CN107677806B - The preparation and detection method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation based on magnetic enrichment - Google Patents
The preparation and detection method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation based on magnetic enrichment Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
Abstract
The present invention relates to the preparations and detection method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of the fluorescent quantitation based on magnetic enrichment；Fe is wrapped up by ultrasonic emulsification fado using polyphosphazene polymer poly lactic coglycolic acid3O4Magnetic nanoparticle, target substance can be made quickly and effectively to be integrated on magnetic microsphere, newly-generated compound magnetic responsiveness in magnetic field is identical, for being enriched with sample to be examined, enhance detection sensitivity, joint-detection is realized simultaneously, rely on immuno-chromatographic test paper strip detection platform, carry out double antibodies sandwich immune response, and it under given conditions can be with the reaction mechanism of quenching fluorescence signal by magnetic nano particle, reach fast qualitative under the conditions of naked eye, wide scope bimodal detection precisely quantitative under dark field test, serum to be checked is enriched with using the superparamagnetism of magnetic nano particle simultaneously, the accuracy of early diagnosing mammary cancer and the universality of community's screening are improved by the joint-detection to breast cancer tumour marker CEA and CA153.
Description
Technical field
The present invention relates to field of medicine diagnostic technology, more particularly to a kind of fluorescent quantitation Gao Ling based on magnetic enrichment
The preparation and detection method of quick visualization joint inspection immuno-chromatographic test paper strip.
Background technique
Breast cancer is a kind of most common most multiple malignant tumour of modern female, has accounted for women's Cancer Mortality at present
First place, and disease incidence is all increasing every year, and according to statistics, in most of developed countries, malignant tumour is in death original
It ranks first or second because in.Wherein the disease incidence of breast cancer occupies domestic eight big cancers the 5th, in the same of rapid economic development
When, breast cancer patients are also being advanced by leaps and bounds ".It is the main of decision survival of patients time that whether Mammary cancer, which occurs DISTANT METASTASES IN,
Factor, early discovery, early diagnosis treatment early are the key that extend the survival of patients time, reduce case fatality rate.However metastases
The clinical manifestation or image analysis that diagnosis is spread by tumour, it is often late.Therefore need a kind of sensibility higher
Diagnostic method come assist clinician judge it is postoperative whether shift, this will be directly related to anaphase and the existence of patient
Time.The research of tumor markers at present help to find as early as possible it is postoperative whether shift, and by ideal breast cancer tumour mark
Will object will have very high clinical value for treating therapeutic evaluation and prognosis evaluation.
Breast cancer antigen (CA153) is a kind of macromolecular mucus glycoprotein, and it is thin to be primarily present in breast epithelium lumen face
On after birth, when malignant change of cell, serum levels are significantly raised, are at present using most common more special to breast cancer one
The variation of tumor markers, content is closely related with therapeutic effect, is Diagnosis of Breast cancer and monitoring postoperative metastasis, observation curative effect
Optimal parameter.Carcinomebryonic antigen (CEA) is a kind of acidoglycoprotein, is a kind of broad-spectrum tumor marker, has document to show serum
Late breast cancer positive rate may be up to 71.43% to CEA, CEA occurs in the patient that breast cancer has DISTANT METASTASES IN phenomenon to occur
The raised ratio of content is up to 50% -70%.The relevant report of current single tumor markers and research are existing many but single
Solely detection is lower for the diagnostic sensitivity of breast cancer, and detection is clinically improved frequently with joint-detection Diagnostic Value of Several Serum Tumor Markers
Sensitivity.Therefore, while using CA153 and CEA as tumor markers, screening is carried out to people at highest risk, to the morning of breast cancer
The work such as phase diagnosis, progression of the disease and Prognosis scoveillance will be very helpful.Therefore, Gao Ling is realized to the intracorporal tumor markers of biology
The detection of sensitivity is an important topic of life science research, and developing the new highly sensitive detection method of one kind is also mesh
The target that preceding researchers make great efforts.
