CN107991484A - A kind of rotary electrical chemo-immunity refill card analyzer and its detection method - Google Patents
A kind of rotary electrical chemo-immunity refill card analyzer and its detection method Download PDFInfo
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- CN107991484A CN107991484A CN201711159820.5A CN201711159820A CN107991484A CN 107991484 A CN107991484 A CN 107991484A CN 201711159820 A CN201711159820 A CN 201711159820A CN 107991484 A CN107991484 A CN 107991484A
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- bracket panel
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- washing disk
- immunity
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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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
Abstract
The present invention relates to electrochemical analysis, biological immune and micro fluidic chip technical field, is more specifically a kind of rotary electrical chemo-immunity refill card analyzer and its detection method.Device includes the bracket panel, reagent disc and washing disk of rotation connection, the bracket panel, reagent disc and washing disk can independently rotate, respective fluid passage is respectively equipped with bracket panel, reagent disc and washing disk, be provided with hole on each fluid passage, hole of the control fluid on the bracket panel, reagent disc and washing disk rotate to when communicating with each other by being flowed through in each hole.Each layer paper chip can independently be rotated around center in apparatus of the present invention, this causes rotary valve is dexterously integrated into be used for the flowing for controlling paper chip upper fluid on refill sheet devices.In the present invention device prepare it is simple and quick, it is easy to operate, provide a kind of new device for quickly and conveniently medical care diagnostic.
Description
Technical field
The present invention relates to electrochemical analysis, biological immune and micro fluidic chip technical field, is more specifically a kind of
Rotary electrical chemo-immunity refill card analyzer and its detection method.
Background technology
Micro-fluidic paper chip is using paper as backing material, has the cost low, easy as a micro-fluidic important branch
The advantages that making, is easy to carry, since the Whiteside seminars by Harvard University in 2007 propose, developing rapidly becomes
A kind of emerging analysis platform.Compared with conventional ELISA test strip, micro-fluidic refill card analyzer can realize multichannel,
The parallel quantitative analysis of multi objective, meets to the quick, cheap of medical diagnosis on disease, food security and environmental contaminants and easily divides
Analysis demand.Current various detection methods are applied to sensitivity and the accuracy that refill sheet devices improve detection, such as colorimetric method, fluorescence
Method, chemiluminescence, electrochemical process etc..Wherein, electrogenerated chemiluminescence is to combine Electrochemical Detection and chemiluminescence, high
Sensitivity, low background value and small-sized detection device make it more and more be applied in biological and chemical context of detection.
And with the development of science and technology, quantitative inspection can be realized by collecting the luminous intensity data for chemically reacting and sending by cell-phone camera
Survey, the combination of the portable detection equipment such as paper chip and mobile phone is more advantageous to detection easily and fast, therefore paper chip analysis dress
The electrochemiluminescdetection detection put provides a kind of new hand to develop rapid, facilitation care diagnostic analysis platform
Section.
The flowing of paper chip upper fluid mainly drives by capillary force, it is not necessary to by external impetus, but this
The manipulation of fluid brings certain difficulty in one advantage paper feeding chip analysis, and it is multiple in multi-step, multicomponent to limit paper chip
Application in miscellaneous system.
The content of the invention
It is an object of the invention to provide a kind of rotary electrical chemo-immunity refill card analyzer and its detection method.
To achieve the above object, the present invention use technical solution for：
A kind of rotary electrical chemo-immunity refill card analyzer, including the bracket panel of rotation connection, reagent disc and washing
Disk, the bracket panel, reagent disc and washing disk can independently rotate, and be respectively equipped with bracket panel, reagent disc and washing disk respective
Fluid passage, hole, hole rotation of the control fluid on the bracket panel, reagent disc and washing disk are provided with each fluid passage
To when communicating with each other by being flowed through in each hole.
The bracket panel, detection plate and washing disk are using cellulosic filter paper as carrier respectively, will be flowed by wax spray printer
Body channel pattern is printed on paper, and hydrophilic region and the alternate paper chip of hydrophobic region are obtained after baking, then passes through screen printing
Brush board, working electrode is printed onto in paper chip electrode and reference electrode.
