CN103018231A - Preparation method and application of composite nano material paper chip electrochemical luminescence immunosensor - Google Patents
Preparation method and application of composite nano material paper chip electrochemical luminescence immunosensor Download PDFInfo
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- CN103018231A CN103018231A CN201210577094XA CN201210577094A CN103018231A CN 103018231 A CN103018231 A CN 103018231A CN 201210577094X A CN201210577094X A CN 201210577094XA CN 201210577094 A CN201210577094 A CN 201210577094A CN 103018231 A CN103018231 A CN 103018231A
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Abstract
The invention discloses a method for researching and applying a paper chip electrochemical luminescence immunosensor for detecting tumor marker by using a composite nano material. The paper chip and sensor preparation method (see the schematic diagram in the attached figure) mainly comprises the steps of printing designed channel patterns on a piece of chromatographic paper by using a printer; heating the wax printed chromatographic paper by using an electronic temperature controller; preparing a graphene-metal composite nano material and a quantum dot-porous silicon nano material; modifying the nano material on the paper chip; connecting an antibody to the paper chip modified by the nano material; after antigen specificity identification, carrying out antigen specificity identification on the antibody marked by a luminescent material so as to make the electrochemical luminescence immunosensor; and detecting an electrochemical luminescence signal with the combination of the processed paper chip with a screen-printed electrode. The sensor is strong in specificity detection and high in sensitivity, and the sensitivity can be of ng grade; the diagnosis and monitoring speed is fast, and the time for accomplishing one basic detection process is shorter; and a film electrode at the bottom of the sensor can be repeatedly used for multiple times, so that the cost is low.
Description
Technical field
The present invention relates to composite nano materials technology, refill chip technology and tumor markers detection technique field, say that more specifically a kind of applying composite nano material detects the electrochemiluminescence immunosensor of tumor markers by means of the refill sheet.
Background technology
Tumour is the killer of human health.Malignant tumour also claims cancer, it is the class disease that current serious threatens human health and life, it be body be subjected to various in or external carcinogenic factor effect under, the cell of local organization has lost normal regulation to himself growth at gene level, causes undesired hyperplasia and the neoformation that forms.Tumour be that long-term, multistage a, polygenes changes accumulation, have the complicacy of multifactor adjusting and controlled by multiple genes.The ascendant trend of tumor incidence and mortality ratio is directly threatening human health, and its early diagnosis and therapy is one of important preface field for the treatment of and prevention of tumour research.Immunoassay is to utilize antibody to be combined with antigentic specificity and the high selectivity biochemical method set up.Detect and the peculiar tumor marker of positioning tumor cell with immunocytochemistry, more and more caused vast oncological pathology research worker's interest.There are now many immunohistochemical methods to can be used for histiocytic qualitative and quantitative analysis and composition detection.At present, main immune analysis method has electrochemical immunoanalytical, chemiluminescence immune assay, portable injection chemiluminescence immunoassay, high performance liquid chromatography chemiluminescence immune assay, capillary electrophoresis chemiluminescence immunoassay etc., therefore but these detection methods are gone back Shortcomings in some aspects, are badly in need of a kind of detection method of new sensitivity.
For adapting to clinical diagnosis and the instant requirement that detects, lab-on-a-chip arises at the historic moment, the importance of lab-on-a-chip and application are by extensive cognition, present chip research mainly concentrate on being easy to get of material, inexpensive, be easy to modify, be easy to store and transportation, and the refill sheet has embodied these advantages well.Have the method for much economical and practical structure refill sheet to be employed, it is most economical wherein printing, and the easiest one of the mass-produced mode of carrying out, and what save time effective structure paper sensor most in printing then is the wax printing.Refill sheet sensor can detect by several different methods, and wherein why the electrochemiluminescence immunosensor causes that the great interest of the public is because this method combines electrochemical analysis and chemiluminescent high selectivity, high sensitivity, needs the advantages such as the sample amount is few.Production and the operation technique of the screen printing electrode that electrochemiluminescence relates to also have been reported.In addition, electrochemiluminescence has presented preferably operation result on the paper; Cellulose fibre is without any diffusion blocking, on the contrary, pastes the chip that soaks at electrode surface and makes detection more not be subject to shake impact with other convection current.
