CN103760209A - Multi-parameter paper-chip electrochemical immunosensor and method for detecting lung cancer markers - Google Patents
Multi-parameter paper-chip electrochemical immunosensor and method for detecting lung cancer markers Download PDFInfo
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- CN103760209A CN103760209A CN201410039635.2A CN201410039635A CN103760209A CN 103760209 A CN103760209 A CN 103760209A CN 201410039635 A CN201410039635 A CN 201410039635A CN 103760209 A CN103760209 A CN 103760209A
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Abstract
The invention provides a multi-parameter paper-chip electrochemical immunosensor which comprises a covering layer, a filtering film and a foldable paperchip, wherein the covering layer is provided with a sample introduction hole, and first comb teeth which are used for exposing the end of a metal lead wire on the foldable paperchip; the filtering film is closely attached to a position below the sample introduction hole to filter and intercept large cells in a sample; the foldable paperchip comprises a first filter paperchip, a second filter paperchip and a broken line located in a middle position, sensitive areas and central drainage holes which are formed in the first filter paper chip, and working electrodes and an Ag-AgCl electrodes which are arranged on the second filter paper chip along the broken line, are symmetrically distributed; craphene-metal composite nano-materials, electronicmediators and various antibodies are decorated in the sensitive areas. After specific recognition with various substances to be detected, the filter paper chip is folded to be used for detecting the electrochemical signal change caused by the substances to be detected in each sensitive area. The invention also provides a method for detecting multiple lung cancer markers.
Description
Technical field
The present invention relates to paper chip manufacturing, nano-modified and technical field of immunoassay, relate to particularly multiparameter refill sheet electrochemical immunosensor and the application in lung cancer detection thereof that a kind of graphene-based nano composite material is modified.
Background technology
Lung cancer is one of modal malignant tumour, and its M & M all ranks first in all kinds of malignant tumours, and how fast early diagnosis is the key that improves lung cancer curative effect and cure rate.Tumor markers be cancer cells secrete be shed to body fluid or tissue in material, its detections is compared with traditional imaging examination, expense is cheap, not damaged, patient are easy to acceptance, but unique identification thing still lacks enough susceptibility and specificity to pulmonary cancer diagnosis.Diagnostic Value of Several Serum Tumor Markers joint-detection has more value to the early diagnosis of lung cancer and examination of curative effect, U.S. clinical biochemical association is according to lung cancer pathology feature, recommendation is by carcinomebryonic antigen (CEA), Cyfra21-1 segment (CYFRA21-1) is combined the detection for gland lung cancer and maxicell lung cancer, by neuronspecific enolase (NSE), gastrin-releasing peptide precursor (ProGRP) is combined the detection for small-cell carcinoma of the lung, by CEA, CYFRA21-1 and squamous cell carcinoma antigen (SCC) three parametric joints are for the detection of squamous cell carcinoma, by CEA, CYFRA21-1, NSE and ProGRP combine the detection for unknown cause lung cancer.There are at present many immunoassays to can be used for the quantitative detection of tumor markers, comprise radiommunoassay, enzyme-linked immuno assay, electrochemiluminescence immunoassay, chemiluminescence immune assay etc., but these methods mostly relate to loaded down with trivial details solution-treated process, need to be equipped with instrumentation, are difficult to realize the quick joint-detection of many index.
Micro-full analytical system is one of sciemtifec and technical sphere of forefront on our times, its core technology is to take microflow control technique as basic micro-fluidic chip, it can be integrated by multiple monotechnics flexible combination, scale on the controlled small platform of integral body, can simplify the operation, increase analysis speed and reduce reagent consumption, when being expected to realize to many indexes, detecting.Common micro-fluidic chip parts are the microchannel that consists of glass, plastics, silica gel materials etc., Micropump, micro-valve etc., and these parts complexity and cost of manufacture are higher, are not suitable for the large-scale promotion application in developing country.For this reason, at the beginning of 2007, the Whitesides of Harvard University group take first filter paper and has built the paper substrate micro-fluidic chip (refill sheet) detecting for glucose, total protein as substrate, this device have cost low, make simple, little without external driving force (as pump), sample volume, be easy to the advantages such as incineration process.Afterwards, researcher has attempted structure and the detection method of multiple refill sheet, wherein, it is save time most effectively and be easy to a large amount of modes of making that wax is printed, electrochemical immunosensor because have, can produce in batches, specificity is good, can in conjunction with miniature instrument, realize the advantages such as quantitatively detection by electrode, and making to develop cost multiparameter refill sheet electrochemical immunosensor low, simple to operate becomes possibility.The sensitivity detecting in order to improve electro-chemistry immunity, researcher generally adopts decorated nanometer material (as golden nanometer particle, carbon nano-tube, metal oxide etc.) to strengthen the transmission of identification electronic signal, as University Of Ji'nan combines and has built the electrochemical immunosensor that detects tumor markers based on carbon nano tube modified and refill sheet in capital group, screen printing electrode need to be equipped with and electrochemical workstation is used, and the cost of decorative material carbon nano-tube is higher.
