CN105954346A - Preparation method of a three-dimensional paper-based electrochemical ratio meter - Google Patents
Preparation method of a three-dimensional paper-based electrochemical ratio meter Download PDFInfo
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- CN105954346A CN105954346A CN201610595195.8A CN201610595195A CN105954346A CN 105954346 A CN105954346 A CN 105954346A CN 201610595195 A CN201610595195 A CN 201610595195A CN 105954346 A CN105954346 A CN 105954346A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
Abstract
The invention discloses a preparation method of a three-dimensional paper-based electrochemical ratio meter and application of the three-dimensional paper-based ratio meter in zinc ion detection. A working area is made on a paper chip through a wax printing technique, three electrodes are printed by virtue of a silk-screen printing technique, therefore, the working area is functionalized, and dual signal amplification of two kinds of electroactive substances including methylene blue and o-phenylenediamine is achieved; mimic enzyme chains are recognized through metal ions, substrate chains are broken through the metal ions, and high-sensitivity portable detection on Zn<2+> is achieved by virtue of an electrochemical workstation.
Description
Technical field
The present invention relates to the preparation method of a kind of three-dimensional paper substrate electrochemistry ratiometer, the structure of a kind of three-dimensional paper substrate electrochemistry ratiometer laboratory technique platform.
Background technology
Preventing and treating water pollution, most important to the health of environmental conservation and the mankind.Some inorganic pollutions, particularly metal ion, such as Pb2+, Mg2+, Zn2+, Hg2+Etc. being material common in water pollution, but expensive equipment needed for now widely used chromatography, fluorescence detection method, and instrument is relatively cumbersome, is unfavorable for instant Site Detection.Therefore, one of the detection method task of becoming researcher finding and setting up a kind of low cost, efficient rapid metal ion.
Paper, as one of four invents by China product, be widely used in write, print, paint, the field such as packaging.The paper fiber of many traversed bies mistakes makes to present inside paper three-dimensional porous structure, and paper fiber surface is rich in abundant oxy radical, thus, it is easy to realize the surface-functionalized of paper fiber.In recent years, functionalization paper substrate experiment detection platform scale expands day by day, tested pregnant rod by simple medical instrument and develop into biosensing device miscellaneous, including paper substrate electrochemica biological sensor, paper substrate Photoelectrochemistrbiosensor biosensor, paper substrate colorimetric bio sensor, paper substrate electrochemiluminescence biosensor etc..
Electrochemica biological sensor, because it has low cost, highly sensitive, it is easy to accomplish miniaturization and integrated, and the advantage such as required instrument is simple and be widely used in bio-sensing field.But traditional electrochemica biological sensor is inconvenient to carry, and it is easily subject to the impact of external environment condition, such as change, the H of electrolyte pH2O2The change etc. of concentration, and make measurement result accuracy.In order to solve this problem, the thought of Ratio-type fluorescence detection method is incorporated in electrochemica biological sensor by first, combined function paper chip, we construct a kind of novel paper substrate electrochemistry ratiometer detection platform, constructed bioanalytical sensing platform not only has low cost, collapsible, the advantage such as be easy to carry about with one, testing result sensitivity simultaneously and accuracy are significantly improved, therefore, constructed laboratory technique platform is expected to be applied in the outlying district that resource-constrained, economic development fall behind relatively and promote.
Summary of the invention
For the problem that presently, there are, the present invention has made a kind of low cost, accuracy three-dimensional paper substrate electrochemistry ratiometer high and easy to carry and has applied it to Zn2+Detection in.