Described device is by being followed successively by washing for bracket panel, the detection plate of immune response and the unnecessary reaction reagent of elution under upper
Disk is washed, is rotatablely connected and assembled by rivet.
Fluid passage is equipped with each layer paper chip, and fluid is controlled in respective passage by the rotation of paper chip
Circulation；Four are dispersed with detection plate and prints the circular immunologic areas of working electrode；Four are dispersed with bracket panel
Circular hole and four are printed with reference electrode and the hydrophilic region to electrode.Under the rotation of bracket panel, sense channel disconnects, detection
Immunologic areas on disk reveals, and reaction reagent adds immunologic areas, and the rotation of another aspect bracket panel connects sense channel
Connect, reference electrode on working electrode and bracket panel in detection plate and three-electrode system is formed to electrode be used for electrochemistry inspection
Survey, washing disk is equipped with four washing channels, and the rotation by washing disk can control the connection of immunologic areas and washing channel
With disconnection, the unreacted reagent in immunologic areas is removed for washing.
A kind of application of the device, described device are quantitatively and/or qualitatively carrying out electrification to Diagnostic Value of Several Serum Tumor Markers
Learn the application in immune detection.
A kind of method using quantitative analysis of the described device to Diagnostic Value of Several Serum Tumor Markers,
1) described device is utilized, capture cross-linking reaction of the antibody through glutaraldehyde is fixed on multi-walled carbon nanotube modification, shell
The immunologic areas of glycan load；
2) signal antibody marked successively with antigen to be detected and ruthenium after bovine serum albumin(BSA) is closed reacts to form folder
The immune complex of cardioid is on such devices；
3) luminous signal is produced as coreaction reagent be used for Diagnostic Value of Several Serum Tumor Markers quantitatively and/or qualitatively by tripropyl amine (TPA)
Analysis.
Reaction process in the above-mentioned immunoassay is by the bracket panel that is alternately rotated in described device, reagent disc and washes
Wash disk and then realize analysis and detection.
Advantage for present invention：
Rotary valve is integrated on refill card analyzer by the present invention constructs the analysis of rotary electrical chemo-immunity paper chip
Detection platform.Rotating to manipulate the connection and disconnection between fluid passage based on paper chip, will arrive involved in immunoassay process
Reaction process, washing process and detection process be integrated on whole refill card analyzer, realize and multi-step be immunized
The manipulation of analytic process, simplifies cumbersome immunoassay process.
The electro-chemistry immunity refill card analyzer of the present invention is easy to operate, the fast response time of valve, with reference to Gao Ling
The electrochemiluminescdetection detection technology of sensitivity, quick diagnosis and the scene that can be widely applied to Diagnostic Value of Several Serum Tumor Markers are instant
Detection.
The quick, high of Diagnostic Value of Several Serum Tumor Markers can be used for using rotary electrical chemo-immunity refill card analyzer of the present invention
The quantitative analysis of sensitivity, meet its multicomponent, multi-step complex system in application.
Brief description of the drawings
Fig. 1 is rotary electrical chemo-immunity refill card analyzer assembling schematic diagram provided in an embodiment of the present invention, wherein
Area is immunized in A- washing disks, B- detection plates, C- bracket panels, 1- washing channels, 2- circular holes, 3- center holes, 4- working electrodes, 5-
Domain, 6- reference electrodes, 7- fix electrode centrally through rivet, it is possible to achieve spinfunction.
Fig. 2 is the pictorial diagram of rotary electrical chemo-immunity refill card analyzer provided in an embodiment of the present invention.
Fig. 3 is provided in an embodiment of the present invention bent to the luminous intensity response of carcino-embryonic antigen assay and standard using device
Line.
Fig. 4 is the luminous intensity measured using device to prostate-specific antigen the response and mark that present example provides
Directrix curve.
Embodiment
The following examples are with reference to the accompanying drawings described in further detail the present invention, but therefore do not limit this hair
It is bright.