Summary of the invention
(1) technical matters that will solve
In order to solve the low technical matters of prior art sensitivity, the object of this invention is to provide the refill sheet electrochemical immunosensor that the graphene-based composite nano materials of a kind of use is modified.
(2) technical scheme
In order to solve the problem of prior art, a first aspect of the present invention provides multiparameter refill sheet electrochemical immunosensor, and this sensor comprises overlayer, filtering membrane and foldable paper chip, wherein:
A sample holes is set on overlayer, at the tectal other end, the first broach is set, the first broach is for exposing the end of a plurality of metal lead wires;
Filtering membrane is close to the below of sample holes, the size of filtering membrane be greater than sample holes and with the center superposition of sample holes, for filtering, hold back the cell that testing sample is larger;
Foldable paper chip is to have the Wax-patterned filter paper chip that hydrophilic and hydrophobic region alternately occurs, comprises the first filter paper chip, the second filter paper chip and is positioned at the broken line in centre position, and the first filter paper chip and the second filter paper chip can be folded up along broken line; Cohesive action by overlayer self is dressed up one by overlayer, filtering membrane and the first filter paper chipset, and the second filter paper chip is folded up along broken line and the first filter paper chip, like this, in sensitizing range, from after different determinand specific recognition, the second filter paper chip and the first filter paper chip are folded up the electrochemical signals that can cause for detection of each sensitizing range identification test substance and are changed;
An end at the first filter paper chip arranges the second broach, and after the first filter paper chip and the second filter paper chip are folding, the second broach is for exposing the end of a plurality of metal lead wires; Between the side near broken line and the second broach, hydrophobic wax region, a plurality of sensitizing range, separatory raceway groove, center conduction hole and a plurality of separatory hole are set; Center conduction hole be positioned at separatory raceway groove straight-line segment center and overlap with the central vertical line of sample holes, the filtering membrane vertical vertical direction along this multiparameter refill sheet immune-electrochemistry sensor; A plurality of separatory hole is positioned at the both sides of center conduction hole, and a plurality of separatory hole is connected with one end of a plurality of V-type sections of separatory raceway groove respectively, and one end of each V-type section arranges sensitizing range, in order to the shunting of guiding liquids sample, enters into sensitizing range and seepage does not occur;
On the second filter paper chip, be provided with center conduction hole, a plurality of conduction hole and a plurality of liquid stream raceway groove; At the opposite side near broken line, be provided with center conduction hole, this center conduction hole and the first filter paper chip Shang center conduction hole are symmetric along broken line; A plurality of sensitizing ranges on a plurality of conduction holes and the first filter paper chip are symmetric; On the second filter paper chip and away from the end of broken line, be provided with a plurality of liquid stream raceway grooves, this liquid stream raceway groove one end is connected with conduction hole, and the other end is connected with the edge of the second filter paper chip and is symmetric along the second broach on broken line and the first filter paper chip;
At the back side of the second filter paper chip, be provided with silver/silver chloride contrast electrode, a plurality of working electrode, a plurality of metal lead wire; The position of silver/silver chloride contrast electrode, a plurality of working electrode, a plurality of metal lead wires is corresponding one by one with center conduction hole above, a plurality of conduction hole and a plurality of liquid stream raceway groove respectively, silver/silver chloride contrast electrode and each working electrode are symmetric with each sensitizing range, center conduction hole respectively along broken line, from after different determinand specific recognition, this filter paper chip is folded up the electrochemical signals that can cause for detection of each sensitizing range identification test substance along broken line and is changed.
In order to solve the problem of prior art, a second aspect of the present invention provides the detection method of the realization of using multiparameter refill sheet immune-electrochemistry sensor to lung cancer marker in human body fluid, to utilize above-mentioned multiparameter refill sheet immune-electrochemistry sensor, in conjunction with small electrical chemical instruments or electrochemical workstation, automatically detect the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of determined antigen, thereby realizes the fast detecting to lung cancer marker in human body fluid.