In order to solve above-mentioned technical problem, the present invention is realized by following measures: the preparation of a kind of three-dimensional paper substrate electrochemistry ratiometer, it is characterized in that comprising the following steps:
(1) the hydrophobic wax bulk print pattern of AI software design Ratio-type electrochemistry paper chip as shown in Figure 1 is utilized on computers；
(2) Ratio-type electrochemistry paper chip is cut into the paper of A4 size, wax printer is utilized to print on A4 paper by the hydrophobic wax bulk print pattern of design in step (1), it is placed in baking oven heating subsequently until wax melts and be impregnated with the thickness of whole paper, forms hydrophobic region；
(3) method using silk screen printing, by working electrode, reference electrode, electrode print pattern is printed onto successively in step (2) on gained paper, pattern is as shown in Figure 2, wherein right side is working area, it is printed on working electrode above, left side is reference region, is printed on reference electrode above and to electrode；
(4) to hydrophilic region functionalization in (3), the Zn of Au nanoparticle-iron porphyrin metal-organic framework material will be modified with2+Specific DNA analogue enztme chain, be defined as chain 1, its base sequence, as shown in nucleotides sequence list, is fixed on hydrophilic region, it is achieved first time of signal amplifies, and blocks avtive spot with sulfydryl hexanol subsequently；
(5) Zn that certain density methylene blue is modified2+Substrate chain, it is defined as chain 2, its base sequence is as shown in nucleotides sequence list, wherein to from left to right, and the tenth base, namely base A represents Adenosine acid, with carry out the primer strand of rolling circle amplification, be defined as chain 3, its base sequence is as shown in nucleotides sequence list, mix by a certain percentage, be then added at 43 DEG C of (4) the electrode obtained surface react 3 h；
(6) by certain density annular template probe, it is defined as chain 4, its base sequence is as shown in nucleotides sequence list, and T4 ligase joins step (5) the electrode obtained surface, under room temperature, react 2 h, be subsequently added phi29 DNA ligase, dNTP and the mixed liquor of rolling circle amplification buffer solution, 1 h is reacted at 37 DEG C, rinse electrode surface by phosphate buffered solution subsequently, complete rolling circle amplification reaction, to realize secondary singal amplification；
(7) by certain density Zn2+Join step (6) the electrode obtained surface, mixing, react 40 min under room temperature；
(8) to the dropping of step (7) the electrode obtained surface containing o-phenylenediamine and H2O2The electrolyte of phosphate buffered solution, connect electrochemical workstation, in-0.8 ~ 0 V potential range, measure methylene blue and the current intensity of o-phenylenediamine；
(9) o-phenylenediamine and the change of methylene blue current intensity ratio and Zn are drawn2+Concentration relationship curve, completes Zn2+Mensuration；
Paper material described in step (2), it is characterised in that it is one-level chromatographic paper；
Hydrophilic region functionalization described in step (4), it is characterized in that: in paper chip, grow graphene conductive layer, then drip chitosan solution: repeat to drip the solution of graphene oxide for three times to paper chip surface, under room temperature after drying, dropping have the paper chip of graphene oxide transfer to containing 0.1 M Na2SO4In solution, under 0.9 V current potential, electroreduction graphene oxide, after having reacted, paper chip is taken out, secondary water cleans surface, and under room temperature, dry can acquisition grows the paper chip having Graphene again, then drips the 0.5 mg mL of 2.0 μ L on paper chip surface-1Chitosan, under room temperature be dried 40 min.
The Zn of Au nanoparticle-iron porphyrin metal-organic framework material it is modified with described in step (4)2+Specific DNA analogue enztme chain, it is characterized in that: first prepare iron porphyrin metal-organic framework material, the 5 of i.e. 39.54 mg, 10,15,20-tetra-(tetracarboxylic)-21H, 23H-porphyrin and 40.55 mg iron chloride, the hydrochloric acid of 0.1 M joins in the dimethylformamide of volume ratio 1:2 mixing and the mixed liquor of ethanol of 12 mL, ultrasonic 40 min, react by mixed liquor 150 DEG C to 50 h subsequently, reaction stop after by product dimethylformamide and ethanol purge three times, then by product 60 DEG C vacuum drying, iron porphyrin metal-organic framework material can be obtained；nullSecondly Au nanoparticle-iron porphyrin metal-organic framework material is prepared: weigh the iron porphyrin metal-organic framework material of the above-mentioned preparation of 6 mg，It is dissolved in the ethanol of 1 mL，It is then dispersed in the ammonia of 0.2 mL、13.8 mL ethanol、In 1.8 mL tetramethoxy-silicane mixed liquors，2 h are stirred under room temperature，Product is centrifuged 10 min in 12000 r/min，Use ethanol purge product，By in the ethanol solution of the product obtained and the trimethoxy mercapto propyl silane of 4 mL 20 mM，65 DEG C of backflow 4 h，Product is centrifuged，Secondary water rinses，Then in solution, add the solution of 4 mL Au nanoparticles，3 h are reacted under room temperature，Product is centrifuged，Rinse with secondary water，Au nanoparticle-iron porphyrin metal-organic framework material can be obtained；Finally by Zn2+Specific DNA analogue enztme chain is connected to Au nanoparticle-iron porphyrin metal-organic framework material surface: pipette the Zn of 90 μ L 10 μMs2+Specific DNA analogue enztme chain, join in Au nanoparticle-iron porphyrin metal-organic framework material solution, 24 h are shaken under room temperature, the product sulfydryl hexanol block avtive spot obtained, is finally distributed to product in three (methylol) aminomethane acetate buffer solution of 0.5 mL 50 mM.
