CN107478631A - 3D fan-fold paper based microfluid fluorescence detection devices that are a kind of while detecting Diagnostic Value of Several Serum Tumor Markers - Google Patents
3D fan-fold paper based microfluid fluorescence detection devices that are a kind of while detecting Diagnostic Value of Several Serum Tumor Markers Download PDFInfo
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- CN107478631A CN107478631A CN201710852160.2A CN201710852160A CN107478631A CN 107478631 A CN107478631 A CN 107478631A CN 201710852160 A CN201710852160 A CN 201710852160A CN 107478631 A CN107478631 A CN 107478631A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The present invention relates to a kind of while detect the 3D fan-fold paper based microfluid fluorescence detection devices of Diagnostic Value of Several Serum Tumor Markers, belong to technical field of biological.The detection means can be used for the detection to Diagnostic Value of Several Serum Tumor Markers simultaneously.The present invention comprises the following steps：(1) making of paper substrate micro-fluid chip；(2) pretreatment of paper substrate micro-fluid chip；(3) the immune response program that three-dimensional paper substrate detects simultaneously；(4) fluorescence analysis detects.The present invention combines paper substrate microfluid analysis platform with fluoroscopic examination, for being detected while Diagnostic Value of Several Serum Tumor Markers.This detection means is simple, cheaply, portable, disposably, easy to use, and favourable platform is provided for developing country and resource-constrained and remote districts.
Description
Technical field
The present invention relates to a kind of detection means, especially a kind of 3D that to Diagnostic Value of Several Serum Tumor Markers can detect simultaneously rolls over
Stacker based microfluid fluorescence detection device, belongs to technical field of biological.
Background technology
" report of world's cancer " issued according to the World Health Organization and international cancer research institution (IARC), cancer is complete
Ball main causes of death, early detection Diagnostic Value of Several Serum Tumor Markers is easy to diagnose cancer and Treatment monitoring, and significant improve is controlled
Curative effect rate and survival rate.However, only detecting single tumor markers is typically not enough to Accurate Diagnosis cancer, because most of marks
Thing does not have specificity to specific tumors.In order to improve the accuracy of cancer diagnosis, it is necessary to reference to Diagnostic Value of Several Serum Tumor Markers
Detection, because it can be with significant raising specificity.
A variety of methods have been proposed in the past few years while to detect Diagnostic Value of Several Serum Tumor Markers.For example, electrochemistry is exempted from
Epidemic disease determines, unmarked method, mediates electrophoretic determination, chemiluminescence analysis, the sweep measuring of biomarker, photon suspension array.
But all these technologies are unsuitable for carrying out point-to-point test, because they need large-scale and complicated instrument, complicated behaviour
Make, the personnel of prolonged analysis time and high professional qualification are operated.Limited equipment and personnel's possibility without enough trainings
Remote and rural area early screening tumour can be limited.Therefore, in the case of without using laboratory or trained personnel,
Can detect simultaneously multiple tumor markerses be easy and fast to and low cost analysis method demand be there is an urgent need to.In recent years
Come, the immune labeled test paper combined with paper substrate microfluid is increasingly taken seriously, because it has easy to operate, analyte volume
The advantages of small, analysis time is short, and cost is low, and more analyses are analyzed, it is not necessary to specialty and the ability for realizing point-of care test.So
And these methods are mostly enzyme linked immunologicals, need to add substrate and terminate liquid in enzyme linked immunoassay, make response procedures more complicated；And
And it is enzyme-linked need suitable temperature with PH in order to avoid influenceing the activity of enzyme, be not suitable for quickly detecting whenever and wherever possible.And fluorescent material is
Selection well, because optical signal is sensitive, and the immunoassay with fluorescence labeling is with the latent of quantization multiple analyte
Power.Fluorescein isothiocynate (FITC) is a kind of dyestuff for sending out yellow-green fluorescence strong, and its quantum yield is high, fluorescence lifetime length, uses
FITC come labelled antibody carry out fluoroimmunoassay detect tumor markers be it is a kind of it is convenient effectively, the high detection side of accuracy
Method.