In order further to improve the sensitivity of electrochemical luminescence method on the paper, here we have adopted composite nano materials to increase detection signal.It is better that we know graphene conductive, find after deliberation the very large raising of having got back of graphene conductive after metal-doped, therefore our design is Graphene-metal composite nano material to be modified be used for fixing primary antibodie can better promote the electronics transmission on working electrode, thereby increases electrochemical luminescence signals.On the other hand, the biocompatibility that can become reconciled by means of the larger specific surface area of porous silicon nano material, CdTe quantum dot light emitting reagent received obtain quantum dot-porous silicon composite nano materials on the porous silicon, and with its be marked at two anti-on, can realize so many times of amplification effects of luminous signal.
This paper combines the refill sheet on the basis of using Graphene-metal and quantum dot-porous silicon for the first time with electrochemiluminescence.Wax printer paper chip is combined with screen printing electrode, make up low-cost, disposable composite nano materials electrochemiluminescence refill sheet sensor.Sensors with auxiliary electrode were comes capture antibody by the refill sheet of Graphene-metal-modified, and quantum dot-porous silicon comes mark two anti-, and Graphene-metal is used for accelerating electron transfer rate, and quantum dot-porous silicon is used for amplifying signal, thus the sensitivity that can improve sensor.With several antigens analyte as an example, the hypersensitive immunosensor has represented fabulous analytical performance.This simple, economy, the range of linearity are wide, favorable reproducibility, highly sensitive immunosensor are having good development prospect aspect the different tumor markers of clinical detection.
Summary of the invention
The technical problem to be solved in the present invention has provided that a kind of diagnostic monitoring speed of applying composite nano material is fast, highly sensitive, preparation and the detection method of the refill sheet immunosensor of high specificity, on-the-spot external care diagnostic monitoring, and the method can realize easily in most laboratories and not need complicated loaded down with trivial details main equipment support.
The present invention's manufacture method of Graphene-metal and quantum dot-porous silicon composite nano materials refill sheet immunosensor, its feature may further comprise the steps
(1) in the channel pattern of chromatographic paper with wax printer printed design, heat with the chromatographic paper of electronic temperature controller device to wax seal, purpose is with roastingization of wax and makes its inside that is penetrated into uniformly chromatographic paper, thereby forms hydrophilic channel at the position of not printing wax;
(2) select the higher tumor markers of clinical onset rate to measure;
(3) utilize nano material, prepare Graphene-metal composite nano material;
(4) prepare quantum dot-porous silicon composite nano materials, and it is anti-to be used for mark two;
(5) Graphene-metal composite nano material is modified on the refill sheet passage after the processing, in order to improve the electric conductivity of refill sheet, improved its sensitivity;
(6) modify afterwards the thionine that is used for catching antibody in step (5);
(7) utilize Ag-Ab specific recognition and assembling surface modification technology etc., two anti-modifications of quantum dot-porous silicon composite material mark are arrived on step (6) the refill sheet surface afterwards, make electrochemical luminous sensor.
(8) the refill sheet of handling well the detection electrochemical luminescence signals that need to combine with screen printing electrode;
(9) screen printing electrode needs to polish smooth with burnishing powder before use, and is for subsequent use.
Graphene-metal composite nano the material that uses of the present invention can be Graphene-Cu, Graphene-Co, Graphene-Pt.
Quantum dot-porous silicon the nano material of using of the present invention can be CdTe quantum dot-porous silicon nano material.