Lateral Flow Strip is that one kind is easy to operate, is not necessarily to professional training, convenient and efficient, the immunology detection skill of visual result
Art.Its testing principle is the specific region that special antibody (or antigen) is fixed on to nitrocellulose filter as capture reagent,
After sample to be tested is added, under the promotion of capillary force, sample to be tested is moved forward along film, is fixed with capture when being moved to
When the region of reagent, the antigen (or antibody) in sample is specifically bound with capture reagent, and labelled reagent then shows one
Fixed color is to realize the immunoassay method of specific detection.The immuno-chromatographic test paper strip being commercialized at present is mostly used glue
Body gold is as marker, although testing result passes through naked eyes as it can be seen that being unable to complete accurate quantitative detection, and antigen concentration is slightly higher
It shallowly often can not with the naked eye be identified very much in the pattern detection band color of critical value；Fluorescence immune chromatography test paper bar detection technique is
To realize the new technology of quantitative detection rise after colloidal gold immuno-chromatography test paper strip technology, but find in the course of the research, background
Fluorescence is a key factor for limiting fluorescence immune chromatography technology detection sensitivity, among these includes coming from nitrocellulose filter
With the autofluorescence of sample itself.Therefore the influence for how reducing background fluorescence in fluorescence immune chromatography detection, becomes raising
The project of fluorescence immune chromatography test paper bar detection technique sensitivity urgent need to resolve.
Immune magnetic Nano microsphere i.e. can be with separation and concentration target under the action of externally-applied magnetic field due to its superparamagnetism
Molecule, in addition, magnetic nano particle has certain fluorescent quenching performance.This technology will combine superparamagnetic nano particle test strips
Visual retrieval characteristic and fluorescence detection high sensitivity advantage, can be quenched under given conditions using magnetic nano particle
The reaction mechanism of fluorescence signal, it is quasi- to construct a kind of visual fluorogenic quantitative detection immuno-chromatographic test paper strip, pass through the letter of fluorescence
The aggregation colour developing of number Strength Changes and magnetic nano particle reaches fast qualitative under the conditions of naked eye, precisely quantitative under dark field test
The detection of wide scope bimodal, while the superparamagnetism of application magnetic nano particle is enriched with serum to be checked, by breast cancer tumour mark
The joint-detection of will object CEA and CA153 improve the accuracy of early diagnosing mammary cancer and the universality of community's screening.
Summary of the invention
In order to solve problems in the prior art, the present invention proposes the highly sensitive visualization connection of kind of the fluorescent quantitation based on magnetic enrichment
Examine the preparation method and detection method of immuno-chromatographic test paper strip；Polyphosphazene polymer poly lactic coglycolic acid (PLGA) is passed through
Ultrasonic emulsification fado wraps up Fe3O4Magnetic nanoparticle, due to the size of the Magnetic Spherical nanoparticle of this coating functional group
There is good homogeneity with shape, therefore target substance can be made quickly and efficiently to be integrated on magnetic microsphere, synkaingenesis at
Compound in magnetic field magnetic responsiveness it is identical, behavior is consistent, can be used to be enriched with sample to be examined, enhance detection sensitivity, together
When may be implemented joint-detection, this project will be in conjunction with the visible and fluorescence of superparamagnetic nano particle test strips testing result naked eyes
Highly sensitive advantage is detected, according to the specific reaction mechanism of antibody antigen, relies on immuno-chromatographic test paper strip detection platform, into
The immune response of row double antibodies sandwich, and can be reached under given conditions with the reaction mechanism of quenching fluorescence signal by magnetic nano particle
Fast qualitative under the conditions of to naked eye, wide scope bimodal detection precisely quantitative under dark field test, while applying magnetic nano particle
Superparamagnetism be enriched with serum to be checked, breast cancer is improved by the joint-detection to breast cancer tumour marker CEA and CA153
The accuracy of early diagnosis and the universality of community's screening.
Technical method of the invention is as follows:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation based on magnetic enrichment, step is such as
Under:
1) preparation of the more coated magnetic nanosphere immunological probes of macromolecule: the water solubility with carboxyl after PLGA is wrapped up
Magnetic nanosphere, breast cancer Research of predicting markers CA153 labelled antibody or CEA labelled antibody, EDC (1- ethyl-(3- dimethylamino
Propyl) phosphinylidyne diimmonium salt hydrochlorate) it is dissolved in reaction solution vortex instrument its mixing is made with the rotation of the speed of 50-60r/min, so
Above-mentioned mixed solution is placed on rotation hybrid frame afterwards, the outstanding training of room temperature, activates the carboxyl of magnetic nanometer ball surface, centrifugation is last even
The magnetic nano particle of antibody is closed overnight with BSA on connection；
2) assembling of test strips: common test strips are by sample pad, bonding pad, nitrocellulose filter, and water absorption pad forms,
Detection technique of the invention first mixes in EP pipe due to using magnetic enrichment, magnetic nano particle and antigen, eliminates bonding pad
Use, thus in the present invention test strips its from sample cell be successively up sample pad, nitrocellulose filter specific region spray
CA153 antibody, that is, detection line 1 of useful fluorescence element Cy5 label, the CEA antibody, that is, detection line 2 marked with fluorescein Cy5 are sprayed with
Region, that is, nature controlling line of IgG antibody and the water absorption pad of end.