The content of the invention
Present invention solves the technical problem that it is：It is proposed that 3D that is a kind of while detecting Diagnostic Value of Several Serum Tumor Markers folds paper substrate miniflow
Body fluorescence detection device, the portable paper substrate fluorescence detection device of low cost, and while be applied to Diagnostic Value of Several Serum Tumor Markers
In detection.
In order to solve the above-mentioned technical problem, technical scheme proposed by the present invention is：It is a kind of to detect kinds of tumors mark simultaneously
The 3D fan-fold paper based microfluid fluorescence detection devices of thing, comprise the following steps：
(1) making of paper substrate micro-fluid chip；
The figure of 3D paper substrates is designed on computers, folding three-dimensional hydrophilic and hydrophobic region is formed, for simultaneously
Diagnostic Value of Several Serum Tumor Markers is detected, the number in two layers of the increase detection zone hole of paper substrate 2,3 is 1-5, you can increase detection simultaneously
Mark species number is 1-5, and is printed with wax spray printer, puts baking oven baking a period of time into, taking-up is cooled to room temperature, by paper
Base is cut along print area, and carries out three dimensional fold；
(2) pretreatment of paper substrate micro-fluid chip；
Oxygen gas plasma processing is carried out with plasma cleaner by the 3rd layer of baked paper substrate chip, makes paper surface
Carbon radicals formed oxygen radical, cause the generation of aldehyde radical, this purpose is easy for fixation of the antibody on paper；
(3) the immune response program that three-dimensional paper substrate detects simultaneously；
The coated antibody of 1-5 kind tumor markerses is fixed respectively at plasma treated the 3rd layer first, then with resistance
Binding site on liquid barrier paper, resists in the marked by fluorescein isothiocyanate of 1-5 kind tumor markerses corresponding to the 2nd layer of addition
Body, by 1,2 two layers pair it is folded, add determined antigen mixed liquor 1 layer of sample area, finally carry out three layers immune anti-all to folded
Should；
(4) fluorescence analysis detects；
By the 3rd layer of paper substrate micro-fluid chip as under specific excitation source, assessed according to visual fluorescence is strong and weak
1-5 kind tumor markers concentration, by the 3rd layer of paper substrate micro-fluid chip fluorescent spectrophotometer assay emission wavelength peak,
The 1-5 kind tumor markers concentration of detection can accurately be drawn.
Preferably, the number in two layers of the increase detection zone hole of paper substrate 2,3 is 3, the mark species number 3 that can be detected simultaneously
Kind.
Preferably, the making of (1) paper substrate micro-fluid chip, graceful No. 1 color of water that specification is 20cm × 20cm is used
Manuscript；
(2) pretreatment of paper substrate micro-fluid chip, the 3rd layer of paper substrate micro-fluid chip is completed into baking, it is clear to be put into plasma
Washing machine carries out surface and is modified 4min, and being handled using oxygen surface plasma, which makes the surface of paper take aldehyde radical, is easy to antibody to fix,
Under plasma conditions, the hydroxyl on the carbon radicals-CH-OH on paper substrate cellulose can lose hydrogen atom, so as to be formed
Oxygen radical-O-CH2-, then, the Single Electron on the oxygen radical are combined with the electronics on carbon, cause the generation of aldehyde radical；
(5) in fluorescence immune reaction program, 4 μ are fixed respectively on the 3rd layer of paper substrate G, H, I that aldehyde radical was modified first
L, the coated antibody of 40 μ g/mL 3 kinds of tumor markerses, J is as blank control, after reacting 30min at room temperature, with 20 μ L,
0.01molPH=7.4 phosphate buffer rinses 3 times, then sequentially add on 0.5% BSA barrier paper not with antibody knot
The specific position of conjunction, react at room temperature after 15min with 20 μ L, 0.01molPH=7.4 phosphate buffer flushing 3 times, the
The marked by fluorescein isothiocyanate that three kinds of tumor markerses corresponding to 4 μ L, 40 μ g/mL are separately added on two layers of paper substrate C, D, E resists
Body, after 2min by 1,2 two layers pair it is folded, 20 μ L tumor markers mixed liquor to be measured is added in 1 layer of A holes, after reacting 2min
By three layers all to folded, and 40 μ L, 0.01molPH=7.4 phosphate buffer are added by antigen in 1 layer of A holes and is marked
Antibody complex is flushed to 3 layers.Rinsed 3 times with 40 μ L, 0.01molPH=7.4 phosphate buffer after reacting 4min, and with absorption
Pad absorbs waste liquid.
