CN104634852B - A kind of preparation method of the triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure and application - Google Patents
A kind of preparation method of the triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure and application Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method and application of the triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure.Belong to new function material and bio-sensing detection technique field.The present invention specifically adopts the electrochemical immunosensor of golden hydridization ZSM-5 molecular sieve load electronic media body tag, achieves the highly sensitive synchronous detection of three tumor markers antigens, has great importance to the early diagnosis of cancer.
Description
Technical field
The preparation method of a kind of triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure of the present invention and application.Specifically adopt golden hydridization ZSM-5 molecular sieve load electron mediator as label, prepare the electrochemical immunosensor of a kind of synchronous detection three tumor markers antigens, belong to new function material and bio-sensing detection technique field.
Background technology
Tumor markers is the specificity substance that tumour cell itself exists or secretes, and in tumor markers known so far, the overwhelming majority is not only present in malignant tumour, and is present in benign tumour, embryonic tissue, even in normal structure.Therefore, these tumor markerses the specific product of non-malignant tumors, but in malignant tumor patient showed increased.The prevention of tumour, Diagnosis and Treat are the key subjects of current medical domain research.It is good that electrochemical immunosensor has selectivity, highly sensitive, the advantages such as detectability is low, is applicable to onlineization, easy and simple to handle, online Electrochemical Detection can be realized, sample pretreatment simply or not needs pre-service, and detect not by the impact of sample physical property, required instrument and equipment is relatively simple, there is the features such as easy, quick, volume is little, be widely used in the detection of tumor markers at present.Therefore the present invention has prepared a kind of triple channel electrochemical immunosensor of the mark of the ZSM-5 molecular sieve based on golden hydridization, achieves the synchronous detection to three tumor markers antigens.First, ZSM-5 molecular sieve has large specific surface area and good absorption property, can a large amount of electron mediator of load; Secondly, the ZSM-5 molecular sieve of golden hydridization has good biocompatibility, can with the effective bonding of detection antibody; Finally, dimethyl diaminophenazine chloride, toluidine blue and ferrocenecarboxylic acid in electrode surface generation redoxomorphism, can produce the peak current with different peak position, are respectively three tumor markers antigens and provide electrochemical signals.The method has the advantages such as cost is low, highly sensitive, specificity is good, Fast synchronization detection, and preparation process is comparatively simple, for effective early warning of cancer provides new method.
Summary of the invention
An object of the present invention is based on golden hydridization ZSM-5 molecular sieve load electron mediator, constructs a kind of without enzyme, quick and overdelicate binary channels sandwich type electrochemical immunosensor.
Two of object of the present invention is the detections this triple channel sandwich type electrochemical immunosensor being applied to three tumor markerses.
Technical scheme of the present invention is as follows:
1. the preparation method of the triple channel biology sensor built based on golden hydridization ZSM-5 molecular sieve load electron mediator
(1) carry out polishing, ultrasonic cleaning in ultrapure water and ethanol respectively with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, nitrogen dries up;
(2) drip at electrode surface the aqueous solution that 6 μ L concentration are the golden hydridization ZSM-5 molecular sieve of 1 ~ 2 mg/mL, dry;
(3) continue the capture antibody solution being three tumor markers antigens of 8 ~ 12 μ g/mL by 6 μ L concentration and be added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5 ~ 15 mg/mL, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(5) be that three tumor markers antigens of a series of variable concentrations of 0.000003 ~ 18 ng/mL are used for and the specific recognition of corresponding capture antibody by 6 μ L concentration, incubated at room temperature 1 h, cleans up;
(6) be that the detection antibody-solutions of three tumor markers that 1 ~ 2 mg/mL is respectively golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark drops on electrode by 6 μ L concentration, specific recognition is carried out with corresponding three tumor marker antigens, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
2. the preparation of the detection antibody-solutions of three tumor markers of gold hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
(1) preparation of golden hydridization ZSM-5 molecular sieve
Get 0.5 ~ 1 gZSM-5 molecular sieve to be dissolved in 10 ~ 20 mL dry toluenes, add 0.5 ~ 1 mL 3-aminopropyl triethoxysilane, reflux 1.5 h at 70 DEG C.After centrifugal at 110 DEG C dry 1 h, obtain the ZSM-5 molecular sieve that powder is amino functional, 50 ~ 100 mg amination ZSM-5 molecular sieves are dissolved in 10 mL ultrapure waters, under agitation slowly join in 100 ~ 200 mL solution of gold nanoparticles, solution becomes opaque darkviolet immediately, centrifugal, removing supernatant, vacuum drying at 35 DEG C, the violet solid powder obtained is golden hydridization ZSM-5 molecular sieve;
(2) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load neutral red marker
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the first tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the neutral red solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load neutral red marker, stores for future use in 4 DEG C of refrigerators;
(3) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load toluidine blue mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the second tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the toluidine blue solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load toluidine blue mark, stores for future use in 4 DEG C of refrigerators;
(4) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the 3rd tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the ferrocenecarboxylic acid solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, stores for future use in 4 DEG C of refrigerators.