(3) beneficial effect
This sensor production is simple, highly sensitive, cost is low and integrated level is high, during use without human body fluid sample is carried out to cell separation processing, do not need to be equipped with the equipment of complex and expensive, by the miniature instrument in conjunction with blood glucose meter on similar existing market, can realize the Site Detection application to multiple lung cancer marker and even other tumor markers.
1, the present invention combines micro-fluidic refill sheet and electrochemical immunoanalytical technology, can realize the high specific of multiple lung cancer marker in sample, highly sensitive while fast detecting; Meanwhile, this technology can also be expanded the fast detecting of the multiple markers that is applied to other tumour.
2, the mode that adopts wax spray printing and making foldable paper graphics chip, thin film sputtering/serigraphy to make detecting electrode, can realize the batch making of each component units of refill sheet immune-electrochemistry sensor, reduce cost of manufacture, improved the repeatability of sensor preparation and determination methods.
3, by metal nanoparticle and Graphene is compound is more conducive to electronics transmission, and fixing electron mediator on the foldable paper chip of modifying at Graphene-metal nanometer composite material, to further improve electronics transfer rate, and make prepared refill sheet immune-electrochemistry sensor there is response and higher sensitivity faster.
4, the method for refill sheet immune-electrochemistry sensor detection of lung cancer mark of the present invention, reagent consumption sample amount few (tens microlitres), highly sensitive (pg level), operation fast simple (can complete the detection of multiple markers in several minutes), cost are low, and testing result is read automatically by instrument, affected by subjective factor little, be convenient to Site Detection application.
Accompanying drawing explanation
Fig. 1 is the structural representation of multiparameter refill sheet electrochemical immunosensor.
Fig. 2 is the sectional view in the vertical view of multiparameter refill sheet electrochemical immunosensor and AA ', BB ' cross section.
Each parts description of reference numerals:
A-overlayer, b-filtering membrane, c-foldable paper chip;
C1-the first filter paper chip, c2-the second filter paper chip,
1-the first broach, 2-sample holes, 3-the second broach,
4-hydrophobic wax region, 5-sensitizing range, 6-separatory raceway groove,
7-center conduction hole, 8-separatory hole, 9-broken line,
10-center conduction hole, 11-conduction hole, 12-liquid stream raceway groove,
10 '-Yin/silver chloride contrast electrode, 11 '-working electrode, 12 '-metal lead wire.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in further detail.
Fig. 1 is the structural representation of multiparameter refill sheet immune-electrochemistry sensor according to an embodiment of the invention, and Fig. 2 is the sectional view in the vertical view of this multiparameter refill sheet electrochemical immunosensor and AA ', BB ' cross section.
As shown in Figure 1, this multiparameter refill sheet immune-electrochemistry sensor comprises overlayer a, filtering membrane b and foldable paper chip c, comprising:
A sample holes 2 is set on overlayer a, at the other end of overlayer a, the first broach 1 is set, for exposing the end of a plurality of metal lead wires 12 ';
Filtering membrane b is close to the below of sample holes 2, and its size is slightly larger than sample holes 2, and with the center superposition of sample holes 2, for filtering, hold back the larger cell of testing sample (as human body fluid);
Foldable paper chip c has Wax-patterned filter paper chip hydrophilic and that hydrophobic region alternately occurs, broken line 9, the first filter paper chip c1 and the second filter paper chip c2 that comprise the first filter paper chip c1, the second filter paper chip c2 and be positioned at centre position can fold up along broken line 9; Cohesive action by overlayer a self is assembled into one by overlayer a, filtering membrane b and the first filter paper chip c1, and the second filter paper chip c2 can fold up along broken line 9 and the first filter paper chip c1 easily, like this, in sensitizing range 5, from after different determinand specific recognition, the second filter paper chip c2 and the first filter paper chip c1 fold up the electrochemical signals that can cause for detection of each sensitizing range 5 identification test substances and change.
