CN107478631B - 3D fan-fold paper based microfluid fluorescence detection device that is a kind of while detecting Diagnostic Value of Several Serum Tumor Markers - Google Patents
3D fan-fold paper based microfluid fluorescence detection device that is a kind of while detecting Diagnostic Value of Several Serum Tumor Markers Download PDFInfo
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- CN107478631B CN107478631B CN201710852160.2A CN201710852160A CN107478631B CN 107478631 B CN107478631 B CN 107478631B CN 201710852160 A CN201710852160 A CN 201710852160A CN 107478631 B CN107478631 B CN 107478631B
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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 3D fan-fold paper based microfluid fluorescence detection devices for detecting Diagnostic Value of Several Serum Tumor Markers simultaneously, belong to technical field of biological.The detection device can be used for the detection to Diagnostic Value of Several Serum Tumor Markers simultaneously.The present invention the following steps are included: (1) paper base micro-fluid chip production；(2) pretreatment of paper base micro-fluid chip；(3) the immune response program that three-dimensional paper base detects simultaneously；(4) fluorescence analysis detects.The present invention combines paper base microfluid analysis platform with fluorescence detection, for detecting while Diagnostic Value of Several Serum Tumor Markers.This detection device is simple, cheaply, portable, disposably, easy to use, limited and from far-off regions provide advantageous platform for developing country and resource.
Description
Technical field
The present invention relates to a kind of detection device, especially a kind of 3D folding that Diagnostic Value of Several Serum Tumor Markers can be carried out while be detected
Stacker based microfluid fluorescence detection device, belongs to technical field of biological.
Background technique
According to " report of world's cancer " that the World Health Organization and international cancer research institution (IARC) issue, cancer is complete
The main reason for ball is dead, early detection Diagnostic Value of Several Serum Tumor Markers is convenient for diagnosis 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 of most of marks
Object does not have specificity to specific tumors.In order to improve the accuracy of cancer diagnosis, it is necessary in conjunction with Diagnostic Value of Several Serum Tumor Markers
Detection, because it can significant raising specificity.
A variety of methods have been proposed in the past few years while detecting Diagnostic Value of Several Serum Tumor Markers.For example, electrochemistry is exempted from
Epidemic disease measurement, unmarked method mediate 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 operate.Limited equipment and without enough trainings personnel may
It will limit remote and rural area early screening tumour.Therefore, in the case where not using laboratory or trained personnel,
Can detect simultaneously multiple tumor markers 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 in conjunction with paper base microfluid is increasingly taken seriously, because it has easy to operate, analyte volume
The advantages of small, analysis time is short, at low cost, more analysis analyses does not need profession and realizes the ability of point-of care test.So
And these methods are mostly enzyme linked immunologicals, need to add substrate and terminate liquid in enzyme linked immunoassay, keep response procedures more complicated；And
And the enzyme-linked temperature for needing to be suitable for and PH are not suitable for quickly detecting whenever and wherever possible in order to avoid influencing the activity of enzyme.And fluorescent material is
Selection well, because optical signal is sensitive, and the immunoassay with fluorescent marker has the latent of quantization multiple analyte
Power.Fluorescein isothiocynate (FITC) is a kind of dyestuff for sending out yellow-green fluorescence strong, its quantum yield is high, fluorescence lifetime is long, is used
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.