(6) paper substrate micro-fluid chip is put under the excitation source that wavelength is 470nm and irradiated, can be according to yellow-green fluorescence
Power vision compared with colorimetric card reads reading；Paper substrate micro-fluid chip is put into sample panel, uses sepectrophotofluorometer
It is scanned in the case where wavelength is 470nm exciting light, various tumour marks can be accurately drawn respectively according to the peak value of fluorescence spectrum
The concentration of will thing.
Beneficial effects of the present invention：
(1) model that paper is folded into 3D is easy to carry, not by condition and territory restriction, available for self-test anywhere or anytime
It is fast to survey.
(2) 3D folds paper substrate respectively in latter two layers plus antibody, directly adds testing sample in top layer, without being added an examination of again after sample-adding
Agent operates, and both simplifies operating procedure, realizes again and adds the testing sample i.e. practical detection means of readable effects.
(3) paper substrate microfluid is combined with fluorescence immunoassay, eliminates large-scale medical detecting Instrument or electrochemical workstation
Etc. large-scale instrument, fluoroimmunoassay can be simple and convenient with Visual readings.
(4) corona treatment makes paper, and surface modification takes aldehyde radical and is easy to antibody binding, simple time saving and effective.
Under condition of plasma, the hydroxyl on carbon radicals (- CH-OH) on paper substrate cellulose may lose hydrogen atom, so as to
Form oxygen radical (- O-CH2-).Then, the Single Electron on the oxygen radical is combined with the electronics on carbon, causes aldehyde radical
Generation.
(5) whole paper substrate microfluid fluorescence of fluorescence detection means preparation process is simple, detects quick and high sensitivity, can be simultaneously right
Diagnostic Value of Several Serum Tumor Markers is detected, available for the quick detection of tumour and the early screening of cancer.
(6) paper substrate microfluid analysis device has simplicity, and portability, disposably, low cost are nontoxic, can be at any time
The advantages that self-test is surveyed soon is carried out everywhere.The present invention combines paper substrate microfluid analysis platform with fluoroscopic examination, for a variety of
Detected while tumor markers.This detection means is simple, cheaply, portable, disposably, easy to use, is developing country
Favourable platform is provided with resource-constrained and remote districts.
Brief description of the drawings
The present invention is described further below in conjunction with the accompanying drawings.
Fig. 1 is the plane outspread drawing of 3D fan-fold paper based microfluid fluorescence detection devices.Wherein 1 layer be sample add layer, 2 layers
Layer is added for labelled antibody, 3 layers are detection layers.A is sample application zone, and B is sample flow area, and C, D, E are respectively three kinds of tumor-markers
The addition area of labelled antibody corresponding to thing, F are blank control check plot.G, H, I are respectively the detection zone of three kinds of marks, and J is
Check plot.