3. the detection method of three tumor markers antigens
(1) use electrochemical workstation to test with three-electrode system, saturated calomel electrode is contrast electrode, and platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, is test in the phosphate buffered solution of 7.4 in the pH value of 10 mL;
(2) select square wave voltammetry to detect three tumor markers antigens, scan under-0.8 ~ 0.8 V, record current changes, drawing curve;
(3) three tumor markers antigen standard solution are replaced by testing sample solution to detect.
4. ZSM-5 molecular sieve described above is the mesoporous nano material of high silicon 3 D intersection straight channel, and the channel diameter that intersects is 0.9 nm, and particle diameter is 50 ~ 100nm.
5. tumor markers antigen described above is selected from lung cancer tumor mark, pancreatic tumour mark, breast cancer tumour mark; Wherein, lung cancer tumor mark is selected from any three in CEA, SCC, Cyfra21-1, TPA, CA125, CA242, NSE; Pancreatic tumour mark is selected from any three in CA19-9, CA242, CA724, CA50, CEA, DU-PAN-2, Span-1, IAP; Breast cancer tumour mark is selected from any three in CA153, CEA, CA549, hCG, calcitonin, ferritin.
6. the detection for three tumor markers antigens described in, refers to any three marks that can simultaneously detect in same class tumor markers.
Useful achievement of the present invention
(1) ZSM-5 molecular sieve specific surface area of the present invention is large, can a large amount of golden nanometer particle of load, is conducive to the fixing of antibody.
(2) pore passage structure of ZSM-5 molecular sieve of the present invention is unique, and aperture is more, and duct is shorter, can stablize the load of electron mediator.
(3) the present invention prepares the golden hydridization ZSM-5 molecular sieve with good adsorption properties and carrys out load electron mediator, achieves the synchronous detection to three tumor markers antigens.
(4) detectability of sandwich electro-chemistry immunity prepared of the present invention is low, the range of linearity is wide, can realize simple, quick, sensitive and specific detection, the present invention is 0.000003 ~ 18 ng/mL for detecting the range of linearity of three tumor markerses, and detectability reaches 0.15 fg/mL.
Embodiment
The preparation method of the triple channel biology sensor that embodiment 1 one kinds builds based on golden hydridization ZSM-5 molecular sieve load electron mediator
(1) carry out polishing, ultrasonic cleaning in ultrapure water and ethanol respectively with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, nitrogen dries up;
(2) drip at electrode surface the aqueous solution that 6 μ L concentration are the golden hydridization ZSM-5 molecular sieve of 1 mg/mL, dry;
(3) continue the capture antibody solution being three tumor markers antigens of 8 μ g/mL by 6 μ L concentration and be added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5 mg/mL, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(5) be that three tumor markers antigens of a series of variable concentrations of 0.000003 ~ 18 ng/mL are used for and the specific recognition of corresponding capture antibody by 6 μ L concentration, incubated at room temperature 1 h, cleans up;
(6) be that the detection antibody-solutions of three tumor markers that 1 mg/mL is respectively golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark drops on electrode by 6 μ L concentration, specific recognition is carried out with corresponding three tumor marker antigens, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
The preparation method of the triple channel biology sensor that embodiment 2 one kinds builds based on golden hydridization ZSM-5 molecular sieve load electron mediator
(1) carry out polishing, ultrasonic cleaning in ultrapure water and ethanol respectively with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, nitrogen dries up;
(2) drip at electrode surface the aqueous solution that 6 μ L concentration are the golden hydridization ZSM-5 molecular sieve of 1.5 mg/mL, dry;
(3) continue the capture antibody solution being three tumor markers antigens of 10 μ g/mL by 6 μ L concentration and be added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 10 mg/mL, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(5) be that three tumor markers antigens of a series of variable concentrations of 0.000003 ~ 18 ng/mL are used for and the specific recognition of corresponding capture antibody by 6 μ L concentration, incubated at room temperature 1 h, cleans up;
(6) be that the detection antibody-solutions of three tumor markers that 1.5 mg/mL are respectively golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark drops on electrode by 6 μ L concentration, specific recognition is carried out with corresponding three tumor marker antigens, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
The preparation method of the triple channel biology sensor that embodiment 3 one kinds builds based on golden hydridization ZSM-5 molecular sieve load electron mediator