Summary of the invention
Technical problem solved by the present invention is proposing that 3D that is a kind of while detecting Diagnostic Value of Several Serum Tumor Markers folds paper base miniflow
Body fluorescence detection device, the portable paper base 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 solution proposed by the present invention is: a kind of while detecting kinds of tumors mark
The 3D fan-fold paper based microfluid fluorescence detection device of object, comprising the following steps:
(1) production of paper base micro-fluid chip；
The figure for designing 3D paper base on computers constitutes folding three-dimensional hydrophilic and hydrophobic region, for simultaneously
Diagnostic Value of Several Serum Tumor Markers is detected, the number for increasing detection zone hole at two layers of paper base 2,3 is 1-5, can be increased while detecting
Marker 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 base micro-fluid chip；
Oxygen gas plasma processing is carried out with plasma cleaner by the 3rd layer of baked paper base chip, makes paper surface
Carbon radicals formed oxygen radical, lead to 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 base detects simultaneously；
The coated antibody of 1-5 kind tumor markers is fixed respectively at plasma treated the 3rd layer first, then with resistance
Binding site on liquid barrier paper, the marked by fluorescein isothiocyanate that corresponding 1-5 kind tumor markers are added at the 2nd layer are anti-
Body, by 1,2 two layers pair it is folded, determined antigen mixed liquor is added 1 layer of sample area, finally carries out three layers immune anti-all to folded
It answers；
(4) fluorescence analysis detects；
By the 3rd layer of paper base micro-fluid chip as under specific excitation light source, assessed according to visual fluorescence power
1-5 kind tumor markers concentration, by the 3rd layer of paper base micro-fluid chip fluorescent spectrophotometer assay emission wavelength peak,
It can accurately obtain the 1-5 kind tumor markers concentration of detection.
Preferably, the number for increasing detection zone hole at two layers of paper base 2,3 is 3, the marker species number 3 that can be detected simultaneously
Kind.
Preferably, the production of (1) paper base micro-fluid chip, using specification is graceful No. 1 color of water of 20cm × 20cm
Manuscript；
(2) baking is completed the 3rd layer of paper base micro-fluid chip, it is clear to be put into plasma by the pretreatment of paper base micro-fluid chip
Washing machine carries out the modified 4min in surface, and being handled using oxygen surface plasma makes the surface of paper take aldehyde radical fixes convenient for antibody,
Under plasma conditions, the hydroxyl on the carbon radicals-CH-OH on paper base cellulose can lose hydrogen atom, to be formed
Oxygen radical-O-CH2-, then, the Single Electron on the oxygen radical lead to the generation of aldehyde radical in conjunction with the electronics on carbon；
(5) in fluorescence immune reaction program, 4 μ are fixed respectively on the 3rd layer of paper base G, H, I that aldehyde radical was modified first
L, the coated antibody of 3 kinds of tumor markers of 40 μ g/mL, J is as blank control, after reacting 30min at room temperature, with 20 μ L,
The phosphate buffer of 0.01molPH=7.4 rinses 3 times, then sequentially add on 0.5% BSA barrier paper not with antibody knot
The specific position of conjunction, reacts after 15min at room temperature with the phosphate buffer of 20 μ L, 0.01molPH=7.4 flushing 3 times, the
The marked by fluorescein isothiocyanate that the corresponding three kinds of tumor markers of 4 μ L, 40 μ g/mL are separately added on two layers of paper base C, D, E is anti-
Body, after 2min by 1,2 two layers pair it is folded, the tumor markers mixed liquor to be measured of 20 μ L is added in 1 layer of the hole A, after reacting 2min
By three layers all to folded, and the phosphate buffer of 40 μ L, 0.01molPH=7.4 is added in 1 layer of the hole A by antigen and label
Antibody complex is flushed to 3 layers.It is rinsed 3 times after reacting 4min with the phosphate buffer of 40 μ L, 0.01molPH=7.4, and with absorption
Pad absorbs waste liquid.
(6) paper base micro-fluid chip is put under the excitation light source that wavelength is 470nm and is irradiated, it can be according to yellow-green fluorescence
Power is compared vision with colorimetric card and reads reading；Paper base micro-fluid chip is put into sample panel, uses sepectrophotofluorometer
It is scanned in the case where wavelength is the exciting light of 470nm, can accurately obtain respectively various tumour marks according to the peak value of fluorescence spectrum
The concentration of will object.
Beneficial effects of the present invention:
(1) model for paper being folded into 3D is easy to carry, not by condition and territory restriction, can be used for self-test anywhere or anytime
It is fast to survey.
(2) 3D folds paper base respectively in latter two layers plus antibody, directly adds sample to be tested in top layer, without adding an examination of again after sample-adding
Agent operation, not only simplifies operating procedure, and realizes and the sample to be tested i.e. practical detection device of readable effects is added.