Fig. 2 is the use fold sequence figure of 3D fan-fold paper based microfluid fluorescence detection devices.First by paper substrate microfluid core
Piece is folded into shown in II figure as I figure, then the fixed coated antibody of 3 layers after plasma treatment and add BSA barrier, the 2 of paper
Layer adds labelled antibody.Paper is folded into as shown in figure III again, antigen mixed liquor to be detected is added toward 1 layer of sample area, makes 1,2
Layer is to folded.Finally it is folded into as shown in figure IV, makes 1,2,3 layer all to folded, progress immune response.
Fig. 3 is the principle schematic of paper substrate Immunofluorescence test.
Fig. 4 is with a kind of 3D fan-fold paper based microfluids fluorescence detection device detection CA724 (CA724) tumor-marker
Thing concentration and signal intensity graph of a relation.
Fig. 5 is with 3D fan-fold paper based microfluids fluorescence detection device detection CA125 (CA125), CA153
(CA153) two kinds of tumor markers concentration and signal intensity graph of a relation.
Fig. 6 is with 3D fan-fold paper based microfluid fluorescence detection devices while detects carcinomebryonic antigen (CEA), alpha-fetoprotein
(AFP), three kinds of tumor markers concentration of CA199 (CA199) and signal intensity graph of a relation.
Embodiment
Embodiment 1
3D fan-fold paper based microfluid detection means detects to a kind of CA724 (CA724) tumor markers.
The specific steps of the present embodiment, including：
(1) 3D paper substrate patterns are designed on computers, and carry out wax spray printing.
(2) paper is put into 150 degree of bakings in baking oven to take out for 150 seconds.
(3) after paper substrate is cooled to room temperature, 3D shape is folded into, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) labelled antibody corresponding to marked by fluorescein isothiocyanate CA724 tumor markerses is used.
Coated antibody corresponding to 4ul, 40ug/ml CA724 tumor markerses is fixed on (5) the 3rd layers of paper substrate G, G, I, J make
For blank control, after reacting 30min at room temperature, rinsed 3 times with 20ul, 0.01molPH=7.4 phosphate buffer.
(6) and then sequentially add 0.5% BSA and obstruct on the 3rd layer of paper the not specific position with antibody binding, room temperature
Rinsed 3 times with 20ul, 0.01molPH=7.4 phosphate buffer after lower reaction 15min.
(7) isothiocyanic acid of the kind tumor markers of CA724 corresponding to 4ul, 40ug/ml is added in second layer paper substrate C holes
Fluorescein labelled antibody, react 2min.
(8) by 1,2 two layers pair of folded, the addition 20ul tumor markers mixed liquors to be measured in 1 layer of A holes, reaction 2min.
(9) by three layers all to folded, and addition 40ul, 0.01molPH=7.4 phosphate buffer will in 1 layer of A holes
Antigen is flushed to 3 layers with labelled antibody compound.40ul, 0.01molPH=7.4 phosphate buffer is used to rinse 3 after reacting 4min
It is secondary, and absorb waste liquid with absorption pad.
(10) paper substrate micro-fluid chip is put under the excitation source that wavelength is 470nm and irradiated, can be according to yellow-green fluorescence
Power with colorimetric card compared with vision reading reading.Paper substrate micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
Count and be scanned in the case where wavelength is 470nm exciting light, tumor-marker can accurately be drawn respectively according to the peak value of fluorescence spectrum
The concentration of thing.
Embodiment 2
3D fan-fold paper based microfluid detection means is simultaneously to CA125 (CA125), CA153 (CA153) two
Kind tumor markers is detected.
The specific steps of the present embodiment, including：
(1) 3D paper substrate patterns are designed on computers, and carry out wax spray printing.
(2) paper is put into 150 degree of bakings in baking oven to take out for 150 seconds.
(3) after paper substrate is cooled to room temperature, 3D shape is folded into, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) labelled antibody corresponding to two kinds of tumor markerses of marked by fluorescein isothiocyanate CA125, CA153 is used.