In an end of the first filter paper chip c1, the second broach 3 is set, after the first filter paper chip c1 and the second filter paper chip c2 are folding, the second broach 3 is for exposing the end of a plurality of metal lead wires 12 '; Between the side near broken line 9 and the second broach 3, hydrophobic wax region 4, a plurality of sensitizing range 5, separatory raceway groove 6, center conduction hole 7 and a plurality of separatory hole 8 are set; Center conduction hole 7 is positioned at the center of the straight-line segment of separatory raceway groove 6, and overlaps with the central vertical line of sample holes 2, the filtering membrane b vertical vertical direction along this multiparameter refill sheet immune-electrochemistry sensor; A plurality of separatory hole 8 is positioned at the both sides of center conduction hole 7, and a plurality of separatory hole 8 is connected with one end of a plurality of V-type sections of separatory raceway groove 6 respectively, and one end of each V-type section arranges sensitizing range 5, in order to the shunting of guiding liquids sample, enters into sensitizing range 5 and seepage does not occur;
On the second filter paper chip c2, be provided with center conduction hole 10, a plurality of conduction hole 11 and a plurality of liquid stream raceway groove 12; In the side near broken line 9, be provided with center conduction hole 10, this center conduction hole 10 and the first filter paper chip c1Shang center conduction hole 7 are symmetric along broken line 9; A plurality of sensitizing ranges 5 on a plurality of conduction holes 11 and the first filter paper chip c1 are symmetric; At the second filter paper chip c2, away from that end of broken line 9, be provided with a plurality of liquid stream raceway grooves 12, these liquid stream raceway groove 12 one end are connected with conduction hole 11, and the other end is connected with the edge of the second filter paper chip c2 and is symmetric along the second broach 3 on broken line 9 and the first filter paper chip c1.
At the back side of the second filter paper chip c2, be provided with silver/silver chloride contrast electrode 10 ', a plurality of working electrode 11 ', a plurality of metal lead wire 12 '; The position of silver/silver chloride contrast electrode 10 ', a plurality of working electrode 11 ', a plurality of metal lead wires 12 ' is corresponding one by one with center conduction hole 10, a plurality of conduction hole 11 and a plurality of liquid stream raceway groove 12 above respectively, and silver/silver chloride contrast electrode 10 ' and each working electrode 11 ' are symmetric with each sensitizing range 5, center conduction hole 7 respectively along broken line 9.
Described overlayer a is conventional medical adhesive tape, and the reagent of going up sensitizing range 5 for the protection of foldable paper chip c does not run off, is not subject to outside contamination, the assembling of filtering membrane b and foldable paper chip c is played to bonding effect simultaneously.
In the GR of described filtering membrane b Shi Cong Pall company, GX filtering membrane, choose a kind of, be mainly used in the cell separation in human body fluid to be trapped on film, all the other materials enter into the sensitizing range 5 of the first filter paper chip c1 after seeing through filtering membrane b again through 6 shuntings of separatory raceway groove, like this, to guarantee sample all to enter the separatory raceway groove 6 of foldable paper chip c after filter membrane b filters, there is not seepage.
Described sensitizing range 5 is to be communicated with the essential elements that filtering membrane b filters out the working electrode on sample and the second filter paper chip c2, finishing has Graphene-metal composite nano material, electron mediator and different antibodies and auxiliary reagent, Graphene-metal composite nano material is in order to promote the immunity identification of test substance and electronics transmission capacity, stability and fixedly adsorptive power, in sensitizing range from after different determinand specific recognition, in conjunction with the working electrode on the second filter paper chip and small electrical chemical instruments or electrochemical workstation, realize the fast detecting to multiparameter disease marker in human body fluid, the port number of described sensitizing range 5 is between 4-8, each sensitizing range 5 is and is equidistantly distributed in the direction parallel with broken line 9, the quantity of separatory raceway groove 6 and shape can be made suitable adjustment according to the port number of sensitizing range 5, to facilitate, realize the batch modification of responsive reagent on each sensitizing range 5.
Described sensitizing range 5 is by physisorption and fixing different Graphene-metal nanometer composite material, electron mediator, antibody and the auxiliary reagents of chemical crosslink technique.Metal in this compound substance can be a kind of or any two combinations in platinum, gold, silver, palladium; It is a kind of that electron mediator is selected by the group from sulphur a beautiful gem, methylene blue, ferricyanate, Prussian blue, ferrocene and derivant thereof and rhodium/osmium ion polymkeric substance form, for improving electronics transfer rate; Antibody comprises monoclonal antibody, how anti-etc., for test substance being carried out to specific recognition and response; Auxiliary reagent forms by being coupled reagent, sealer, damping fluid and surfactant etc.
Described antibody is the monoclonal antibody that lung cancer tumor markers is corresponding or resists more, its lung cancer tumor markers is the tumor markers combination of recommending for lung cancer pathology feature in the world, is a kind of combination of selecting from the marks such as CEA, NSE, CYFRA21-1, ProGRP, SCC.