Tumor markers of the present invention is alpha-fetoprotein (AFP), carcinomebryonic antigen, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, squama cancer associated antigen, prostate specific antigen (PSA), acid phosphatase (ACP), alkaline phosphatase (ALP), neuronspecific enolase (NSE), CEA, CA19-9, SCC, CA15-3, CA12-5, HCG, F-PSA, PAP, TMA, human chorionic gonadotrophin (hCG), Catecholamine matter.
Description of drawings
Fig. 1. the zone of a-micro-fluidic refill sheet immune modification process;
The working electrode of b-corresponding a;
C-silver/silver chloride contrast electrode;
D-graphite is to electrode;
E-polyamide fibre film;
Fig. 2. a zone of the refill sheet among (1)-Fig. 1;
(2)-the first synoptic diagram behind grapheme modified-metal in a zone;
(3)-after step (2), modify again thionine and glutaraldehyde;
(4)-after step (3), catch and the corresponding antibody of antigen;
(5)-with bovine serum albumin(BSA) sealing may conjugated antigen the site;
(6)-the corresponding antigen of antibody capture;
(7)-be combined with antigen with the antibody of quantum dot-porous silicon mark.
Tumor markers of the present invention is alpha-fetoprotein (AFP), carcinomebryonic antigen, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, squama cancer associated antigen, prostate specific antigen (PSA), acid phosphatase (ACP), alkaline phosphatase (ALP), neuronspecific enolase (NSE), CEA, CA19-9, SCC, CA15-3, CA12-5, HCG, F-PSA, PAP, TMA, human chorionic gonadotrophin (hCG), Catecholamine matter.
A kind of method of fast detecting tumor markers is characterized in that comprising the steps: that the refill sheet electrochemiluminescence immunosensor of the applying composite nano material that will make as stated above cooperates the electrochemiluminescence instrument, and tumor markers is detected.
Embodiment 1(The embryonal antigen class is such as alpha-fetoprotein, AFP)
The refill sheet electrochemiluminescence immunosensor preparation method of a kind of applying composite nano material tests AFP may further comprise the steps:
(1) select the higher AFP of clinical onset to measure;
(2) preparation electrode inspector: design bipolar electrode screen printing template with screen process press or hand-printing graphite electrode and silver silver chloride electrode, and polishes smooth for subsequent use.Design the refill sheet corresponding with the film electrode model, print the pattern of design with wax printing machine, and be baked to wax infiltration refill sheet, the perform region of preparation suction,
(3) Graphene-Cu composite nano materials preparation: 2 mL graphene oxides are dissolved in the aqueous solution that ultrasonic 1h in the 100 mL water obtains graphene oxide, add in the solution to be dissolved with 2.5 g CuSO
45H
20 and 0.8 g EDTA2NaH
2The 20 mL aqueous solution of O, ultrasonic 10 minutes.Then, the solution that the 20 mL aqueous solution of 2.24 g KOH and 0.27 g KBH4 obtain more than slowly adding, this solution add 1.0 g KBH after stirring the 30min reaction again
4, then, this mixed liquor is transferred in the round-bottomed flask back flow reaction 1 h under the infrared lamp irradiation;
(4) CdTe quantum dot solution preparation: at N
2Protection is lower, and will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(5) the porous silicon nano material of amino functional preparation: the mixed solution of cetyl trimethyl ammonium bromide (CTAB), NaOH and water is heated to 80 ℃, then add ethyl orthosilicate (TEOS), aminopropyl trimethoxysilane (APTMS) stirring reaction obtains the porous silicon crude product, rear heat filtering, washing, drying.Then product joins the HCl-methanol solution and reacts to remove CTAB, and rear filtration, washing, drying obtain final porous silica material.
(6) CdTe quantum dot-porous silicon composite nano materials preparation: the amination porous silicon that in the CdTe quantum dot solution of step (4) preparation, adds step (5) preparation, with coupling activator ethyl-(3-dimethyl propyl) carbodiimide hydrochloride (EDC), N-hydroxy-succinamide (NHS), centrifuge washing obtains final product behind the stirring reaction.