Corresponding to the two kinds of tumor markerses of CA125, CA153 for fixing 4ul, 40ug/ml on (5) the 3rd layers of paper substrate G, H respectively
Coated antibody, I, J are as blank control, after reacting 30min at room temperature, with 20ul, 0.01molPH=7.4 phosphoric acid buffer
Liquid rinses 3 times.
(6) and then sequentially add 0.5% BSA and obstruct on the 3rd layer of paper the not specific position with antibody binding, room temperature
Rinsed 3 times with 20ul, 0.01molPH=7.4 phosphate buffer after lower reaction 15min.
(7) two kinds of tumour marks of CA125, CA153 corresponding to 4ul, 40ug/ml are separately added on the second layer paper substrate C, D
The marked by fluorescein isothiocyanate antibody of will thing, react 2min.
(8) by 1,2 two layers pair of folded, the addition 20ul tumor markers mixed liquors to be measured in 1 layer of A holes, reaction 2min.
(9) by three layers all to folded, and addition 40ul, 0.01molPH=7.4 phosphate buffer will in 1 layer of A holes
Antigen is flushed to 3 layers with labelled antibody compound.40ul, 0.01molPH=7.4 phosphate buffer is used to rinse 3 after reacting 4min
It is secondary, and absorb waste liquid with absorption pad.
(10) paper substrate micro-fluid chip is put under the excitation source that wavelength is 470nm and irradiated, can be according to yellow-green fluorescence
Power with colorimetric card compared with vision reading reading.Paper substrate micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
Count and be scanned in the case where wavelength is 470nm exciting light, various tumours can accurately be drawn respectively according to the peak value of fluorescence spectrum
The concentration of mark.
Embodiment 3
3D fan-fold paper based microfluid detection means is simultaneously to carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), CA199
(CA199) three kinds of tumor markerses are detected.
The specific steps of the present embodiment, including：
(1) 3D paper substrate patterns are designed on computers, and carry out wax spray printing.
(2) paper is put into 150 degree of bakings in baking oven to take out for 150 seconds.
(3) after paper substrate is cooled to room temperature, 3D shape is folded into, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) labelled antibody corresponding to tri- kinds of tumor markerses of marked by fluorescein isothiocyanate CEA, AFP, CA199 is used.
4ul, 40ug/ml tri- kinds of tumor markerses pair of CEA, AFP, CA199 are fixed on (5) the 3rd layers of paper substrate G, H, I respectively
The coated antibody answered, J after reacting 30min at room temperature, are delayed as blank control with 20ul, 0.01molPH=7.4 phosphoric acid
Fliud flushing is rinsed 3 times.
(6) and then sequentially add 0.5% BSA and obstruct on the 3rd layer of paper the not specific position with antibody binding, room temperature
Rinsed 3 times with 20ul, 0.01molPH=7.4 phosphate buffer after lower reaction 15min.
(7) three kinds that CEA, AFP, CA199 corresponding to 4ul, 40ug/ml are separately added on the second layer paper substrate C, D, E are swollen
The marked by fluorescein isothiocyanate antibody of tumor markers, react 2min.
(8) by 1,2 two layers pair of folded, the addition 20ul tumor markers mixed liquors to be measured in 1 layer of A holes, reaction 2min.
(9) by three layers all to folded, and addition 40ul, 0.01molPH=7.4 phosphate buffer will in 1 layer of A holes
Antigen is flushed to 3 layers with labelled antibody compound.40ul, 0.01molPH=7.4 phosphate buffer is used to rinse 3 after reacting 4min
It is secondary, and absorb waste liquid with absorption pad.
(10) paper substrate micro-fluid chip is put under the excitation source that wavelength is 470nm and irradiated, can be according to yellow-green fluorescence
Power with colorimetric card compared with vision reading reading.Paper substrate micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
Count and be scanned in the case where wavelength is 470nm exciting light, various tumours can accurately be drawn respectively according to the peak value of fluorescence spectrum
The concentration of mark.