Chain 1 described in step (4), it is characterised in that: its specific base sequence and its 5 ' terminal modified upper amino, its 3 ' terminal modified upper sulfydryl.
DNA 4 described in step (6), it is characterised in that: its specific base sequence and its 5 ' terminal modified upper phosphate group.
Beneficial effects of the present invention
(1) use Ratio-type electrochemical method to Zn2+Detect so that measurement result accuracy improves.
(2) functionalization paper chip is as Electrochemical Detection substrate, and active site is many, be prone to the feature of batch production.
(3) whole Ratio-type paper substrate electrochemical device preparation process is simple, and required expense is low, is expected to realize Zn2+Low cost, portable inspectiont.
Accompanying drawing explanation
Fig. 1: the hydrophobic wax bulk print pattern of three-dimensional paper chip；
Fig. 2: working electrode on silk screen printing on hydrophobic wax print pattern, reference electrode and to electrode, wherein, right side is working electrode, and left side is reference and to electrode.
Detailed description of the invention
In order to be more fully understood that the present invention, it is further elucidated with present disclosure below by embodiment, but the present invention is not limited solely to the following examples.
Embodiment
1
A kind of three-dimensional paper substrate electrochemistry ratiometer preparation and at Zn2+Application in detection:
(1) the hydrophobic wax bulk print pattern of AI software design Ratio-type electrochemistry paper chip is utilized on computers；
(2) Ratio-type electrochemistry paper chip is cut into the paper of A4 size, wax printer is utilized to print on A4 paper by the hydrophobic wax bulk print pattern of design in step (1), it is placed in baking oven heating subsequently until wax melts and be impregnated with the thickness of whole paper, forms hydrophobic region；
(3) method using silk screen printing, by working electrode, reference electrode, being printed onto electrode print pattern successively in step (2) on gained paper, wherein right side is working area, is printed on working electrode above, left side is reference region, is printed on reference electrode above and to electrode；
(4) to hydrophilic region functionalization in (3), the Zn of Au nanoparticle-iron porphyrin metal-organic framework material will be modified with2+Specific DNA analogue enztme chain, be defined as chain 1, be fixed on hydrophilic region, subsequently with sulfydryl hexanol block avtive spot；
(5) Zn that certain density methylene blue is modified2+Substrate chain, be defined as chain 2, wherein to from left to right, the tenth base, namely base A represents Adenosine acid, and primer strand, is defined as chain 3, mixes by a certain percentage, react 3 h at 43 DEG C, is then added to (4) the electrode obtained surface；
(6) by certain density annular template probe, it is defined as chain 4, and T4 ligase joins step (5) the electrode obtained surface, 2 h are reacted under room temperature, it is subsequently added phi29 DNA ligase, dNTP and the mixed liquor of rolling circle amplification buffer solution, react 1 h at 37 DEG C, rinse electrode surface by phosphate buffered solution subsequently；
(7) by certain density Zn2+Join in step (6) gained mixed liquor, mixing, react 40 min under room temperature；
(8) to the dropping of step (7) the electrode obtained surface containing o-phenylenediamine and H2O2The electrolyte of phosphate buffered solution, connect electrochemical workstation, in-0.8 ~ 0 V potential range, measure methylene blue and the current intensity of o-phenylenediamine；
(9) o-phenylenediamine and the change of methylene blue current intensity ratio and Zn are drawn2+Concentration relationship curve, completes Zn2+Mensuration.