In the step 1), macromolecule PLGA and Fe3O4The mass fraction proportion of magnetic nanoparticle dosage is 10-20:1.
In the step 1), the mass fraction proportion of magnetic nano particle and EDC dosage is 40-80:1.
In the step 1), after 1.5~2h of the outstanding training of magnetic nano particle coupled antibody, it is centrifuged, washing.
In the step 1), BSA confining liquid dosage is 1%~3%.
In the step 2), bonding pad pretreatment fluid is sucrose, bovine serum albumin(BSA) BSA, polyethylene glycol PEG, polyoxy second
The mixed solution of alkene sorbitan monolaurate Tween-20.
The detection method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation based on magnetic enrichment:
Qualitative checking method: the principle being immunoreacted according to double antibodies sandwich, with the more coated magnetic nanospheres of above-mentioned macromolecule
It is enriched with serum to be checked, the object to be checked after enrichment is dripped into sample cell, is exempted under the action of capillary force to water absorption pad direction
Epidemic disease chromatography reaction, when logistics to be checked is after detection zone and control area, reaction 2-3min can be qualitatively judged visually, once sample
When containing target antigen in this, compound will be captured by T line and C line simultaneously, and magnetic nano particle is in detection zone and control area
Aggregation colour developing, testing result naked eyes are visible as the positive；Conversely, detection sample in be free of target antigen when, then magnetic nano particle only
Assemble in C line position and develop the color, testing result is feminine gender；Illustrate that testing result is invalid if T line and C line do not develop the color.
Quantitative detecting method: reaction step is same as above, i.e., after enrichment is added dropwise in the sample cell of assembled plastic bottom board
The mixture of magnetic nano-probe and antigen is put into analyzer after persistent immunological 15-20min and takes pictures, then uses
Photoshop calculates photo fluorescence intensity value, is fluorescence difference as read output signal according to the fluorescence intensity value variation of reaction front and back.
Establish the standard curve of the invention detection technique.
In the standard curve: CEA standard curve antigen various concentration selection are as follows: 0.01-10ng/ml；CA153 standard is bent
Line antigen various concentration is selected as 0.01-10ng/ml.
The advantage of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of fluorescent quantitation prepared by the present invention based on magnetic enrichment
It is:
1. the superparamagnetism of magnetic nano particle can be enriched with serum to be checked, it is expected to realize the detection to whole blood.
2. magnetic nano particle can be disappeared with quenching fluorescence by the reaction mechanism to quenching fluorescence signal under given conditions
Except the interference of background fluorescence, highly sensitive detection is realized.
3. magnetic nano particle develops the color in the aggregation of detection line and control line, fast qualitative under the conditions of naked eye may be implemented.
4. early diagnosing mammary cancer can be improved by the joint-detection to breast cancer Research of predicting markers CEA and CA153
Accuracy.
Detailed description of the invention
The transmission electricity of the more coated magnetic nanospheres of joint inspection immuno-chromatographic test paper strip macromolecule based on magnetic enrichment of Fig. 1 preparation
Mirror photo.
The magnetic enrichment process of the joint inspection immuno-chromatographic test paper strip based on magnetic nano particle of Fig. 2 preparation.
The specificity experiments numerical value of the joint inspection immuno-chromatographic test paper strip based on magnetic enrichment of Fig. 3 preparation.
Sensitivity experiments numerical value of the joint inspection immuno-chromatographic test paper strip based on magnetic enrichment of Fig. 4 preparation about CA153.
Sensitivity experiments numerical value of the joint inspection immuno-chromatographic test paper strip based on magnetic enrichment of Fig. 5 preparation about CEA.
Specific embodiment
The invention will be further elaborated in following case study on implementation, however, the present invention is not limited thereto.