(3) paper base microfluid is combined with fluorescence immunoassay, eliminates large-scale medical detecting Instrument or electrochemical workstation
Deng large-scale instrument, fluoroimmunoassay can be simple and convenient with Visual readings.
(4) corona treatment makes paper, and the modified aldehyde radical that takes in surface is simple time saving and effective convenient for antibody combination.?
Under condition of plasma, the hydroxyl on carbon radicals (- CH-OH) on paper base cellulose may lose hydrogen atom, thus
It is formed oxygen radical (- O-CH2-).Then, the Single Electron on the oxygen radical leads to aldehyde radical in conjunction with the electronics on carbon
Generation.
(5) entire paper base microfluid fluorescence of fluorescence detection device preparation process is simple, detects quick and high sensitivity, can be right simultaneously
Diagnostic Value of Several Serum Tumor Markers is detected, and can be used for the early screening of the quick detection and cancer of tumour.
(6) paper base microfluid analysis device has simplicity, and portability, disposably, low cost are nontoxic, can be at any time
The advantages that self-test is surveyed fastly is carried out everywhere.The present invention combines paper base microfluid analysis platform with fluorescence detection, for a variety of
It is detected while tumor markers.This detection device is simple, cheaply, portable, disposably, easy to use, is developing country
It is limited and from far-off regions provide advantageous platform with resource.
Detailed description of the invention
Of the invention is described further with reference to the accompanying drawing.
Fig. 1 is the plane outspread drawing of 3D fan-fold paper based microfluid fluorescence detection device.Wherein 1 layer for sample be added 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 the corresponding labelled antibody of object, F are blank control check plot.G, H, I are respectively the detection zone of three kinds of markers, and J is
Check plot.
Fig. 2 is the use fold sequence figure of 3D fan-fold paper based microfluid fluorescence detection device.First by paper base 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
Labelled antibody is added in layer.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.It is finally folded into as shown in figure IV, makes 1,2,3 layer all to folding, be immunoreacted.
Fig. 3 is the schematic illustration of paper base Immunofluorescence test.
Fig. 4 is to detect a kind of carbohydrate antigen 724 (CA724) tumor-marker with 3D fan-fold paper based microfluid fluorescence detection device
Object concentration and signal strength relational graph.
Fig. 5 is to detect carbohydrate antigen 125 (CA125), Carbohydrate Antigen 153 with 3D fan-fold paper based microfluid fluorescence detection device
(CA153) two kinds of tumor markers concentration and signal strength relational graph.
Fig. 6 is with 3D fan-fold paper based microfluid fluorescence detection device while to detect carcinomebryonic antigen (CEA), alpha-fetoprotein
(AFP), three kinds of tumor markers concentration of carbohydrate antigen 199 (CA199) and signal strength relational graph.
Specific embodiment
Embodiment 1
3D fan-fold paper based microfluid detection device detects a kind of carbohydrate antigen 724 (CA724) tumor markers.
The specific steps of the present embodiment, comprising:
(1) 3D paper base pattern is designed on computers, and carries out wax spray printing.
(2) paper is put into baking oven and is taken out within baking 150 seconds for 150 degree.
(3) after paper base is cooled to room temperature, it is folded into 3D shape, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) the corresponding labelled antibody of marked by fluorescein isothiocyanate CA724 tumor markers is used.
The corresponding coated antibody of CA724 tumor markers of 4ul, 40ug/ml are fixed on (5) the 3rd layers of paper base G, G, I, J make
For blank control, after reacting 30min at room temperature, rinsed 3 times with the phosphate buffer of 20ul, 0.01molPH=7.4.
(6) BSA for then sequentially adding 0.5% obstructs specific position on the 3rd layer of paper not in conjunction with antibody, room temperature
It is rinsed 3 times after lower reaction 15min with the phosphate buffer of 20ul, 0.01molPH=7.4.
(7) isothiocyanic acid of the kind tumor markers of the corresponding CA724 of 4ul, 40ug/ml is added in the second layer hole paper base C
Fluorescein labelled antibody reacts 2min.