The preparation of described multiparameter refill sheet immune-electrochemistry sensor, is to adopt wax seal technology, thin film sputtering, serigraphy and nano-modified technology batch machining to obtain, and comprises the steps:
(1) take cellulose filter paper as carrier, utilize the layout on wax spray printer bulk print with the first filter paper chip c1, the second filter paper chip c2 and broken line 9, in electron temperature-control instrument, add hot melt wax, to form the alternately foldable paper chip c of appearance of hydrophilic area, hydrophobic wax region 4;
(2), according to the pattern of design, utilize engraving machine to produce with double faced adhesive tape overlayer a, the filtering membrane b of sample holes 2 and with the thin plastics mask of electrode pattern;
(3) combination film sputter and silk-screen printing technique, at the some working metal electrodes 11 ' of back side batch making and metal lead wire 12 ' and the silver/silver chloride contrast electrode 10 ' of the second filter paper chip c2, to form the array detection electrode of multiplexing two electrode systems of contrast electrode;
(4) take graphene oxide as nanometer substrate, utilize solution reaction original position carried metal particle, in conjunction with chemical reduction method, prepare Graphene-metal nanometer composite material;
(5) utilize physisorption and chemical crosslink technique, Graphene-metal nanometer composite material, electron mediator and different antibody on fixing successively on the sensitizing range 5 of foldable paper chip c, and with bovine serum albumin(BSA), unnecessary avtive spot is sealed to processing, stand-by after low temperature drying;
(6) utilize the adhesive effect of double faced adhesive tape overlayer a that overlayer a, filtering membrane b and foldable paper chip c from top to down are assembled, and cutting obtain single hyperchannel refill sheet immune-electrochemistry sensor.
A kind of method of fast detecting lung cancer marker for the present invention, it is characterized in that the multiparameter refill sheet immune-electrochemistry sensor that utilizes above-mentioned graphene-based composite nano materials to modify, in conjunction with multiple tracks electrochemical workstation or make the changing value that portable multiple tracks galvanochemistry instrument detects multi-channel electric signal automatically by oneself, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of determined antigen, thereby realize the quantitatively detection fast to lung cancer marker in sample to be tested (human body fluid), specific embodiment comprises the steps:
(1) pipette the testing sample of 4-40 μ L, join in the sample holes 2 of refill sheet immune-electrochemistry sensor standing 2-5 minute under room temperature;
(2) by refill sheet immune-electrochemistry sensor along broken line 9 doublings, its a plurality of metal lead wire 12 ' ends are inserted into the interface of the portable multiple tracks galvanochemistry instrument of self-control or are connected with the wire of multiple tracks electrochemical workstation, by instrument, automatically detected the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of determined antigen.
In the present invention, under the driving of capillary force, sample solution first automatically filters and holds back wherein larger cell through filter membrane b, and all the other materials shunt through conduction hole 7 and separatory raceway groove 6 sensitizing range 5 that enters into sensor; Each antibody generation specific recognition on determined antigen in sample and sensitizing range 5, causes the change in electric of corresponding working electrode 11 '; Then in conjunction with instrument, complete Electrochemical Detection.
The joint-detection of embodiment 1:NSE and ProGRP two parameters
Using mineral carbon thiazolinyl composite nano materials carries out a refill sheet electrochemical immunosensor preparation and application for small-cell carcinoma of the lung detection, comprises the following steps:
(1) according to the biochemical tumor markers combination that can recommend of the U.S., select NSE and ProGRP two parameters to carry out simultaneous determination;
(2) prepare foldable paper chip c: at Whatman1# filter paper, with wax spray printer, print the design configuration with four-way sensitizing range 5 and four working electrodes 11 ', in electron temperature-control instrument, add hot melt wax, obtain containing the alternately Wax-patterned foldable paper chip c of appearance of hydrophilic area, hydrophobic wax region 4; Utilize engraving machine to make double faced adhesive tape overlayer a, filtering membrane b and with the thin plastics mask of electrode pattern, the combination film sputtering technology preparation working electrode 11 ' based on golden on foldable paper refill sheet c, and in serigraphy silver/silver chloride as multiplexing contrast electrode 10 '; Utilize the double faced adhesive tape of overlayer a, assemble with filtering membrane b and foldable paper refill sheet c bonding.
(3) preparation of Graphene-Jin composite nano materials: by the ultrasonic 1h of potpourri of 4ml graphene oxide (1mg/mL) and 25 μ L gold chlorides (0.2%), more dropwise add 0.94mL sodium citrate (1%); Then potpourri is transferred to 80 ℃ of reaction 2h, is chilled to room temperature, centrifugal, and with ultrapure water washing 3 times, be resuspended in 2mL chitosan solution (1%) standby.