Novel test paper detection technique of the invention wraps up oil-soluble magnetic by ultrasonic emulsification fado with macromolecule (PLGA)
Nano particle, the dispersion of nano particle in aqueous solution after wrapping up is good, and size is substantially uniform, and partial size is on the left side 80nm
The right side, as shown in Figure 1；Secondly with the nanoparticle coupled antibody of Fig. 1 and be enriched with object to be checked, enrichment process as shown in Fig. 2, outside plus
Target molecule is separated under the action of magnetic field；Variety classes antigen is finally added dropwise in the sample cell of assembled plastic bottom board, instead
It is put into progress signal reading in analyzer after answering a period of time, specificity experiments numerical value is as shown in figure 3, containing specific mesh
Heterogenetic antigen (CA125, HCG, PSA, AFP) will be much higher than by marking fluorescence intensity change in the experimental group of molecule CEA/CA153
Control group change in signal strength；It is real to the detection linear fit of CA153 antigen that Fig. 4 illustrates this immuno-chromatographic test paper strip technology
Numerical value is tested, sensitivity can achieve 0.09U/mL, be far below clinical threshold 28U/mL；Fig. 5 illustrates this immunity-chromatography test
Paper slip technology is to the detection linear fit empirical value of CEA antigen, and sensitivity can achieve 0.06ng/mL, far below clinic
Critical value 5ng/mL.
Steps are as follows for implementation process of the invention:
1) accurately weigh quantitative EDC, magnetic nanoparticle and breast cancer Research of predicting markers CEA that macromolecule wraps up more and
The mixing of CA153 labelled antibody is dissolved in the EP pipe of 2ml, and EP pipe is placed on room temperature on rotary incubator and rotates about 1.5h.It then will be upper
It states liquid and is transferred to tip EP pipe, sufficiently collection sediment, setting speed centrifuge washing is at least three times, molten with the BSA of 1%-3%
Liquid and 4 DEG C of refrigerator closings are overnight.
2) breast cancer Research of predicting markers CA153 coated antibody and CEA coated antibody are marked with Cy5 fluorescein, by fluorescence mark
Antibody after note is sprayed on nitrocellulose filter, respectively as detection line 1 and detection line 2, the sheep anti-mouse antibody of 1mg/ml is molten
Liquid is sprayed on the top of detection line, and as nature controlling line, detection line and nature controlling line spray and be put into 37 DEG C of baking ovens drying after getting well；Glass-cutting is fine
Tieing up plain film width is about 2cm, and the Tris-HCl buffer for being immersed in the PH=7.4 of the 0.01M containing 0.5% Tween-20 is made
For the treatment fluid of sample pad, after infiltrating 10-15min, it is put into 37 DEG C of baking oven drying；The sample pad that will be handled well, is pasted onto test paper
On item, loading is assembled with the plastic bottom board that company customizes.
3) the antigen addition CEA and CA153 label probe that the various concentration of 200 μ L is taken with liquid-transfering gun, uses alnico magnets
It is enriched with serum to be checked, 160 μ L supernatants is discarded, surplus solution is added drop-wise in the sample cell of assembled plastic bottom board, reacts 2-3
Minute after, according to detection line region whether be magnetic nanoparticle aggregation carry out naked eyes fast qualitative detection.
4) it after the reaction was continued 15-20min, is put into gel analysis instrument and is taken pictures and counted and calculated fluorescence change.
The above reaction is the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection of fluorescent quantitation of the preparation based on magnetic enrichment
The general step of technology, is not described further in embodiment.
Case study on implementation 1:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 15:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:60, and BSA confining liquid dosage is 2%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 115231, and detection line 2 (CA153) fluorescence difference is 86741；After
After continuous reaction 18min, fluorescence difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is before and after sample is added dropwise in statistics
115366, detection line 2 (CA153) fluorescence difference is 86931；After the reaction was continued 20min, fluorescence difference before and after sample is added dropwise in statistics
(Δ F) result is that detection line 1 (CEA) fluorescence difference is 115803, and detection line 2 (CA153) fluorescence difference is 87002.
Case study on implementation 2:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 10:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:60, and BSA confining liquid dosage is 2%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 95231, and detection line 2 (CA153) fluorescence difference is 83007；Continue
It is 96248 that fluorescence difference (Δ F) result, which is detection line 1 (CEA) fluorescence difference, after reacting 18min, before and after statistics dropwise addition sample, inspection
Survey line 2 (CA153) fluorescence difference is 83919；After the reaction was continued 20min, fluorescence difference (Δ F) result before and after sample is added dropwise in statistics
It is 96904 for detection line 1 (CEA) fluorescence difference, detection line 2 (CA153) fluorescence difference is 84047.