(8) by 1,2 two layers pair of folded, the tumor markers mixed liquors to be measured of addition 20ul in 1 layer of the hole A, reaction 2min.
(9) by three layers all to folded, and the phosphate buffer that 40ul, 0.01molPH=7.4 is added in 1 layer of the hole A will
Antigen and labelled antibody compound are flushed to 3 layers.The phosphate buffer of 40ul, 0.01molPH=7.4 is used to rinse 3 after reacting 4min
It is secondary, and waste liquid is absorbed with absorption pad.
(10) paper base micro-fluid chip is put under the excitation light source that wavelength is 470nm and is irradiated, it can be according to yellow-green fluorescence
Power be compared with colorimetric card vision read read.Paper base micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
It counts and is scanned in the case where wavelength is the exciting light of 470nm, can accurately obtain respectively tumor-marker according to the peak value of fluorescence spectrum
The concentration of object.
Embodiment 2
3D fan-fold paper based microfluid detection device is simultaneously to carbohydrate antigen 125 (CA125), Carbohydrate Antigen 153 (CA153) two
Kind tumor markers are detected.
The specific steps of the present embodiment, comprising:
(1) 3D paper base pattern is designed on computers, and carries out wax spray printing.
(2) paper is put into baking oven and is taken out within baking 150 seconds for 150 degree.
(3) after paper base is cooled to room temperature, it is folded into 3D shape, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) the corresponding labelled antibody of two kinds of tumor markers of marked by fluorescein isothiocyanate CA125, CA153 is used.
The two kinds of tumor markers of CA125, CA153 for fixing 4ul, 40ug/ml on (5) the 3rd layers of paper base G, H respectively are corresponding
Coated antibody, I, J are as blank control, after reacting 30min at room temperature, with the phosphoric acid buffer of 20ul, 0.01molPH=7.4
Liquid rinses 3 times.
(6) BSA for then sequentially adding 0.5% obstructs specific position on the 3rd layer of paper not in conjunction with antibody, room temperature
It is rinsed 3 times after lower reaction 15min with the phosphate buffer of 20ul, 0.01molPH=7.4.
(7) two kinds of tumour marks of 4ul, 40ug/ml corresponding CA125, CA153 are separately added on the second layer paper base C, D
The marked by fluorescein isothiocyanate antibody of will object reacts 2min.
(8) by 1,2 two layers pair of folded, the tumor markers mixed liquors to be measured of addition 20ul in 1 layer of the hole A, reaction 2min.
(9) by three layers all to folded, and the phosphate buffer that 40ul, 0.01molPH=7.4 is added in 1 layer of the hole A will
Antigen and labelled antibody compound are flushed to 3 layers.The phosphate buffer of 40ul, 0.01molPH=7.4 is used to rinse 3 after reacting 4min
It is secondary, and waste liquid is absorbed with absorption pad.
(10) paper base micro-fluid chip is put under the excitation light source that wavelength is 470nm and is irradiated, it can be according to yellow-green fluorescence
Power be compared with colorimetric card vision read read.Paper base micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
It counts and is scanned in the case where wavelength is the exciting light of 470nm, can accurately obtain respectively various tumours according to the peak value of fluorescence spectrum
The concentration of marker.
Embodiment 3
3D fan-fold paper based microfluid detection device is simultaneously to carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 199
(CA199) three kinds of tumor markers are detected.
The specific steps of the present embodiment, comprising:
(1) 3D paper base pattern is designed on computers, and carries out wax spray printing.
(2) paper is put into baking oven and is taken out within baking 150 seconds for 150 degree.
(3) after paper base is cooled to room temperature, it is folded into 3D shape, and third layer is carried out at oxygen surface plasma
Reason 4 minutes, makes aldehyde radical in the surface modification of paper.
(4) the corresponding labelled antibody of tri- kinds of tumor markers of marked by fluorescein isothiocyanate CEA, AFP, CA199 is used.
Tri- kinds of tumor markers pair of CEA, AFP, CA199 of 4ul, 40ug/ml are fixed on (5) the 3rd layers of paper base G, H, I respectively
The coated antibody answered, J is as blank control, slow with the phosphoric acid of 20ul, 0.01molPH=7.4 after reacting 30min at room temperature
Fliud flushing is rinsed 3 times.