Each layer paper chip can independently be rotated around center in apparatus of the present invention, this causes rotary valve dexterously to integrate
It is used for the flowing for controlling paper chip upper fluid on to refill sheet devices.Company's through and off based on the rotation of paper chip to fluid passage
The control opened, reaction process, washing process and the detection process of multistep are simplified on the apparatus in immunoassay process,
With reference to highly sensitive electrochemical luminescence detection technique, realize to the quick quantitative analysis of Diagnostic Value of Several Serum Tumor Markers.In the present invention
Preparing for device is simple and quick, easy to operate, and a kind of new device is provided for quickly and conveniently medical care diagnostic.
Embodiment 1：A kind of preparation of rotary electrical chemo-immunity refill card analyzer
As shown in Figure 1, rotary paper chip analysis device is led to by bracket panel C, detection plate B and washing disk A three-ply paper chips
Cross rivet rotation connection composition.Detection plate B has four immunologic areas 5, wherein the immunologic areas print of the wherein one side in the disk
Brushed with working electrode 4.Four circular holes 2 are dispersed with bracket panel C and are printed with reference electrode 6 and the circular hydrophilic area to electrode 7
Domain.Detection plate B and bracket panel C constitutes the upper strata valve of the device, when rotation bracket panel B disconnects sense channel, by detection plate
On immunologic areas 5 be exposed and add reaction reagent immune response, the rotation of another aspect bracket panel B can also lead to detection
Road connects, and working electrode 4 and reference electrode 6, be used for Electrochemical Detection to the formation three-electrode system of electrode 7, washs on disk and has on A
There are four circular holes 2 and hydrophilic washing channel 1, washing disk A and detection plate B forms lower floor's valve, and rotation washing disk A, which can be controlled, to exempt from
Epidemic-stricken area domain 5 and the connection of washing channel 1 are used to remove the unreacted reagent in immunologic areas with disconnecting.Wash disk A, detection plate B and
The circular hole 3 of 3mm is designed with the center of bracket panel C, three-ply paper chip is assembled into rotary paper chip point by rivet through the circular hole
Analysis apparatus so that each layer paper chip can independently rotate the connection and disconnection of control fluid passage around the rivet at center.
Washing disk A, detection plate B and washing disk C three-ply papers chip in the device are using fiber filter paper as carrier, are passed through
The pattern that designs in the printing of wax spray printer, the back side of paraffin heated melt infiltration to paper obtain containing different hydrophilic, hydrophobic
The paper chip in region.It is hydrophobic region that colored region, which is printed with paraffin, in 1 in figure, and the region of white does not print paraffin,
Still keep the original hydrophily of paper.Reference electrode 6 and auxiliary electrode 7 are logical on working electrode 4 and bracket panel on detection plate B
The method for crossing silk-screen printing is printed onto in paper chip.Using card punch respectively bracket panel, detection plate and wash disk center at
Stamp circular hole 3 and stamp circular hole 2 in bracket panel and washing disk.As shown in Figure 1, by the washing disk A, the detection plate B that make and auxiliary
Disk C is helped to be used for the making of rotary paper chip apparatus, detection plate B is had working electrode first one is placed on washing disk A down
On, then bracket panel C is had the one side of electrode and reference electrode is placed on detection plate A upward, make washing disk A, detection plate B
And bracket panel C is stacked in an orderly manner, then rivet is sequentially passed through to the center hole of each layer paper chip, each disk is rotatablely connected；
Under the beaing of hammer, the tip dimensions of rivet expand about 1.5 times compared with original size, make bracket panel, detection plate and washing disk
Fixed assembling obtains rotary paper chip apparatus (referring to Fig. 2).
Embodiment 2：The operating process of immunoassay on the device
Each paper chip disk can be rotated around center in the rotary paper chip analysis device of the gained of above-described embodiment 1,
The connection and disconnection of paper chip upper fluid passage are manipulated by rotating bracket panel and washing disk, realizes how rapid immunoassay mistake
Journey.