The concrete technical scheme being not limited to described in above-described embodiment of the present invention, all technologies formed using equivalent substitution
Scheme is the protection domain of application claims.
Claims (3)
1. 3D fan-fold paper based microfluid fluorescence detection devices that are a kind of while detecting Diagnostic Value of Several Serum Tumor Markers, it is characterised in that：Bag
Include following steps：
(1) making of paper substrate micro-fluid chip；
The figure of 3D paper substrates is designed on computers, folding three-dimensional hydrophilic and hydrophobic region is formed, for detecting simultaneously
Diagnostic Value of Several Serum Tumor Markers, the number in two layers of the increase detection zone hole of paper substrate 2,3 is 1-5, you can the mark of increase detection simultaneously
Species number is 1-5, and is printed with wax spray printer, puts baking oven baking a period of time into, taking-up is cooled to room temperature, by paper substrate edge
Print area to cut, and carry out three dimensional fold；
(2) pretreatment of paper substrate micro-fluid chip；
Oxygen gas plasma processing is carried out with plasma cleaner by the 3rd layer of baked paper substrate chip, makes the carbon on paper surface
Free radical forms oxygen radical, causes the generation of aldehyde radical, this purpose is easy for fixation of the antibody on paper；
(3) the immune response program that three-dimensional paper substrate detects simultaneously；
The coated antibody of 1-5 kind tumor markerses is fixed respectively at plasma treated the 3rd layer first, then with barrier liquid
Obstruct paper on binding site, the 2nd layer add corresponding to 1-5 kind tumor markerses marked by fluorescein isothiocyanate antibody,
By 1,2 two layers pair it is folded, add determined antigen mixed liquor 1 layer of sample area, finally carry out immune response all to folded by three layers；
(4) fluorescence analysis detects；
By the 3rd layer of paper substrate micro-fluid chip as under specific excitation source, 1-5 kinds are assessed according to visual fluorescence is strong and weak
Tumor markers concentration, can be accurate by the 3rd layer of paper substrate micro-fluid chip fluorescent spectrophotometer assay emission wavelength peak
Draw the 1-5 kind tumor markers concentration of detection.
2. 3D fan-fold paper based microfluids fluoroscopic examination dress that is according to claim 1 while detecting Diagnostic Value of Several Serum Tumor Markers
Put, it is characterised in that：Number in two layers of the increase detection zone hole of paper substrate 2,3 is 3, the mark species number 3 that can be detected simultaneously
Kind.
3. 3D fan-fold paper based microfluids fluoroscopic examination dress that is according to claim 2 while detecting Diagnostic Value of Several Serum Tumor Markers
Put, it is characterised in that：
(1) making of paper substrate micro-fluid chip, graceful No. 1 chromatographic paper of water that specification is 20cm × 20cm is used；
(2) pretreatment of paper substrate micro-fluid chip, the 3rd layer of paper substrate micro-fluid chip is completed into baking, is put into plasma cleaner
Carry out surface and be modified 4min, being handled using oxygen surface plasma, which makes the surface of paper take aldehyde radical, is easy to antibody to fix, is waiting
Under the conditions of gas ions, the hydroxyl on carbon radicals-CH-OH on paper substrate cellulose can lose hydrogen atom, so as to form oxygen certainly
By base-O-CH2-, then, the Single Electron on the oxygen radical is combined with the electronics on carbon, causes the generation of aldehyde radical；
(3) in fluorescence immune reaction program, 4 μ L, 40 μ are fixed respectively on the 3rd layer of paper substrate G, H, I that aldehyde radical was modified first
The coated antibody of g/mL 3 kinds of tumor markerses, J is as blank control, after reacting 30min at room temperature, with 20 μ L,
0.01molPH=7.4 phosphate buffer rinses 3 times, then sequentially add on 0.5% BSA barrier paper not with antibody knot
The specific position of conjunction, react at room temperature after 15min with 20 μ L, 0.01molPH=7.4 phosphate buffer flushing 3 times, the
The marked by fluorescein isothiocyanate that three kinds of tumor markerses corresponding to 4 μ L, 40 μ g/mL are separately added on two layers of paper substrate C, D, E resists
Body, after 2min by 1,2 two layers pair it is folded, 20 μ L tumor markers mixed liquor to be measured is added in 1 layer of A holes, after reacting 2min
By three layers all to folded, and 40 μ L, 0.01molPH=7.4 phosphate buffer are added by antigen in 1 layer of A holes and is marked
Antibody complex is flushed to 3 layers.Rinsed 3 times with 40 μ L, 0.01molPH=7.4 phosphate buffer after reacting 4min, and with absorption
Pad absorbs waste liquid.