(7) 150 microlitres Graphene-Cu composite nano materials is added drop-wise to perform region corresponding to working electrode and modifies, and the thionine and the glutaraldehyde that further drip respectively 50 microlitres modify the refill sheet, ready for connecing antigen-antibody.
(8) primary antibodie of corresponding 5 microlitres of selection in the step (1) is modified on the refill sheet of handling well in the step (7), the avtive spot that may exist with the sealing of 30 microlitre bovine serum albumin(BSA)s subsequently, connect subsequently the antigen of corresponding 5 microlitres, after reacting completely, connect 10 microlitres two anti-of CdTe quantum dot-porous silicon composite nano materials mark, each step connects the back of antigen-antibody and all uses the PBS buffer solution of pH 7.4 to wash, to wash away the unsteady antigen of adsorbability or antibody, make detection sensitiveer, accurate.
(9) as shown in the figure, the refill sheet immunosensor that is modified with AFP is placed on the film electrode of bottom, electrode is connected with the electrogenerated chemiluminescence instrument, the certain pH of dropping is 7.4 PBS buffer solution during detection, this moment is with the luminous signal that shows under the instrument record, according to the amount of the big or small Accurate Diagnosis detection material of luminous intensity.
Embodiment 2(The carbohydrate mark is such as CA19-9)
The refill sheet electrochemiluminescence immunosensor preparation method of a kind of applying composite nano material tests CA19-9 may further comprise the steps:
(1) select the higher CA19-9 of clinical onset to measure;
(2) preparation electrode inspector: design bipolar electrode screen printing template with screen process press or hand-printing graphite electrode and silver silver chloride electrode, and polishes smooth for subsequent use.Design the refill sheet corresponding with the film electrode model, print the pattern of design with wax printing machine, and be baked to wax infiltration refill sheet, the perform region of preparation suction,
(3) Graphene-Co composite nano materials preparation: 0.2 g Co (NO
3)
26H
2The O stirring and dissolving is in 7mL water, add 0.005g Graphene ultrasonic dissolution, the ammoniacal liquor that slowly adds 28mL 28-30%, transfer to 100 ℃ of lower reaction 8h in the autoclave after ultrasonic 10 minutes, behind the cool to room temperature, sediment centrifuge washing final vacuum is dry, and then at 623K, quenching 1h obtains Graphene-Co compound under the Ar flow conditions;
(4) the porous silicon nano material of amino functional preparation: the mixed solution of cetyl trimethyl ammonium bromide (CTAB), NaOH and water is heated to 80 ℃, then add ethyl orthosilicate (TEOS), aminopropyl trimethoxysilane (APTMS) stirring reaction obtains the porous silicon crude product, rear heat filtering, washing, drying.Then product joins the HCl-methanol solution and reacts to remove CTAB, and rear filtration, washing, drying obtain final porous silica material.
(5) CdTe quantum dot-porous silicon composite nano materials preparation: the amination porous silicon that in the CdTe quantum dot solution, adds step (4) preparation, with coupling activator ethyl-(3-dimethyl propyl) carbodiimide hydrochloride (EDC), N-hydroxy-succinamide (NHS), centrifuge washing obtains final product behind the stirring reaction.
(6) 150 microlitres Graphene-Co composite nano materials is added drop-wise to perform region corresponding to working electrode and modifies, and the thionine and the glutaraldehyde that further drip respectively 50 microlitres modify the refill sheet, ready for connecing antigen-antibody.
(7) primary antibodie of corresponding 5 microlitres of selection in the step (1) is modified on the refill sheet of handling well in the step (6), the avtive spot that may exist with the sealing of 30 microlitre bovine serum albumin(BSA)s subsequently, connect subsequently the antigen of corresponding 5 microlitres, after reacting completely, connect 10 microlitres two anti-of CdTe quantum dot-porous silicon composite nano materials mark, each step connects the back of antigen-antibody and all uses the PBS buffer solution of pH 7.4 to wash, to wash away the unsteady antigen of adsorbability or antibody, make detection sensitiveer, accurate.