(6) BSA for then sequentially adding 0.5% obstructs specific position on the 3rd layer of paper not in conjunction with antibody, room temperature
It is rinsed 3 times after lower reaction 15min with the phosphate buffer of 20ul, 0.01molPH=7.4.
(7) three kinds that 4ul, 40ug/ml corresponding CEA, AFP, CA199 are separately added on the second layer paper base C, D, E are swollen
The marked by fluorescein isothiocyanate antibody of tumor markers reacts 2min.
(8) by 1,2 two layers pair of folded, the tumor markers mixed liquors to be measured of addition 20ul in 1 layer of the hole A, reaction 2min.
(9) by three layers all to folded, and the phosphate buffer that 40ul, 0.01molPH=7.4 is added in 1 layer of the hole A will
Antigen and labelled antibody compound are flushed to 3 layers.The phosphate buffer of 40ul, 0.01molPH=7.4 is used to rinse 3 after reacting 4min
It is secondary, and waste liquid is absorbed with absorption pad.
(10) paper base micro-fluid chip is put under the excitation light source that wavelength is 470nm and is irradiated, it can be according to yellow-green fluorescence
Power be compared with colorimetric card vision read read.Paper base micro-fluid chip is put into sample panel, uses fluorescence spectrophotometry
It counts and is scanned in the case where wavelength is the exciting light of 470nm, can accurately obtain respectively various tumours according to the peak value of fluorescence spectrum
The concentration of marker.
Of the invention is not limited to the above embodiment the specific technical solution, all technologies formed using equivalent replacement
Scheme be the present invention claims protection scope.
Claims (3)
1. a kind of 3D fan-fold paper based microfluid fluorescence detection device for detecting Diagnostic Value of Several Serum Tumor Markers simultaneously, it is characterised in that: packet
Include following steps:
(1) production of paper base micro-fluid chip；
The figure for designing 3D paper base on computers constitutes folding three-dimensional hydrophilic and hydrophobic region, for detecting simultaneously
Diagnostic Value of Several Serum Tumor Markers, the number for increasing detection zone hole at two layers of paper base 2,3 is 2-5, the mark that can be increased while detecting
Species number is 2-5, 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
It is cut along print area, and carries out three dimensional fold；
(2) pretreatment of paper base micro-fluid chip；
Oxygen gas plasma processing is carried out with plasma cleaner by the 3rd layer of baked paper base micro-fluid chip, makes paper table
The carbon radicals in face form oxygen radical, lead to 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 base detects simultaneously；
Fix the coating of 2-5 kind tumor markers respectively at the 3rd layer of plasma treated paper base micro-fluid chip first
Antibody, it is swollen in the corresponding 2-5 kind of the 2nd layer of addition of paper base micro-fluid chip then with the binding site on barrier liquid barrier paper
The marked by fluorescein isothiocyanate antibody of tumor markers, by the 1 of paper base micro-fluid chip, 2 two layers pair it is folded, in paper base microfluid core
Determined antigen mixed liquor is added in 1st layer of sample area of piece, is finally immunoreacted three layers all to folding；
(4) fluorescence analysis detects；
The 3rd of paper base micro-fluid chip is placed under specific excitation light source, 2-5 kind is assessed according to visual fluorescence power
Tumor markers concentration can be accurate by the 3rd layer of paper base micro-fluid chip fluorescent spectrophotometer assay emission wavelength peak
Obtain the 2-5 kind tumor markers concentration of detection.
2. 3D fan-fold paper based microfluid fluorescence detection dress that is according to claim 1 while detecting Diagnostic Value of Several Serum Tumor Markers
It sets, it is characterised in that: the number for increasing detection zone hole at two layers of paper base 2,3 is 3, the marker species number 3 that can be detected simultaneously
Kind.