Case study on implementation 3:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 20:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:60, and BSA confining liquid dosage is 2%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 90507, and detection line 2 (CA153) fluorescence difference is 80725；Continue
It is 90978 that fluorescence difference (Δ F) result, which is detection line 1 (CEA) fluorescence difference, after reacting 18min, before and after statistics dropwise addition sample, inspection
Survey line 2 (CA153) fluorescence difference is 81019；After the reaction was continued 20min, fluorescence difference (Δ F) result before and after sample is added dropwise in statistics
It is 91914 for detection line 1 (CEA) fluorescence difference, detection line 2 (CA153) fluorescence difference is 81337.
Case study on implementation 4:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 15:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:40, and BSA confining liquid dosage is 2%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 98915, and detection line 2 (CA153) fluorescence difference is 81165；Continue
It is 99211 that fluorescence difference (Δ F) result, which is detection line 1 (CEA) fluorescence difference, after reacting 18min, before and after statistics dropwise addition sample, inspection
Survey line 2 (CA153) fluorescence difference is 81791；After the reaction was continued 20min, fluorescence difference (Δ F) result before and after sample is added dropwise in statistics
It is 99984 for detection line 1 (CEA) fluorescence difference, detection line 2 (CA153) fluorescence difference is 82004.
Case study on implementation 5:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 15:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:80, and BSA confining liquid dosage is 2%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 92833, and detection line 2 (CA153) fluorescence difference is 82721；Continue
It is 93088 that fluorescence difference (Δ F) result, which is detection line 1 (CEA) fluorescence difference, after reacting 18min, before and after statistics dropwise addition sample, inspection
Survey line 2 (CA153) fluorescence difference is 82974；After the reaction was continued 20min, fluorescence difference (Δ F) result before and after sample is added dropwise in statistics
It is 93774 for detection line 1 (CEA) fluorescence difference, detection line 2 (CA153) fluorescence difference is 83001.
Case study on implementation 6:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 15:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:60, and BSA confining liquid dosage is 1%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe, detection line 1 after 2.5min is presented
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 123431, and detection line 2 (CA153) fluorescence difference is 88711；After
After continuous reaction 18min, fluorescence difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is before and after sample is added dropwise in statistics
123977, detection line 2 (CA153) fluorescence difference is 88997；After the reaction was continued 20min, fluorescence difference before and after sample is added dropwise in statistics
(Δ F) result is that detection line 1 (CEA) fluorescence difference is 124284, and detection line 2 (CA153) fluorescence difference is 89002.
Case study on implementation 7:
The preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip detection technique of fluorescent quantitation based on magnetic enrichment:
Breast cancer Research of predicting markers CEA and the CA153 coated antibody that fluorescein Cy5 is marked is sprayed on to the detection line position of nitrocellulose filter
It sets, sheep anti-mouse antibody solution is sprayed on to the Quality Control line position of nitrocellulose filter, the quality of macromolecule PLGA and magnetic nano particle
Score proportion is 15:1, and the mass fraction proportion of EDC and magnetic nano particle dosage is 1:60, and BSA confining liquid dosage is 3%, rich
Integrating concentration as the CA153 of 50U/mL and concentration is the 200 μ L of hybrid antigen of the CEA of 10ng/mL in sample cell, does three repetitions.
Qualitative checking method is as follows: after being added dropwise in above-mentioned sampling test strips reaction 2min, visually observe reading frame, detection line 1 (CEA) and
Detection line 2 (CA153) and control line have the aggregation of black magnetic nanometer that black stripe is presented.Detection line 1 after 2.5min
(CEA) band color when and detection line 2 (CA153) and control line region band color are close to 2min, detection line 1 after 3min
(CEA) color burn is little by little when and detection line 2 (CA153) and control line region band color are compared with 2min.Quantitative detection side
Method is as follows: after above-mentioned sample the reaction was continued 15min, being put into progress signal reading in analyzer, fluorescence before and after sample is added dropwise in statistics
Difference (Δ F) result is that detection line 1 (CEA) fluorescence difference is 93006, and detection line 2 (CA153) fluorescence difference is 81545.Continue
It is 93778 that fluorescence difference (Δ F) result, which is detection line 1 (CEA) fluorescence difference, after reacting 18min, before and after statistics dropwise addition sample, inspection
Survey line 2 (CA153) fluorescence difference is 81974；After the reaction was continued 20min, fluorescence difference (Δ F) result before and after sample is added dropwise in statistics
It is 94021 for detection line 1 (CEA) fluorescence difference, detection line 2 (CA153) fluorescence difference is 82107.