SEQUENCE LISTING
<110>
University Of Ji'nan
<120>
The preparation method of three-dimensional paper substrate electrochemistry ratiometer
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> DNA
<213>
Artificial sequence
<400> 1
catctcttct ccgagccggt
cgaaatagtg agtcatgcat gcatgtacat c
51
<210> 2
<211> 20
<212> DNA
<213>
Artificial sequence
<400> 2
actcactata ggaagagatg
20
<210> 3
<211> 36
<212> DNA
<213>
Artificial sequence
<400> 3
acagagccta tagagctgat
gatgtacatg catgca
36
<210> 4
<211> 45
<212> DNA
<213>
Artificial sequence
<400> 4
atctcgacta aaaacccaac
ccgccctacc caaaatgtct cggat
45
Claims (4)
1. the preparation method of three-dimensional paper substrate electrochemistry ratiometer, is characterized in that comprising the following steps:
(1) the hydrophobic wax bulk print pattern of AI software design Ratio-type electrochemistry paper chip is utilized on computers；
(2) Ratio-type electrochemistry paper chip is cut into the paper of A4 size, wax printer is utilized to print on A4 paper by the hydrophobic wax bulk print pattern of design in step (1), it is placed in baking oven heating subsequently until wax melts and be impregnated with the thickness of whole paper, forms hydrophobic region；
(3) method using silk screen printing, by working electrode, reference electrode, being printed onto electrode print pattern successively in step (2) on gained paper, wherein right side is working area, is printed on working electrode above, left side is reference region, is printed on reference electrode above and to electrode；
(4) to hydrophilic region functionalization in (3), the Zn of Au nanoparticle-iron porphyrin metal-organic framework material will be modified with2+Specific DNA analogue enztme chain, be defined as chain 1, be fixed on hydrophilic region, it is achieved first time of signal amplifies, block avtive spot with sulfydryl hexanol subsequently；
(5) Zn that certain density methylene blue is modified2+Substrate chain, be defined as chain 2, wherein to from left to right, tenth base, namely base A represents Adenosine acid, and carries out the primer strand of rolling circle amplification, it is defined as chain 3, mixes by a certain percentage, be then added at 43 DEG C of (4) the electrode obtained surface react 3 h；
(6) by certain density annular template probe, it is defined as chain 4, and T4 ligase joins step (5) the electrode obtained surface, 2 h are reacted under room temperature, it is subsequently added phi29 DNA ligase, dNTP and the mixed liquor of rolling circle amplification buffer solution, reacts 1 h at 37 DEG C, rinse electrode surface by phosphate buffered solution subsequently, complete rolling circle amplification reaction, to realize secondary singal amplification；
(7) by certain density Zn2+Join step (6) the electrode obtained surface, mixing, react 40 min under room temperature；
(8) to the dropping of step (7) the electrode obtained surface containing o-phenylenediamine and H2O2The electrolyte of phosphate buffered solution, connect electrochemical workstation, in-0.8 ~ 0 V potential range, measure methylene blue and the current intensity of o-phenylenediamine；
(9) o-phenylenediamine and the change of methylene blue current intensity ratio and Zn are drawn2+Concentration relationship curve, completes Zn2+Mensuration；
Paper material described in step (2), it is characterised in that it is one-level chromatographic paper；
Hydrophilic region functionalization described in step (4), it is characterized in that: in paper chip, grow graphene conductive layer, then drip chitosan solution: repeat to drip the solution of graphene oxide for three times to paper chip surface, under room temperature after drying, dropping have the paper chip of graphene oxide transfer to containing 0.1 M Na2SO4In solution, under 0.9 V current potential, electroreduction graphene oxide, after having reacted, paper chip is taken out, secondary water cleans surface, and under room temperature, dry can acquisition grows the paper chip having Graphene again, then drips the 0.5 mg mL of 2.0 μ L on paper chip surface-1Chitosan, under room temperature be dried 40 min.
2. according to the preparation of the three-dimensional paper substrate electrochemistry ratiometer described in claim 1, it is characterised in that: it is modified with the Zn of Au nanoparticle-iron porphyrin metal-organic framework material described in claim 1 step (4)2+Specific DNA analogue enztme chain: first prepare iron porphyrin metal-organic framework material, the 5 of i.e. 39.54 mg, 10,15,20-tetra-(tetracarboxylic)-21H, 23H-porphyrin and 40.55 mg iron chloride, the hydrochloric acid of 0.1 M joins in the dimethylformamide of volume ratio 1:2 mixing and the mixed liquor of ethanol of 12 mL, ultrasonic 40 min, subsequently mixed liquor 150 DEG C is reacted 50 h, reaction stop after by product dimethylformamide and ethanol purge three times, then by product 60 DEG C vacuum drying, iron porphyrin metal-organic framework material can be obtained；Secondly Au nanoparticle-iron porphyrin metal-organic framework material is prepared: weigh the iron porphyrin metal-organic framework material of the above-mentioned preparation of 6 mg, be dissolved in the ethanol of 1 mL, be then dispersed in 0.2
In the ammonia of mL, 13.8 mL ethanol, 1.8 mL tetramethoxy-silicane mixed liquors, 2 h are stirred under room temperature, product is centrifuged 10 min in 12000 r/min, use ethanol purge product, by in the ethanol solution of the product obtained and the trimethoxy mercapto propyl silane of 4 mL 20 mM, 65 DEG C of backflow 4 h, product is centrifuged, secondary water rinses, then in solution, add the solution of 4 mL Au nanoparticles, react 3 h under room temperature, product is centrifuged, rinse with secondary water, Au nanoparticle-iron porphyrin metal-organic framework material can be obtained；Finally by Zn2+Specific DNA analogue enztme chain is connected to Au nanoparticle-iron porphyrin metal-organic framework material surface: pipette the Zn of 90 μ L 10 μMs2+Specific DNA analogue enztme chain, join in Au nanoparticle-iron porphyrin metal-organic framework material solution, 24 h are shaken under room temperature, the product sulfydryl hexanol block avtive spot obtained, is finally distributed to product in three (methylol) aminomethane acetate buffer solution of 0.5 mL 50 mM.