(1) carry out polishing, ultrasonic cleaning in ultrapure water and ethanol respectively with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, nitrogen dries up;
(2) drip at electrode surface the aqueous solution that 6 μ L concentration are the golden hydridization ZSM-5 molecular sieve of 2 mg/mL, dry;
(3) continue the capture antibody solution being three tumor markers antigens of 12 μ g/mL by 6 μ L concentration and be added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 15 mg/mL, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(5) be that three tumor markers antigens of a series of variable concentrations of 0.000003 ~ 18 ng/mL are used for and the specific recognition of corresponding capture antibody by 6 μ L concentration, incubated at room temperature 1 h, cleans up;
(6) be that the detection antibody-solutions of three tumor markers that 2 mg/mL are respectively golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark drops on electrode by 6 μ L concentration, specific recognition is carried out with corresponding three tumor marker antigens, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
The preparation of the detection antibody-solutions of three tumor markers of embodiment 4 gold medal hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
(1) preparation of golden hydridization ZSM-5 molecular sieve
Get 0.5 gZSM-5 molecular sieve to be dissolved in 10 mL dry toluenes, add 0.5 mL 3-aminopropyl triethoxysilane, reflux 1.5 h at 70 DEG C.After centrifugal at 110 DEG C dry 1 h, obtain the ZSM-5 molecular sieve that powder is amino functional, 50 mg amination ZSM-5 molecular sieves are dissolved in 10 mL ultrapure waters, under agitation slowly join in 100 mL solution of gold nanoparticles, solution becomes opaque darkviolet immediately, centrifugal, removing supernatant, vacuum drying at 35 DEG C, the violet solid powder obtained is golden hydridization ZSM-5 molecular sieve;
(2) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load neutral red marker
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 mg/mL is the first tumor markers of 10 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the neutral red solution that 1 mL concentration is 1 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load neutral red marker, stores for future use in 4 DEG C of refrigerators;
(3) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load toluidine blue mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 mg/mL is the second tumor markers of 10 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the toluidine blue solution that 1 mL concentration is 1 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load toluidine blue mark, stores for future use in 4 DEG C of refrigerators;
(4) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 mg/mL is the 3rd tumor markers of 10 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the ferrocenecarboxylic acid solution that 1 mL concentration is 1 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, stores for future use in 4 DEG C of refrigerators.
The preparation of the detection antibody-solutions of three tumor markers of embodiment 5 gold medal hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
(1) preparation of golden hydridization ZSM-5 molecular sieve
Get 0.75 gZSM-5 molecular sieve to be dissolved in 15 mL dry toluenes, add 0.75 mL 3-aminopropyl triethoxysilane, reflux 1.5 h at 70 DEG C.After centrifugal at 110 DEG C dry 1 h, obtain the ZSM-5 molecular sieve that powder is amino functional, 75 mg amination ZSM-5 molecular sieves are dissolved in 10 mL ultrapure waters, under agitation slowly join in 150 mL solution of gold nanoparticles, solution becomes opaque darkviolet immediately, centrifugal, removing supernatant, vacuum drying at 35 DEG C, the violet solid powder obtained is golden hydridization ZSM-5 molecular sieve;
(2) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load neutral red marker
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1.5 mg/mL is the first tumor markers of 15 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the neutral red solution that 1 mL concentration is 1.5 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load neutral red marker, stores for future use in 4 DEG C of refrigerators;
(3) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load toluidine blue mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1.5 mg/mL is the second tumor markers of 15 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the toluidine blue solution that 1 mL concentration is 1.5 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load toluidine blue mark, stores for future use in 4 DEG C of refrigerators;
(4) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1.5 mg/mL is the 3rd tumor markers of 15 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the ferrocenecarboxylic acid solution that 1 mL concentration is 1.5 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, stores for future use in 4 DEG C of refrigerators.