3. 3D fan-fold paper based microfluid fluorescence detection dress that is according to claim 2 while detecting Diagnostic Value of Several Serum Tumor Markers
It sets, it is characterised in that:
(1) production of paper base micro-fluid chip, using specification is graceful No. 1 chromatographic paper of water of 20cm × 20cm；
(2) baking is completed the 3rd layer of paper base micro-fluid chip, is put into plasma cleaner by the pretreatment of paper base micro-fluid chip
The modified 4min in surface is carried out, handling using oxygen surface plasma, which makes the surface of paper take aldehyde radical, fixes convenient for antibody, waiting
Under the conditions of gas ions, the hydroxyl on carbon radicals-CH-OH on paper base cellulose can lose hydrogen atom, to form oxygen certainly
By base-O-CH2-, then, the Single Electron on the oxygen radical leads to the generation of aldehyde radical in conjunction with the electronics on carbon；
(3) in fluorescence immune reaction program, first on the paper base micro-fluid chip that aldehyde radical was modified the 3rd layer of G, H, I respectively
The coated antibody of 3 kinds of tumor markers of 4 μ L, 40 μ g/mL is fixed, J is as blank control, after reacting 30min at room temperature, uses
20 μ L, 0.01molPH=7.4 phosphate buffer rinse 3 times, then sequentially add 0.5% BSA barrier paper on not with antibody
In conjunction with specific position, reacted after 15min at room temperature with the phosphate buffers of 20 μ L, 0.01molPH=7.4 flushing 3 times,
The isothiocyanic acid of the corresponding three kinds of tumor markers of 4 μ L, 40 μ g/mL is separately added on paper base micro-fluid chip the 2nd layer of C, D, E
Fluorescein labelled antibody, after 2min by the 1 of paper base micro-fluid chip, 2 two layers pair it is folded, in the 1st layer of the hole A of paper base micro-fluid chip
The middle tumor markers mixed liquor to be measured that 20 μ L are added reacts three layers after 2min all to folded, and in paper base micro-fluid chip
40 μ L are added in 1st layer of the hole A, antigen and labelled antibody compound are flushed to 3 layers by the phosphate buffer of 0.01molPH=7.4；
It is rinsed 3 times after reacting 4min with the phosphate buffer of 40 μ L, 0.01molPH=7.4, and absorbs waste liquid with absorption pad；
(4) paper base micro-fluid chip is put under the excitation light source that wavelength is 470nm and is irradiated, it can be according to the power of yellow-green fluorescence
It is compared vision with colorimetric card and reads reading；Paper base micro-fluid chip is put into sample panel, with sepectrophotofluorometer in wave
It is scanned under the exciting light of a length of 470nm, can accurately obtain respectively various tumor markers according to the peak value of fluorescence spectrum
Concentration.
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| CN107884380B (en) * | 2017-12-19 | 2020-05-12 | 西北工业大学 | Full-automatic rapid fluorescence analysis method of micro-reaction cavity array chip |
| KR102088277B1 (en) * | 2018-04-27 | 2020-03-13 | 주식회사 유니언스진 | 3D paper based Microfluidic device for Thioredoxin detection by Enzyme linked immunosorbent assay |
| 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 |
| CN109012770B (en) * | 2018-07-12 | 2021-05-14 | 中国科学院成都生物研究所 | Multilayer paper chip structure, manufacturing apparatus and method, and fluid interlayer flow method |
| 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 |
| CN110031617B (en) * | 2019-04-17 | 2020-11-24 | 厦门大学 | One-dimensional paper chip for immunodiagnosis and preparation method and application thereof |
| 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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Citations (10)
| 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 |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100128340A (en) * | 2008-03-27 | 2010-12-07 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Paper-based microfluidic systems |
-
2017
- 2017-09-19 CN CN201710852160.2A patent/CN107478631B/en active Active
Patent Citations (10)
| 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 |
| 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 |
|---|
| 3D打印微流控芯片技术研究进展;范一强等;《分析化学》;20160430;第44卷(第4期);第551-561页 * |
| Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays;Syrena C.Fernandes等;《Journal of Visualized Experiments》;20170309;第1-10页 * |
| Paper-Based Solid一Phase Nucleic Acid Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer;M.Omair Noor等;《Analytical Chemistry》;20121209;第1860-1867页 * |
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