(8) as shown in the figure, the refill sheet immunosensor that is modified with AFP is placed on the film electrode of bottom, with electrode with cause Chemiluminescence Apparatus and be connected, the certain pH of dropping is 7.4 PBS buffer solution during detection, this moment is with the luminous signal that shows under the instrument record, according to the amount of the big or small Accurate Diagnosis detection material of luminous intensity.
Embodiment 3(Steroids is such as human chorionic gonadotrophin, HCG)
A kind of refill sheet electrochemiluminescence immunosensor preparation method of applying composite nano material tests cytokeratin may further comprise the steps:
(1) select the higher HCG of clinical onset to measure;
(2) preparation electrode inspector: design bipolar electrode screen printing template with screen process press or hand-printing graphite electrode and silver silver chloride electrode, and polishes smooth for subsequent use.Design the refill sheet corresponding with the film electrode model, print the pattern of design with wax printing machine, and be baked to wax infiltration refill sheet, the perform region of preparation suction,
(3) Graphene-Pt composite nano materials preparation: 0.4g graphene oxide ultrasonic dissolution is in 50mL water, then added the chloroplatinic acid stirring reaction 2 hours, product is lower dry at 60 ℃, getting the 0.2g product is dispersed in the glass culture dish, place under the sunshine, and come focusing sunlight irradiation product to promote its reduction reaction with a convex mirror;
(4) the porous silicon nano material of amino functional preparation: the mixed solution of cetyl trimethyl ammonium bromide (CTAB), NaOH and water is heated to 80 ℃, then add ethyl orthosilicate (TEOS), aminopropyl trimethoxysilane (APTMS) stirring reaction obtains the porous silicon crude product, rear heat filtering, washing, drying.Then product joins the HCl-methanol solution and reacts to remove CTAB, and rear filtration, washing, drying obtain final porous silica material.
(5) CdTe quantum dot-porous silicon composite nano materials preparation: the amination porous silicon that in the CdTe quantum dot solution, adds step (4) preparation, with coupling activator ethyl-(3-dimethyl propyl) carbodiimide hydrochloride (EDC), N-hydroxy-succinamide (NHS), centrifuge washing obtains final product behind the stirring reaction.
(6) 150 microlitres Graphene-Co composite nano materials is added drop-wise to perform region corresponding to working electrode and modifies, and the thionine and the glutaraldehyde that further drip respectively 50 microlitres modify the refill sheet, ready for connecing antigen-antibody.
(7) primary antibodie of corresponding 5 microlitres of selection in the step (1) is modified on the refill sheet of handling well in the step (6), the avtive spot that may exist with the sealing of 30 microlitre bovine serum albumin(BSA)s subsequently, connect subsequently the antigen of corresponding 5 microlitres, after reacting completely, connect 10 microlitres two anti-of CdTe quantum dot-porous silicon composite nano materials mark, each step connects the back of antigen-antibody and all uses the PBS buffer solution of pH 7.4 to wash, to wash away the unsteady antigen of adsorbability or antibody, make detection sensitiveer, accurate.
(8) as shown in the figure, the refill sheet immunosensor that is modified with HCG is placed on the film electrode of bottom, electrode is connected with the electrochemiluminescence instrument, the certain pH of dropping is 7.4 PBS buffer solution during detection, this moment is with the luminous signal that shows under the instrument record, according to the amount of the big or small Accurate Diagnosis detection material of luminous intensity.