Rotary valve manipulation immunoassay process be：For the reaction process in immunoassay, bracket panel C is rotated first and is closed
Sense channel is closed, reveals the immunologic areas 5 of detection plate B, then rotation washing disk A, the washing channel 1 on disk will be washed
Disconnected with the immunologic areas 5 in detection plate, reaction reagent can be added to immunologic areas 5 and be reacted at this time.Question response complete with
Afterwards, it is necessary to which washing is carried out to immunologic areas removes unreacted reagent.For washing process, rotation washing disk A makes on washing disk
Washing channel 1 connected with the immunologic areas 5 in detection plate, and the position of bracket panel C remains unchanged.30 μ L washing buffers are added
Enter to immunologic areas 5, washing buffer solution, which rapidly flows toward washing channel and takes away unreacted reagent, under capillarity driving reaches
To the effect of washing.
The rotary valve integrated on the device, which can be dried or dried up by room temperature, recovers original capillary force and repetition
Use, therefore wash three times to reduce the non-specific adsorption of immunologic areas by rotation washing disk after every single step reaction.
Immunoassay process is related to the fixation of capture antibody, the closing of non-specific sites, antigen and signal antibody it is anti-
The reaction process of multistep should be waited, and respectively also needs to multiple washing between step reaction and removes unreacted reagent, in the present invention
Refill sheet devices in rotation by washing disk reaction process and washing process can be made alternately to build exempting from for interlayer type
Epidemic disease is reacted.Then washing disk A is removed from device, rotation bracket panel C makes on working electrode and bracket panel C on detection plate B
Three-electrode system, the luminous signal that measure electrochemistry produces are formed to electrode and reference electrode.
Embodiment 3：Quantitative determination of the device to tumor markers
Using detection of the above device to tumor markers：
1) capture antibody is fixed on the modification of multi-walled carbon nanotube by the cross-linking reaction of glutaraldehyde, the load of chitosan
Working region.The immunologic areas in detection plate is revealed specifically, rotating bracket panel and washing disk first, then by 5 μ L
Multi-walled carbon nanotube be added to immunologic areas and be used to improve the electric conductivity of electrode, again by 5 μ L after spontaneously drying at room temperature
The chitosan of 0.25mg/mL is added to immunologic areas, and dries in atmosphere；The glutaraldehyde of 5 μ L 2.5% is then added to exempting from
Epidemic-stricken area domain carries out the activation of 2h；Rotation washing disk, after adding the unreacted glutaraldehyde of lavation buffer solution removal, CEA and PSA are caught
Obtain antibody and be added separately to immunologic areas, rotation washing disk is washed after hatching 40min at room temperature；Then by 5 μ L 0.5%
BSA solution be added to working region carry out non-specific sites closing and washing.
2) after antibody to be captured is successfully fixed to working region, which can be used for the measure of tumor markers.Specifically
, the CEA and PSA of the various concentrations of 5 μ L are added separately to working region and are incubated.By 5 μ L ruthenium marks after rotation washing disk washing
Note CEA and PSA signal antibodies are added separately to working region, be incubated at room temperature, and capture antibody-anti-is formd in working region
The immune complex of the interlayer type of original-signal antibody.After rotation washing disk washing removes unnecessary signal antibody, bracket panel is rotated
Make thereon three-electrode system is formed to electrode and reference electrode and the working electrode in detection plate.Then add 40 μ L 10mM
Tripropyl amine (TPA) solution, the intensity of electrochemical luminescence is measured, according to luminous intensity and the relation of antigen concentration, you can realize to CEA
With quickly detecting (referring to Fig. 3 and Fig. 4) for PSA.
Claims (6)
- A kind of 1. rotary electrical chemo-immunity refill card analyzer, it is characterised in that:Bracket panel, reagent including rotation connection Disk and washing disk, the bracket panel, reagent disc and washing disk can independently rotate, and be set respectively on bracket panel, reagent disc and washing disk There is respective fluid passage, hole is provided with each fluid passage, control fluid is on the bracket panel, reagent disc and washing disk Hole rotate to when communicating with each other by being flowed through in each hole.
