherein content of CEA, obtain detected value.Recovery %=(detected value-background values)/addition × 100%.From the result of table two, the recovery, 91.80%～114.10%, illustrates that the present invention is stable, sensitive, accurately, is suitable for the detection of CEA in blood serum sample.
Table two: the detection of CEA and recovery of standard addition in human serum sample
The present invention is described in conjunction with the embodiments.Should be understood that these embodiment, only for illustration purpose, limit the scope of the invention and be not used in.In addition should be understood that and reading after foregoing of the present invention, those skilled in the art can do various changes and amendment to the present invention, and these equivalent form of values fall within the application's book appended claims limited range equally.
Claims (1)
1. carcinomebryonic antigen (CEA) detection kit, is characterized in that described kit comprises:
The magnetic Nano material of antibody function is as antigen capture and magnetic separation agent; Finishing detect antibody up-conversion fluorescence nano material form fluorescence probe; Positive control; Negative control; Antigen standard items;
Described antigen capture and magnetic separation agent are surface amination Fe
3o
4magnetic Nano microsphere and anti-CEA first antibody covalent bond are assembled the magnetic Nano material of the CEA capture antibody functionalization obtaining; Described fluorescence probe is NaY
0.78f
4: Yb
0.20, Ho
0.02the finishing that up-conversion fluorescence nano material and anti-CEA second antibody obtain in conjunction with assembling CEA detect the up-conversion fluorescence nano material of antibody; Described surface amination Fe
3o
4magnetic Nano microsphere is the magnetic Nano material that adopts the surface amination prepared of single stage method; Described up-conversion fluorescence nano material is that mass percent is 28%Y
2o
3, 70%Yb
2o
3and 2%Ho
2o
3the potpourri NaY that uses hydro-thermal-solvent heat technology to prepare
0.78f
4: Yb
0.20, Ho
0.02; Described anti-CEA first antibody concentration is 0.1mgmL
-1, itself and Fe
3o
4the mass ratio of magnetic Nano microsphere is 1: 20, and described anti-CEA second antibody concentration is 0.15mgmL
-1, with NaY
0.78f
4: Yb
0.20, Ho
0.02the mass ratio of up-conversion fluorescence nano material is 3: 40.
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| CN104422770A (en) * | 2013-08-23 | 2015-03-18 | 上海凯璟生物科技有限公司 | Up-conversion luminescence-based chip detection system and reagent for gynecological tumor markers CA125, CA153, SCCA, HE4 |
| CN103808923A (en) * | 2013-11-25 | 2014-05-21 | 珠海迪尔生物工程有限公司 | Movable type magnetic separation fluorescence immunoassay detection and analysis method and movable type magnetic separation fluorescence immunoassay detection and analysis device |
| CN105241869A (en) * | 2015-09-29 | 2016-01-13 | 江南大学 | Bisphenol A electrochemiluminescent aptamer sensor based on upper conversion nano material |
| CN105424662A (en) * | 2015-11-24 | 2016-03-23 | 江南大学 | Fluorescent biological detection system |
| CN105651989A (en) * | 2016-01-28 | 2016-06-08 | 天津科技大学 | Blue-light-emitting up-conversion material and preparation and application thereof |
| CN107525937A (en) * | 2017-08-25 | 2017-12-29 | 苏州优函信息科技有限公司 | Based on up-conversion luminescence label, protein chip and detection method |
| KR102098030B1 (en) * | 2017-12-11 | 2020-04-07 | 주식회사 딕스젠 | Composition for Tuberculosis Diagnosis and Diagnostic Method for Tuberculosis based on Optical Properties |
| CN111017978A (en) * | 2019-12-27 | 2020-04-17 | 江西鑫泰功能材料科技有限公司 | Preparation method of nano holmium oxide for dielectric ceramic capacitor |
| CN112462056B (en) * | 2020-11-19 | 2023-02-28 | 武汉大学 | Urine test platform for detecting bacteria in urine on site and using method thereof |
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| CN101776683A (en) * | 2010-01-06 | 2010-07-14 | 东华大学 | Luminescent and magnetic nano material-based method for detecting carcinoembryonic antigen |
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