The preparation of the detection antibody-solutions of three tumor markers of embodiment 6 gold medal hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
(1) preparation of golden hydridization ZSM-5 molecular sieve
Get 0.5 ~ 1 gZSM-5 molecular sieve to be dissolved in 20 mL dry toluenes, add 1 mL 3-aminopropyl triethoxysilane, reflux 1.5 h at 70 DEG C.After centrifugal at 110 DEG C dry 1 h, obtain the ZSM-5 molecular sieve that powder is amino functional, 100 mg amination ZSM-5 molecular sieves are dissolved in 10 mL ultrapure waters, under agitation slowly join in 200 mL solution of gold nanoparticles, solution becomes opaque darkviolet immediately, centrifugal, removing supernatant, vacuum drying at 35 DEG C, the violet solid powder obtained is golden hydridization ZSM-5 molecular sieve;
(2) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load neutral red marker
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 2 mg/mL is the first tumor markers of 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the neutral red solution that 1 mL concentration is 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load neutral red marker, stores for future use in 4 DEG C of refrigerators;
(3) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load toluidine blue mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 2 mg/mL is the second tumor markers of 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the toluidine blue solution that 1 mL concentration is 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load toluidine blue mark, stores for future use in 4 DEG C of refrigerators;
(4) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 2 mg/mL is the 3rd tumor markers of 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the ferrocenecarboxylic acid solution that 1 mL concentration is 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, stores for future use in 4 DEG C of refrigerators.
The detection method of embodiment 7 lung cancer tumor mark CEA, SCC and CA125
(1) use electrochemical workstation to test with three-electrode system, saturated calomel electrode is contrast electrode, and platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, is test in the phosphate buffered solution of 7.4 in the pH value of 10 mL;
(2) select square wave voltammetry to detect CEA, SCC and CA125, scan under-0.8 ~ 0.8 V, record current changes, drawing curve;
(3) CEA, SCC and CA125 standard solution is replaced by testing sample solution to detect.
(4) the detection range of linearity of this two-channel electrochemical immunosensor to CEA, SCC and CA125 is 0.000003 ~ 18 ng/mL, and detectability reaches 0.15 fg/mL.
Claims (6)
1., based on a preparation method for the triple channel biology sensor of golden hydridization ZSM-5 molecular sieve load electron mediator structure, it is characterized in that, comprise the following steps:
(1) carry out polishing, ultrasonic cleaning in ultrapure water and ethanol respectively with the alumina powder foot couple glass-carbon electrode of 1.0,0.3,0.05 μm successively, nitrogen dries up;
(2) drip at electrode surface the aqueous solution that 6 μ L concentration are the golden hydridization ZSM-5 molecular sieve of 1 ~ 2 mg/mL, dry;
(3) continue the capture antibody solution being three tumor markers antigens of 8 ~ 12 μ g/mL by 6 μ L concentration and be added drop-wise to modified electrode surface, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L concentration are 5 ~ 15 mg/mL, in 4 DEG C of refrigerators, hatch 1 h, clean up;
(5) be that three tumor markers antigens of a series of variable concentrations of 0.000003 ~ 18 ng/mL are used for and the specific recognition of corresponding capture antibody by 6 μ L concentration, incubated at room temperature 1 h, cleans up;
(6) be that the detection antibody-solutions of three kinds of tumor markers that 1 ~ 2 mg/mL is respectively golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark drops on electrode by 6 μ L concentration, specific recognition is carried out with corresponding three kinds of tumor marker antigens, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
2. the preparation method of a kind of triple channel biology sensor built based on golden hydridization ZSM-5 molecular sieve load electron mediator as claimed in claim 1, the preparation of the detection antibody-solutions of three kinds of tumor markers of described golden hydridization ZSM-5 molecular sieve load neutral red, golden hydridization ZSM-5 molecular sieve load toluidine blue and golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, it is characterized in that, comprise the following steps:
(1) preparation of golden hydridization ZSM-5 molecular sieve
Get 0.5 ~ 1 gZSM-5 molecular sieve to be dissolved in 10 ~ 20 mL dry toluenes, add 0.5 ~ 1 mL 3-aminopropyl triethoxysilane, reflux 1.5 h at 70 DEG C; After centrifugal at 110 DEG C dry 1 h, obtain the ZSM-5 molecular sieve that powder is amino functional, 50 ~ 100 mg amination ZSM-5 molecular sieves are dissolved in 10 mL ultrapure waters, under agitation slowly join in 100 ~ 200 mL solution of gold nanoparticles, solution becomes opaque darkviolet immediately, centrifugal, removing supernatant, vacuum drying at 35 DEG C, the violet solid powder obtained is golden hydridization ZSM-5 molecular sieve;