The present invention wraps up Fe by ultrasonic emulsification fado using polyphosphazene polymer poly lactic coglycolic acid3O4Magnetic Nano
Particle, and the size and shape of the Magnetic Spherical nanoparticle of this coating functional group has good homogeneity, can make target
Substance is quickly and effectively integrated on magnetic microsphere, and newly-generated compound magnetic responsiveness in magnetic field is identical, and behavior is consistent, can
To be used to be enriched with sample to be examined, enhance detection sensitivity, while joint-detection may be implemented, this project will be received in conjunction with superparamagnetism
The visible and highly sensitive fluorescence detection advantage of rice grain test strips testing result naked eyes, it is anti-according to the specificity of antibody antigen
Mechanism is answered, immuno-chromatographic test paper strip detection platform is relied on, carries out double antibodies sandwich immune response, and by magnetic nano particle specific
Under the conditions of can reach fast qualitative under the conditions of naked eye with the reaction mechanism of quenching fluorescence signal, it is precisely quantitative under dark field test
The detection of wide scope bimodal, while the superparamagnetism of application magnetic nano particle is enriched with serum to be checked, by breast cancer tumour mark
The joint-detection of will object CEA and CA153 improve the accuracy of early diagnosing mammary cancer and the universality of community's screening.
Claims (6)
1. the preparation method of the highly sensitive visualization joint inspection immuno-chromatographic test paper strip of the fluorescent quantitation based on magnetic enrichment, it is characterized in that step
It is rapid as follows:
1) macromolecule wraps up the preparation of magnetic nanosphere immunological probe more: after PLGA (poly lactide-glycolide acid) package
The water-soluble magnetic nanosphere with carboxyl, breast cancer Research of predicting markers CA153 labelled antibody or Carcinoembryonic Antigen CEA label are anti-
Body, EDC hydrochloride (1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate) are dissolved in reaction solution, vortex instrument
On rotated and mix with the speed of 50-60r/min, then mixed solution is placed on rotation hybrid frame, the outstanding training of room temperature, activation magnetic is received
The carboxyl of rice ball surface, centrifugation, the magnetic nanosphere for being finally coupled upper antibody are closed overnight with BSA；
2) assembling of test strips: it is successively up sample pad, is sprayed with glimmering in the specific region of nitrocellulose filter from sample cell
CA153 antibody, that is, detection line 1 of light element Cy5 label, the CEA antibody, that is, detection line 2 marked with fluorescein Cy5 are sprayed with IgG antibody
Region, that is, nature controlling line and end water absorption pad.
2. the method as described in claim 1, it is characterized in that magnetic nanosphere is Fe in the step 1)3O4Magnetic nanoparticle is high
Molecule PLGA and Fe3O4The mass fraction proportion of magnetic nanoparticle dosage is 10-20:1.
3. the method as described in claim 1, it is characterized in that in the step 1), the matter of magnetic nanosphere and EDC hydrochloride dosage
Measuring score proportion is 40-80:1.
4. the method as described in claim 1, it is characterized in that in the step 1), the matter of magnetic nanosphere and CA153 antibody dosage
Measuring score proportion is 15-35:1；Magnetic nanosphere and the mass fraction of CEA antibody dosage proportion are 8-20:1.
5. the method as described in claim 1, it is characterized in that in the step 1), 1.5~2h of the outstanding training of magnetic nanosphere coupled antibody
Afterwards, it is centrifuged, washing.
6. the method as described in claim 1, it is characterized in that BSA confining liquid dosage is 1%~3% in the step 1).
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| CN109270269A (en) * | 2018-09-05 | 2019-01-25 | 河南省生物工程技术研究中心 | A kind of fluorescence immune chromatography detection card, kit for the multi-joint detection of early-stage breast cancer |
| CN109382052B (en) * | 2018-12-04 | 2020-09-01 | 郑州大学 | Visual full-automatic coupling reaction device |
| CN109813692A (en) * | 2019-01-02 | 2019-05-28 | 北京科技大学 | A kind of capillary analysis detection method based on ultrasound aggregation |
| CN111157738A (en) * | 2019-12-28 | 2020-05-15 | 王贤俊 | Method for improving detection sensitivity of detecting serum α 1-acid glycoprotein |
| CN115684580B (en) * | 2022-09-27 | 2023-08-25 | 郑州大学 | Microsphere array chip based on magnetic control, detection assembly, detection system and method |
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