(4) modification of the sensitizing range 5 of foldable paper chip c: Xian Yong six road pipettors pipette the above-mentioned Graphene-Jin of 5 μ L composite nano materials and are added drop-wise on the four-way sensitizing range 5 of foldable paper chip c, drying at room temperature; Sulphur a beautiful gem/shitosan the mixed liquor and the glutaraldehyde that further drip 5 μ L are modified sensitizing range 5; Then, the antibody-solutions of 5 μ L NSE, ProGRP is added drop-wise to respectively on four sensitizing ranges 5 of foldable paper chip c, incubated at room 30min, with PBST damping fluid washing 3 times; Finally, with 1% bovine serum albumin solution, seal possible avtive spot, with PBST damping fluid washing 3 times, drying at room temperature, completes the preparation of refill sheet electrochemical immunosensor.
(5) use of refill sheet electrochemical immunosensor: pipette the clinical sample of 15 μ L, join in the sample holes 2 of refill sheet immune-electrochemistry sensor, under room temperature standing 2 minutes; Then, by refill sheet immune-electrochemistry sensor along broken line 9 doublings, the wire that its metal lead wire 12 ' end is inserted into Gamry multiple tracks electrochemical workstation connects, by instrument, automatically detected the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of NSE in sample to be tested, ProGRP.
The joint-detection of embodiment 2:CEA, CYFRA21-1 and SCC tri-parameters
Using mineral carbon thiazolinyl composite nano materials carries out a refill sheet electrochemical immunosensor preparation and application for squamous cell carcinoma detection, comprises the following steps:
(1) according to the biochemical tumor markers combination that can recommend of the U.S., select CEA, CYFRA21-1 and SCC tri-parameters to measure;
(2) prepare foldable paper chip c: at Whatman1# filter paper, with wax spray printer, print the design configuration with six passage sensitizing ranges 5 and six working electrodes 11 ', in electron temperature-control instrument, add hot melt wax, obtain containing the alternately foldable paper chip c of appearance of hydrophilic area, hydrophobic wax region 4; Utilize engraving machine to make double faced adhesive tape overlayer a, filtering membrane b and with the thin plastics mask of electrode pattern, the combination film sputtering technology preparation a plurality of working electrodes 11 ' based on gold electrode on foldable paper chip c, and in serigraphy silver/silver chloride as multiplexing contrast electrode 10 '; Utilize assembling with filtering membrane b and foldable paper chip c bonding of double faced adhesive tape overlayer a general.
(3) preparation of Graphene-platinum composite nano materials: by the ultrasonic dispersion 1h of 25mL graphene oxide (0.2mg/mL); Then, add 1ml K
2ptCl
66H
2o (20mM), stirs, and is 10 with NaOH adjusting pH; Slowly add 400mg NaBH
4, after stirring at room 24h, to reaction product centrifuge washing, and be resuspended in 2mL chitosan solution (1%) standby.
(4) modification of the sensitizing range 5 of foldable paper chip c: Xian Yong six road pipettors pipette the above-mentioned Graphene-Jin of 5 μ L composite nano materials and are added drop-wise on the six passage sensitizing ranges 5 of foldable paper chip c, drying at room temperature; Methylene blue/shitosan the mixed liquor and the glutaraldehyde that further drip 5 μ L are modified sensitizing range 5; Then, by the antibody-solutions of 5 μ L CEA, CYFRA21-1 and SCC be added drop-wise to respectively can six sensitizing ranges 5 of paper folding chip c on, incubated at room 30min, with PBST damping fluid washing 3 times; Finally, with 1%BSA, seal possible avtive spot, with PBST damping fluid washing 3 times, drying at room temperature, completes the preparation of refill sheet electrochemical immunosensor.
(5) use of refill sheet electrochemical immunosensor: pipette the clinical sample of 25 μ L, join in the sample holes 2 of refill sheet immune-electrochemistry sensor, under room temperature standing 3 minutes; Then, by refill sheet immune-electrochemistry sensor along broken line 9 doublings, its metal lead wire 12 ' end is inserted into the interface of the portable multiple tracks galvanochemistry instrument of self-control, by instrument, automatically detected the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of CEA to be measured, CYFRA21-1 and SCC.