(4) paper substrate micro-fluid chip is put under the excitation source that wavelength is 470nm and irradiated, can be according to the power of yellow-green fluorescence
Vision reads reading compared with colorimetric card；Paper substrate micro-fluid chip is put into sample panel, with sepectrophotofluorometer in ripple
It is scanned under a length of 470nm exciting light, various tumor markerses can be accurately drawn respectively according to the peak value of fluorescence spectrum
Concentration.
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| CN108802356A (en) * | 2018-07-03 | 2018-11-13 | 济南大学 | A kind of preparation method of double mode three-dimensional paper chip device for cancer cell detection |
| CN109012770A (en) * | 2018-07-12 | 2018-12-18 | 中国科学院成都生物研究所 | Multi-ply paper chip structure, manufacturing equipment and method and fluid interlayer current method |
| CN109839502A (en) * | 2019-03-15 | 2019-06-04 | 中国科学院武汉物理与数学研究所 | One kind being used for on-site diagnosis MBP enzyme linked immuno-adsorbent assay device and method |
| CN110031617A (en) * | 2019-04-17 | 2019-07-19 | 厦门大学 | A kind of one-dimensional paper chip and the preparation method and application thereof for immunodiagnosis |
| CN110568192A (en) * | 2019-08-28 | 2019-12-13 | 千藤（南京）环保科技有限公司 | Zinc oxide nanowire fluorescence enhancement-based paper base analysis device and method |
| CN112088308A (en) * | 2018-04-27 | 2020-12-15 | 株式会社优尼恩斯真 | Microchip having three-dimensional structure of paper base for detecting target antigen using immunochemical diagnostic method and target antigen detecting method using the same |
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004038413A1 (en) * | 2002-10-24 | 2004-05-06 | Shanghai Healthdigit Co., Ltd. | A protein chips detecting system which can simultaneously detect multi target |
| CN101661034A (en) * | 2008-08-28 | 2010-03-03 | 上海领潮生物科技有限公司 | Method for synchronously performing parallel detection of various biomarkers and chip test paper |
| US20110111517A1 (en) * | 2008-03-27 | 2011-05-12 | President And Fellows Of Harvard College | Paper-based microfluidic systems |
| CN102980998A (en) * | 2012-11-21 | 2013-03-20 | 济南大学 | High-flux microfluidics paper chip for instantly and quickly detecting multiple human tumor markers |
| CN103364550A (en) * | 2013-07-16 | 2013-10-23 | 天津大学 | Method and device for rapidly and quantitatively detecting tumor marker with immunochromatography test strip marked by quantum dots |
| CN103760209A (en) * | 2014-01-27 | 2014-04-30 | 中国科学院电子学研究所 | Multi-parameter paper-chip electrochemical immunosensor and method for detecting lung cancer markers |
| CN103954751A (en) * | 2014-04-29 | 2014-07-30 | 成都君亚科技有限公司 | Paper-based micro-fluidic immunosensor chip and timely field detection immunoassay platform |
| CN104267182A (en) * | 2014-09-27 | 2015-01-07 | 成都领御生物技术有限公司 | Quantum dot immunochromatographic strip for synchronously quantifying multiple tumor markers and method of quantum dot immunochromatographic strip |
| CN106226523A (en) * | 2016-07-04 | 2016-12-14 | 福建广生堂药业股份有限公司 | Detection method, reagent and the detection kit of a kind of immunological marker thing |