Embodiment
(1) manually printed book is tested used film electrode, and with the alundum (Al2O3) burnishing powder electrode is polished smooth, and is for subsequent use;
(2) use the wax printer to print pattern shown in Fig. 1-A, and use the electron temperature-control instrument that the chromatographic paper of seal wax is heated, make wax be penetrated into uniformly chromatographic paper inside, the zone of not printing wax forms refill sheet passage;
(3) prepare Graphene-metal and quantum dot-porous silicon composite nano materials;
(4) 100-200 microlitre Graphene-metallic solution being dripped to a zone shown in Figure 1 is passage area;
(5) until Graphene-metallic solution behind bone dry on the paper, drip the thionine solution of 10-100 microlitre in a zone;
(6) treat that thionine solution is combined fully with carbon nano-tube on paper, and stable be adsorbed on the paper after, a zone drips the glutaraldehyde solution of 10-100 microlitre, and etc. it is natural drying;
(7) with ultrapure water flushing three times, purging method is: the positive buffer solution that drips, in reverse side thieving paper diversion;
(8) after water parches, drip the primary antibodie of 1-10 microlitre in a zone;
(9) repeating step (7), flush away does not adsorb or adsorbs unstable antibody
(10) until primary antibodie solution after a zone absorption is firm, drip again the bovine serum albumin solution of 20-100 microlitre, may get avtive spot with sealing;
(11) repeating step (7);
(12) at the antigen of a zone dropping 1-10 microlitre corresponding with difference in the step (8), it is reacted completely;
(13) after reacting completely, repeating step (7) immediately;
(14) can drip in a zone the two anti-of the quantum dot of corresponding 5-20 microlitre-porous silicon mark behind the buffer solution wash-out, it is fully reacted;
(15) repeating step (7) immediately afterwards;
(16) after finishing the final step washing, refill sheet passage is positioned on the film electrode for preparing, and drips the buffer solution of pH 7.4, measure in conjunction with the electrogenerated chemiluminescence instrument again.So far get final product the concentration of antigen in the Accurate Determining sample.
The present invention is easy, economical, be usefulness, good development prospect is arranged.
Black alkene-metal composite nano the material that uses of the present invention can be Graphene-Cu, Graphene-Co, Graphene-Pt.
Quantum dot-porous silicon the nano material of using of the present invention is CdTe quantum dot-porous silicon nano material.
Beneficial effect of the present invention:
1. the film electrode of bottom of the present invention can be designed as the 2-8 electrode system, corresponding refill sheet also needs to be designed to the perform region of counter electrode number like this, utilize the specific binding of the antigen-antibody at different interfaces, tumor cell can detect simultaneously; The sample of modifying like this can be identified blood sample simultaneously, and corresponding antigen-antibody combination makes the diagnosis detecting step simpler, is easier to layman's operation; The film electrode of this sensor bottom is Reusability repeatedly, greatly reduces the diagnostic monitoring cost.
1. the present invention can realize high specific, high sensitivity, the simultaneously quick diagnosis monitoring of various diseases antigen in the sample by means of the refill chip technology.
2. can more be conducive to the electronics transmission after receiving metal on the Graphene, thereby enhancing electrochemical luminescence signals, Graphene-metal composite nano material is modified preparation electrochemiluminescence immunosensor on the refill sheet, so that prepared sandwich tumor markers electrochemiluminescence immunosensor has higher sensitivity and and sensing range.
3. can effectively increase luminous signal after receiving a lot of quantum dots on the porous silicon, quantum dot-porous silicon composite nano materials is marked on the signal antibody as luminescent material, has improved sensitivity and the selectivity of electrochemiluminescence immunosensor.
4. field rapid in-vitro care diagnostic monitoring refill sheet electrochemiluminescimmunosensor immunosensor of the present invention detects the method for tumor markers, highly sensitive, favorable reproducibility, electrode life is long, antijamming capability is strong, operation is simple fast, reaction and result finish and record automatically by instrument, avoided the impact of subjective factor, and ensure good repeatability, be convenient to Site Detection.
5. the high specificity of the refill sheet electrochemiluminescence immunosensor of applying composite nano material of the present invention, since with the antigen recognizing antibody modification to electrode, and antigen carried out specific recognition, then, the signal antibody of luminescent material mark is to the antigen specific recognition, in the sample other non-specific molecules on testing result without impact; Highly sensitive, can reach the ng level; Reagent dosage is few, and detecting a sample only needs tens microlitre reagent; Cost is low.And diagnostic monitoring speed is fast, finishes the time that a basic diagnosis observation process only needs 1-2 minute, can realize at short notice the high flux screening of great amount of samples, and cost is low.