3. according to the preparation of the three-dimensional paper substrate electrochemistry ratiometer described in claim 1, it is characterised in that: its specific base sequence of chain 1 described in claim 1 step (4) and its 5 ' terminal modified upper amino, its 3 ' terminal modified upper sulfydryl.
4. according to the preparation of the three-dimensional paper substrate electrochemistry ratiometer described in claim 1, it is characterised in that: its specific base sequence of DNA 4 described in claim 1 step (6) and its 5 ' terminal modified upper phosphate group.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106323934A (en) * | 2016-11-18 | 2017-01-11 | 重庆工商大学 | Fluorescent biological probe for simultaneous determination of three ions of Cu2 +, Mg2 + and Pb2 + and detecting method thereof |
| CN107024523A (en) * | 2017-04-25 | 2017-08-08 | 济南大学 | A kind of preparation and application of paper substrate optical electro-chemistry molecular engram sensor |
| CN107236790A (en) * | 2017-05-09 | 2017-10-10 | 济南大学 | A kind of paper substrate Sensitive Detection miRNA method |
| CN107393980A (en) * | 2017-08-28 | 2017-11-24 | 济南大学 | A kind of preparation method of paper substrate graphene p-type cuprous oxide composite |
| CN107727714A (en) * | 2017-09-10 | 2018-02-23 | 福建师范大学 | One kind is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN107860805A (en) * | 2017-11-03 | 2018-03-30 | 青岛大学 | A kind of preparation method of ratio electrochemistry dopamine aptamer sensor |
| CN110487871A (en) * | 2019-08-28 | 2019-11-22 | 济南大学 | A kind of preparation of electrochemistry Ratio-type paper chip and its application in detection of nucleic acids |
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| CN106323934A (en) * | 2016-11-18 | 2017-01-11 | 重庆工商大学 | Fluorescent biological probe for simultaneous determination of three ions of Cu2 +, Mg2 + and Pb2 + and detecting method thereof |
| CN106323934B (en) * | 2016-11-18 | 2019-06-14 | 重庆工商大学 | It is a kind of to detect Cu simultaneously2+、Mg2+And Pb2+The fluorescent bio-probes and its detection method of three kinds of ions |
| CN107024523A (en) * | 2017-04-25 | 2017-08-08 | 济南大学 | A kind of preparation and application of paper substrate optical electro-chemistry molecular engram sensor |
| CN107236790A (en) * | 2017-05-09 | 2017-10-10 | 济南大学 | A kind of paper substrate Sensitive Detection miRNA method |
| CN107393980A (en) * | 2017-08-28 | 2017-11-24 | 济南大学 | A kind of preparation method of paper substrate graphene p-type cuprous oxide composite |
| CN107727714A (en) * | 2017-09-10 | 2018-02-23 | 福建师范大学 | One kind is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN107727714B (en) * | 2017-09-10 | 2019-06-28 | 福建师范大学 | One kind being based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN107860805A (en) * | 2017-11-03 | 2018-03-30 | 青岛大学 | A kind of preparation method of ratio electrochemistry dopamine aptamer sensor |
| CN107860805B (en) * | 2017-11-03 | 2019-08-30 | 青岛大学 | A kind of preparation method of ratio electrochemistry dopamine aptamer sensor |
| CN110487871A (en) * | 2019-08-28 | 2019-11-22 | 济南大学 | A kind of preparation of electrochemistry Ratio-type paper chip and its application in detection of nucleic acids |
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