The joint-detection of embodiment 3:CEA, CYFRA21-1, NSE and ProGRP tetra-parameters
Using mineral carbon thiazolinyl composite nano materials carries out a refill sheet electrochemical immunosensor preparation and application for unknown cause lung cancer detection, comprises the following steps:
(1) according to the biochemical tumor markers combination that can recommend of the U.S., select CEA, CYFRA21-1, NSE and ProGRP tetra-parameters to measure;
(2) prepare foldable paper chip c: at Whatman1# filter paper, with wax spray printer, print the design configuration with eight passage sensitizing ranges 5 and eight working electrodes 11 ', in electron temperature-control instrument, add hot melt wax, obtain containing the alternately foldable paper chip c of appearance of hydrophilic area, hydrophobic wax region 4; Utilize engraving machine to make double faced adhesive tape overlayer a, filtering membrane b and with the thin plastics mask of electrode pattern, the combination film sputtering technology preparation array detection electrode based on platinum electrode on foldable paper chip c, and in serigraphy silver/silver chloride as multiplexing contrast electrode; Utilize assembling with filtering membrane b and foldable paper chip c bonding of double faced adhesive tape overlayer a general.
(3) preparation of Graphene-gold and silver composite nano materials: the ultrasonic dispersion 1h of 2mL graphene oxide (1mg/mL); Add 2ml AgNO
3(20mM), slowly add 1mL ascorbic acid (100mM), rapid stirring 30min; Then, continue dropwise to add 1mL HAuCl under stirring
4(80mM) also keep stirring 30min; Standing 2 days of reaction product, after centrifuge washing 3-4 time, is resuspended in 2mL chitosan solution (1%) standby.
(4) modification of the sensitizing range 5 of foldable paper chip c: Xian Yong eight road pipettors pipette the above-mentioned Graphene-Jin of 5 μ L composite nano materials and are added drop-wise on the eight passage sensitizing ranges 5 of foldable paper chip c, drying at room temperature; The osmium ion polymkeric substance and the glutaraldehyde that further drip 5 μ L are modified sensitizing range 5; Then, the antibody-solutions of 5ul CEA, CYFRA21-1, NSE and ProGRP is added drop-wise to respectively on the sensitizing range 5 of foldable paper chip c, incubated at room 30min, with PBST damping fluid washing 3 times; Finally, with the BSA (1%) of 10 μ L, seal possible avtive spot, with PBST damping fluid washing 3 times, drying at room temperature, completes the preparation of refill sheet electrochemical immunosensor.
(5) use of refill sheet electrochemical immunosensor: pipette the clinical sample of 40uL, join in the sample holes 2 of refill sheet immune-electrochemistry sensor, under room temperature standing 5 minutes; Then, by refill sheet immune-electrochemistry sensor along broken line 9 doublings, its metal lead wire 12 ' end is inserted into the interface of the portable multiple tracks galvanochemistry instrument of self-control, by instrument, automatically detected the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of CEA to be measured, CYFRA21-1, NSE and ProGRP.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any modification of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a multiparameter refill sheet electrochemical immunosensor, is characterized in that, comprises overlayer, filtering membrane and foldable paper chip, wherein:
A sample holes is set on overlayer, at the tectal other end, the first broach is set, the first broach is for exposing the end of a plurality of metal lead wires on foldable paper chip;
Filtering membrane is close to the below of sample holes, the size of filtering membrane be greater than sample holes and with the center superposition of sample holes, for filtering, hold back the cell that testing sample is larger;
Foldable paper chip is to have the Wax-patterned filter paper chip that hydrophilic and hydrophobic region alternately occurs, comprises the first filter paper chip, the second filter paper chip and is positioned at the broken line in centre position, and the first filter paper chip and the second filter paper chip can be folded up along broken line; Cohesive action by overlayer self is dressed up one by overlayer, filtering membrane and the first filter paper chipset, and the second filter paper chip is folded up along broken line and the first filter paper chip, like this, in sensitizing range, from after different determinand specific recognition, the second filter paper chip and the first filter paper chip are folded up the electrochemical signals that can cause for detection of each sensitizing range identification test substance and are changed;
An end at the first filter paper chip arranges the second broach, and after the first filter paper chip and the second filter paper chip are folding, the second broach is for exposing the end of a plurality of metal lead wires; Between the side near broken line and the second broach, hydrophobic wax region, a plurality of sensitizing range, separatory raceway groove, center conduction hole and a plurality of separatory hole are set; Center conduction hole be positioned at separatory raceway groove straight-line segment center and overlap with the central vertical line of sample holes, the filtering membrane vertical vertical direction along this multiparameter refill sheet immune-electrochemistry sensor; A plurality of separatory hole is positioned at the both sides of center conduction hole, and a plurality of separatory hole is connected with one end of a plurality of V-type sections of separatory raceway groove respectively, and one end of each V-type section arranges sensitizing range, in order to the shunting of guiding liquids sample, enters into sensitizing range and seepage does not occur;
On the second filter paper chip, be provided with center conduction hole, a plurality of conduction hole and a plurality of liquid stream raceway groove; At the opposite side near broken line, be provided with center conduction hole, this center conduction hole and the first filter paper chip Shang center conduction hole are symmetric along broken line; A plurality of sensitizing ranges on a plurality of conduction holes and the first filter paper chip are symmetric; On the second filter paper chip and away from the end of broken line, be provided with a plurality of liquid stream raceway grooves, this liquid stream raceway groove one end is connected with conduction hole, and the other end is connected with the edge of the second filter paper chip and is symmetric along the second broach on broken line and the first filter paper chip;
At the back side of the second filter paper chip, be provided with silver/silver chloride contrast electrode, a plurality of working electrode, a plurality of metal lead wire; The position of silver/silver chloride contrast electrode, a plurality of working electrode, a plurality of metal lead wires is corresponding one by one with center conduction hole above, a plurality of conduction hole and a plurality of liquid stream raceway groove respectively, and silver/silver chloride contrast electrode and each working electrode are symmetric with each sensitizing range, center conduction hole respectively along broken line.