| CN206082558U (en) * | 2016-04-27 | 2017-04-12 | 浙江工业大学 | Microfluid self -powered dynamic formula paper base micro -fluidic chip |
| CN106706916A (en) * | 2017-01-07 | 2017-05-24 | 孙翠敏 | Microfluidic chip for detection of tumor marker group |
-
2017
- 2017-09-19 CN CN201710852160.2A patent/CN107478631B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004038413A1 (en) * | 2002-10-24 | 2004-05-06 | Shanghai Healthdigit Co., Ltd. | A protein chips detecting system which can simultaneously detect multi target |
| US20110111517A1 (en) * | 2008-03-27 | 2011-05-12 | President And Fellows Of Harvard College | Paper-based microfluidic systems |
| CN101661034A (en) * | 2008-08-28 | 2010-03-03 | 上海领潮生物科技有限公司 | Method for synchronously performing parallel detection of various biomarkers and chip test paper |
| CN102980998A (en) * | 2012-11-21 | 2013-03-20 | 济南大学 | High-flux microfluidics paper chip for instantly and quickly detecting multiple human tumor markers |
| CN103364550A (en) * | 2013-07-16 | 2013-10-23 | 天津大学 | Method and device for rapidly and quantitatively detecting tumor marker with immunochromatography test strip marked by quantum dots |
| CN103760209A (en) * | 2014-01-27 | 2014-04-30 | 中国科学院电子学研究所 | Multi-parameter paper-chip electrochemical immunosensor and method for detecting lung cancer markers |
| CN103954751A (en) * | 2014-04-29 | 2014-07-30 | 成都君亚科技有限公司 | Paper-based micro-fluidic immunosensor chip and timely field detection immunoassay platform |
| CN104267182A (en) * | 2014-09-27 | 2015-01-07 | 成都领御生物技术有限公司 | Quantum dot immunochromatographic strip for synchronously quantifying multiple tumor markers and method of quantum dot immunochromatographic strip |
| CN206082558U (en) * | 2016-04-27 | 2017-04-12 | 浙江工业大学 | Microfluid self -powered dynamic formula paper base micro -fluidic chip |
| CN106226523A (en) * | 2016-07-04 | 2016-12-14 | 福建广生堂药业股份有限公司 | Detection method, reagent and the detection kit of a kind of immunological marker thing |
| CN106706916A (en) * | 2017-01-07 | 2017-05-24 | 孙翠敏 | Microfluidic chip for detection of tumor marker group |
Non-Patent Citations (3)
| Title |
|---|
| M.OMAIR NOOR等: "Paper-Based Solid一Phase Nucleic Acid Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer", 《ANALYTICAL CHEMISTRY》 * |
| SYRENA C.FERNANDES等: "Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| 范一强等: "3D打印微流控芯片技术研究进展", 《分析化学》 * |
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| CN112088308A (en) * | 2018-04-27 | 2020-12-15 | 株式会社优尼恩斯真 | Microchip having three-dimensional structure of paper base for detecting target antigen using immunochemical diagnostic method and target antigen detecting method using the same |
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| CN109839502B (en) * | 2019-03-15 | 2020-09-11 | 中国科学院武汉物理与数学研究所 | Enzyme-linked immunosorbent assay device and method for on-site diagnosis |
| CN110031617A (en) * | 2019-04-17 | 2019-07-19 | 厦门大学 | A kind of one-dimensional paper chip and the preparation method and application thereof for immunodiagnosis |
| CN110568192A (en) * | 2019-08-28 | 2019-12-13 | 千藤（南京）环保科技有限公司 | Zinc oxide nanowire fluorescence enhancement-based paper base analysis device and method |
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