(2) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load neutral red marker
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the first tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the neutral red solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load neutral red marker, stores for future use in 4 DEG C of refrigerators;
(3) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load toluidine blue mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the second tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the toluidine blue solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load toluidine blue mark, stores for future use in 4 DEG C of refrigerators;
(4) preparation of the detection antibody of the tumor marker of golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark
The detection antibody that to get 1 mL concentration be golden hydridization ZSM-5 molecular sieve aqueous solution and the 1 mL concentration of 1 ~ 2 mg/mL is the 3rd tumor markers of 10 ~ 20 μ g/mL mixes, shake 12 h, after centrifuge washing, continue to add the ferrocenecarboxylic acid solution that 1 mL concentration is 1 ~ 2 mg/mL, continue concussion 12 h, after centrifuge washing, sediment is dispersed in again in 1 mL ultrapure water, the detection antibody-solutions of the tumor marker of obtained golden hydridization ZSM-5 molecular sieve load ferrocenecarboxylic acid mark, stores for future use in 4 DEG C of refrigerators.
3. the preparation method of a kind of triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure according to claim 1, described ZSM-5 molecular sieve is the mesoporous nano material of high silicon 3 D intersection straight channel, the channel diameter that intersects is 0.9 nm, and particle diameter is 50 ~ 100 nm.
4. the preparation method of a kind of triple channel biology sensor based on golden hydridization ZSM-5 molecular sieve load electron mediator structure according to claim 1, described tumor markers antigen is selected from lung cancer tumor mark, pancreatic tumour mark, breast cancer tumour mark; Wherein, lung cancer tumor mark is selected from any three in CEA, SCC, Cyfra21-1, TPA, CA125, CA242, NSE; Pancreatic tumour mark is selected from any three in CA19-9, CA242, CA724, CA50, CEA, DU-PAN-2, Span-1, IAP; Breast cancer tumour mark is selected from any three in CA153, CEA, CA549, hCG, calcitonin, ferritin.
5. a kind of triple channel biology sensor of preparing of preparation method as claimed in claim 1 is for the detection of three tumor markers antigens, and step is as follows:
(1) use electrochemical workstation to test with three-electrode system, saturated calomel electrode is contrast electrode, and platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, is test in the phosphate buffered solution of 7.4 in the pH value of 10 mL;
(2) select square wave voltammetry to detect three tumor markers antigens, scan under-0.8 ~ 0.8 V, record current changes, drawing curve;
(3) three tumor markers antigen standard solution are replaced by testing sample solution to detect.
6., as claimed in claim 5 for the detection of three tumor markers antigens, refer to three marks that can simultaneously detect in same class tumor markers.
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| US4746539A (en) * | 1983-11-23 | 1988-05-24 | The Ohio State University Research Foundation | Purification of cancer-associated protein and preparation of antibody thereto |
| CN102854235A (en) * | 2012-09-15 | 2013-01-02 | 济南大学 | Preparation method and application of gynecological tumor marker immune sensor constructed with ordered mesoporous carbon |
| CN104133059A (en) * | 2014-08-20 | 2014-11-05 | 济南大学 | Preparation method of electrochemical immunosensor adopting alloy-loaded molecular sieve and application thereof |
| CN104155357A (en) * | 2014-05-23 | 2014-11-19 | 济南大学 | Preparation method and application of three-dimensional cubic duct based mesoporous silica sensor |
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| US4746539A (en) * | 1983-11-23 | 1988-05-24 | The Ohio State University Research Foundation | Purification of cancer-associated protein and preparation of antibody thereto |
| CN102854235A (en) * | 2012-09-15 | 2013-01-02 | 济南大学 | Preparation method and application of gynecological tumor marker immune sensor constructed with ordered mesoporous carbon |
| CN104155357A (en) * | 2014-05-23 | 2014-11-19 | 济南大学 | Preparation method and application of three-dimensional cubic duct based mesoporous silica sensor |
| CN104133059A (en) * | 2014-08-20 | 2014-11-05 | 济南大学 | Preparation method of electrochemical immunosensor adopting alloy-loaded molecular sieve and application thereof |
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