Claims (1)
1. the present invention is with the manufacture method of Graphene-metal and quantum dot-porous silicon composite nano materials refill sheet electrochemiluminescence immunosensor, and its feature may further comprise the steps:
(1) in the channel pattern of chromatographic paper with wax printer printed design, heat with the chromatographic paper of electronic temperature controller device to wax seal, purpose is with roastingization of wax and makes its inside that is penetrated into uniformly chromatographic paper, thereby forms hydrophilic channel at the position of not printing wax;
(2) select the higher tumor markers of clinical onset rate to measure;
(3) utilize nano material, prepare Graphene-metal composite nano material;
(4) prepare quantum dot-porous silicon composite nano materials, and it is anti-to be used for mark two;
(5) Graphene-metal composite nano material is modified on the refill sheet passage after the processing, in order to improve the electric conductivity of refill sheet, improved its sensitivity;
(6) modify afterwards the thionine that is used for catching antibody in step (5);
(7) utilize Ag-Ab specific recognition and assembling surface modification technology etc., two anti-modifications of quantum dot-porous silicon composite material mark are arrived on step (6) the refill sheet surface afterwards, make electrochemical luminous sensor;
(8) the refill sheet of handling well the detection electrochemical luminescence signals that need to combine with screen printing electrode;
(9) screen printing electrode needs to polish smooth with burnishing powder before use, and is for subsequent use;
Graphene-metal composite nano the material that uses of the present invention can be Graphene-Cu, Graphene-Co, Graphene-Pt, quantum dot-porous silicon the nano material of using of the present invention can be CdTe quantum dot-porous silicon nano material, and the implementation step comprises:
(1) manually printed book is tested used film electrode, and with the alundum (Al2O3) burnishing powder electrode is polished smooth, and is for subsequent use;
(2) use the wax printer to print pattern shown in Fig. 1-A, and use the electron temperature-control instrument that the chromatographic paper of seal wax is heated, make wax be penetrated into uniformly chromatographic paper inside, the zone of not printing wax forms refill sheet passage;
(3) prepare Graphene-metal and quantum dot-porous silicon composite nano materials;
(4) 100-200 microlitre Graphene-metallic solution being dripped to a zone shown in Figure 1 is passage area;
(5) until Graphene-metallic solution behind bone dry on the paper, drip the thionine solution of 10-100 microlitre in a zone;
(6) treat that thionine solution is combined fully with carbon nano-tube on paper, and stable be adsorbed on the paper after, a zone drips the glutaraldehyde solution of 10-100 microlitre, and etc. it is natural drying;
(7) with ultrapure water flushing three times, purging method is: the positive buffer solution that drips, in reverse side thieving paper diversion;
(8) after water parches, drip the primary antibodie of 1-10 microlitre in a zone;
(9) repeating step (7), flush away does not adsorb or adsorbs unstable antibody
(10) until primary antibodie solution after a zone absorption is firm, drip again the bovine serum albumin solution of 20-100 microlitre, may get avtive spot with sealing;
(11) repeating step (7);
(12) at the antigen of a zone dropping 1-10 microlitre corresponding with difference in the step (8), it is reacted completely;
(13) after reacting completely, repeating step (7) immediately;
(14) can drip in a zone the two anti-of the quantum dot of corresponding 5-20 microlitre-porous silicon mark behind the buffer solution wash-out, it is fully reacted;
(15) repeating step (7) immediately afterwards;
(16) after finishing the final step washing, refill sheet passage is positioned on the film electrode for preparing, and drips the buffer solution of pH 7.4, the combined with electrochemical light-emitting appearance is measured again, so far gets final product the concentration of antigen in the Accurate Determining sample.
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