2. multiparameter refill sheet electrochemical immunosensor according to claim 1; it is characterized in that; described overlayer is medical adhesive tape; reagent for the protection of sensitizing range on foldable paper chip does not run off, is not subject to outside contamination, the assembling of filtering membrane and foldable paper chip is played to bonding effect simultaneously.
3. multiparameter refill sheet electrochemical immunosensor according to claim 1, it is characterized in that, described filtering membrane is chosen a kind of in GR filtering membrane, GX filtering membrane, for the cell separation of human body fluid is trapped in to filtering membrane, all the other materials enter into the sensitizing range of the first filter paper chip after seeing through filtering membrane again through the shunting of separatory raceway groove, in order to guarantee sample all to enter the separatory raceway groove of foldable paper chip after membrane filtrations after filtration, seepage does not occur.
4. multiparameter refill sheet electrochemical immunosensor according to claim 1, is characterized in that, described sensitizing range is to be communicated with the essential elements that filtering membrane filters out the working electrode on sample and the second filter paper chip; Surface, sensitizing range is by physisorption and fixing different Graphene-metal composite nano material, electron mediator, different antibodies and the auxiliary reagents of chemical crosslink technique, Graphene-metal composite nano material is in order to promote the immunity identification of test substance and electronics transmission capacity, stability and fixing adsorptive power, in sensitizing range from after different determinand specific recognition, in conjunction with the working electrode on the second filter paper chip and small electrical chemical instruments or electrochemical workstation, realize the fast detecting to multiparameter disease marker in human body fluid.
5. multiparameter refill sheet electrochemical immunosensor according to claim 1, is characterized in that, the port number of described sensitizing range is between 4-8, and each sensitizing range is and is equidistantly distributed in the direction parallel with broken line.
6. multiparameter refill sheet electrochemical immunosensor according to claim 1, is characterized in that, the quantity of described separatory raceway groove and shape adjust according to the port number of sensitizing range, in order to conveniently to realize responsive reagent, at the batch of each sensitizing range, modifies.
7. multiparameter refill sheet electrochemical immunosensor according to claim 4, is characterized in that, the metal in described Graphene-metal composite nano material is a kind of or any two combinations in platinum, gold, silver, palladium.
8. multiparameter refill sheet electrochemical immunosensor according to claim 4, it is characterized in that, described electron mediator is select from the group that sulphur a beautiful gem, methylene blue, ferricyanate, Prussian blue, ferrocene and derivant thereof and rhodium/osmium ion polymkeric substance form a kind of, for improving electronics transfer rate.
9. multiparameter refill sheet electrochemical immunosensor according to claim 4, is characterized in that, described antibody comprises monoclonal antibody body, multispecific antibody, for test substance is carried out to specific recognition and response; Auxiliary reagent forms by being coupled reagent, sealer, damping fluid and surfactant.
10. a right to use requires the multiparameter refill sheet electrochemical immunosensor described in 1 to realize the method that lung cancer marker in human body fluid is detected, it is characterized in that, to utilize above-mentioned multiparameter refill sheet immune-electrochemistry sensor, in conjunction with small electrical chemical instruments or electrochemical workstation, automatically detect the changing value of multi-channel electric signal, according to the relation between change in electric value and antigen concentration, inverse goes out the concentration of determined antigen, thereby realizes the fast detecting to lung cancer marker in human body fluid.
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