- 2. rotary electrical chemo-immunity refill card analyzer described in accordance with the claim 1, it is characterised in that the auxiliary Disk, detection plate and washing disk are using cellulosic filter paper as carrier respectively, are printed to fluid channel pattern by wax spray printer Hydrophilic region and the alternate paper chip of hydrophobic region are obtained on paper, after baking, then by plates for screen printing, by working electrode, Electrode and reference electrode are printed onto in paper chip.
- 3. according to the rotary electrical chemo-immunity refill card analyzer described in claim 1 or 2, it is characterised in that the dress The washing disk by being followed successively by bracket panel, the detection plate of immune response and the unnecessary reaction reagent of elution under upper is put, passes through rivet Rotation connection assembling.
- A kind of 4. application of the device described in claim 1, it is characterised in that：Described device is quantified to Diagnostic Value of Several Serum Tumor Markers And/or the qualitative application carried out in electro-chemistry immunity detection.
- A kind of 5. method of quantitative analysis of the device to Diagnostic Value of Several Serum Tumor Markers described in claim 1, it is characterised in that：1) described device is utilized, capture cross-linking reaction of the antibody through glutaraldehyde is fixed on multi-walled carbon nanotube modification, chitosan The immunologic areas of load；2) signal antibody marked successively with antigen to be detected and ruthenium after bovine serum albumin(BSA) is closed reacts to form interlayer type Immune complex on such devices；3) luminous signal is produced as coreaction reagent be used for quantitatively and/or qualitatively dividing for Diagnostic Value of Several Serum Tumor Markers by tripropyl amine (TPA) Analysis.
- 6. according to the method using quantitative analysis of the device to Diagnostic Value of Several Serum Tumor Markers described in claim 5, it is characterised in that： Reaction process in the above-mentioned immunoassay by the bracket panel, reagent disc and washing disk that are alternately rotated in described device and then Realize analysis and detection.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711159820.5A CN107991484B (en) | 2017-11-20 | 2017-11-20 | Rotary electrochemical immune paper chip analysis device and detection method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711159820.5A CN107991484B (en) | 2017-11-20 | 2017-11-20 | Rotary electrochemical immune paper chip analysis device and detection method thereof |
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| CN107991484A true CN107991484A (en) | 2018-05-04 |
| CN107991484B CN107991484B (en) | 2020-09-11 |
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Cited By (6)
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| 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 |
| CN109061194A (en) * | 2018-09-12 | 2018-12-21 | 南京工业大学 | A kind of paper base device and the preparation method and application thereof of quick detection immunoglobulin |
| CN109142329A (en) * | 2018-07-24 | 2019-01-04 | 中山大学 | A kind of electrochemical light-emitting detector |
| CN110389229A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院烟台海岸带研究所 | A kind of manual centrifugal formula rotary valve paper chip immunoassay apparatus and its application |
| CN110389228A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院烟台海岸带研究所 | A kind of paper-based devices based on mobile valve and molecular imprinting technology and preparation method thereof and application |
| CN115228517A (en) * | 2022-03-17 | 2022-10-25 | 烟台大学 | Rotary micro-fluidic paper chip for detecting virus based on frame nucleic acid and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| CN109142329A (en) * | 2018-07-24 | 2019-01-04 | 中山大学 | A kind of electrochemical light-emitting detector |
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| CN109061194A (en) * | 2018-09-12 | 2018-12-21 | 南京工业大学 | A kind of paper base device and the preparation method and application thereof of quick detection immunoglobulin |
| CN110389229A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院烟台海岸带研究所 | A kind of manual centrifugal formula rotary valve paper chip immunoassay apparatus and its application |
| CN110389228A (en) * | 2019-07-24 | 2019-10-29 | 中国科学院烟台海岸带研究所 | A kind of paper-based devices based on mobile valve and molecular imprinting technology and preparation method thereof and application |
| CN110389228B (en) * | 2019-07-24 | 2022-04-12 | 中国科学院烟台海岸带研究所 | Paper base device based on movable valve and molecular imprinting technology and manufacturing method and application thereof |
| CN115228517A (en) * | 2022-03-17 | 2022-10-25 | 烟台大学 | Rotary micro-fluidic paper chip for detecting virus based on frame nucleic acid and preparation method thereof |
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