CN110501405A - A kind of method of integrated paper base dual-mode biological sensor detection miRNA-155 - Google Patents
A kind of method of integrated paper base dual-mode biological sensor detection miRNA-155 Download PDFInfo
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- CN110501405A CN110501405A CN201910929688.4A CN201910929688A CN110501405A CN 110501405 A CN110501405 A CN 110501405A CN 201910929688 A CN201910929688 A CN 201910929688A CN 110501405 A CN110501405 A CN 110501405A
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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
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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
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The present invention provides the methods of integrated paper base dual-mode biological sensor detection miRNA-155 a kind of, belong to the detection technique field of miRNA.During the invention, a kind of integrated paper base dual-mode biological sensor is constructed, the equipment is with S1@CuCo-CeO2As signal amplifier, the hypersensitive for miRNA-155 senses nano material.In order to achieve this goal, CuCo-CeO has been synthesized first2Nanosphere is as signal probe, then by gold nano grain growth in situ on paper base working electrode surface, to improve conductivity and promote to modify miRNA-155 modification hairpin probe；By cross chain reaction, CuCo-CeO2 can successfully be fixed on paper base working electrode surface, be used for quantitative detection miRNA-155.Finally, can realize that double-mode signal is read by simply converting the space configuration of paper base biosensor.
Description
Technical field
The present invention relates to the methods of integrated paper base dual-mode biological sensor detection miRNA-155 a kind of, belong to miRNA's
Detection technique field.
Background technique
Microrna (miRNA) is a kind of endogenous and single stranded RNA for being about 22 nucleotide, for post-transcriptional control base
It is most important and related to the generation of kinds cancer because expressing.MicroRNA-155 is as the typical case for participating in a variety of bioprocess
One of nonprotein coding miRNA, it has also become early screening is latent with diagnosis cancer (such as liver cancer, leukaemia and cardiovascular disease)
In biomarker.Therefore, it explores quickly, the strategy for detecting to accurate and hypersensitive miRNA-155 is of great significance.
In order to realize the super sensitivity detection to miRNA-155, some analyses for detecting miRNA-155 have been developed
Technology, including plasma resonance, electrochemical luminescence, fluorescence etc..However, due to miRNA micro in biological sample and right
The sensibility of degradation and these methods are usually directed to complicated operation, and sensitivity is insufficient, and expensive equipment and big background are dry
It disturbs, keeps their application range very limited.
Paper base biosensor apparatus inherits low cost, batch production, convenient for functionalization and has biggish surface area
With porous advantage, while having many advantages, such as that manufacturing process simple, biocompatibility and biodegradability are good.It is so far
Only, paper base biosensor is in real-time disease diagnostic, and environmental monitoring, food safety etc. has to be applied well.
Summary of the invention
For presently, there are the above problem, technical problem to be solved by the invention is to provide it is a kind of design rationally, at
This cheap, construction method of integrated paper base dual mode device easy to operate and high sensitivity, it is characterized in that including following step
It is rapid:
(1) CuCo-CeO is synthesized using hydro-thermal method2Nano material, and by itself and S1 chain link, it is defined as chain 1, base sequence
As shown in nucleotides sequence list, as signal probe, it to be used for H2O2Catalysis oxidation；Firstly, by 500 mg Ce(NO3)3·6H2O
It is dissolved in 14 mL ethylene glycol with 200 mg PVP, and 30 min is stirred at room temperature；Then, by 20 mg of 0.5 mL/
The CuCl of mL2· 2H220 mg of O and 0.5 mL/mL CoCl2·6H2O solution is added to above-mentioned solution；Next, will
Mixed solution is transferred in the autoclave for the teflon lined that capacity is 20 mL, and 8 h are heated under 160 °C；Reaction
After the completion, it by autoclave cooled to room temperature, collects product and is washed with absolute alcohol difference three times with distilled water；Finally,
Product is dried overnight under 60 °C, 1 h is then calcined with the speed of 1 °C/min under 300 °C, obtains product copper cobalt
Codope cerium dioxide nano material；Then the S1 chain that 300 μ L concentration are 2 μM is added drop-wise to CuCo-CeO2Solution in, and
12 h are stirred under 4 °C；Then, unbonded S1 chain is removed by being centrifuged and washing；Finally, by the S1@CuCo- of acquisition
CeO2It is dispersed in 2 mL ultrapure waters, and saved under 4 °C；
(2) one hydrophobic wax print pattern of Adobe Illustrator CS6 software design is utilized on computers and utilizes spray
It is then melted on the A4 size filter paper of its bulk print to cutting to wax and is permeated whole in heater plate by wax printer
The thickness of a paper forms hydrophobic region, and pattern and size are as shown in Fig. 1, wherein I being working electrode label, II being hollow area
Domain, III be reference and to electrode label, IV be colour developing area, V be assisted tag, VI be cleaning label;
(3) method for using silk-screen printing, is printed onto I region for carbon working electrode, Ag/AgCl reference electrode and carbon are to electrode
It is printed onto III region, carbon electrode is printed onto reaction zone, and the round hydrophilic region that hydrophobic wax in hollow region surrounds is carried out between two parties
Punching；The solid line of color development area is cut, then folds these paper chips, forms a stereochemical structure；
(4) functionalization is carried out to the hydrophilic working region of circle of detection zone, gold nano is grown by seed solution growth method first
Particle, specific growth step are as follows: 80 mL secondary waters are poured into three-necked flask be heated to 90 DEG C first, and 800 μ L matter are added
The chlorauric acid solution that score is 1% is measured, continues to be heated to 96 DEG C of 1 min of holding, being eventually adding 2.8 mL mass fractions is 1%
Sodium citrate continues to heat 15 min, and natural cooling obtains gold seeds solution, and the gold seeds solution for taking 60 μ L to obtain is added dropwise
Working region, standing are dried, and in triplicate, are rinsed 3 times with secondary water, will are then 2 μM of H1 chain by 50 μ L concentration, determine
Justice is chain 2, and base sequence is added drop-wise to the working region of gold nanoparticle modification as shown in nucleotides sequence list, and in room temperature
18 h of lower incubation；Later, the electrode of preparation is washed with distilled water, and with 50 μ L, concentration is the 6- sulfydryl -1- of 1.0 mM
Hexanol closes 1 h, to remove non-specific adsorption；Then, by the miRNA- of the working region modified and 50 μ L various concentrations
155 chains are defined as chain 3, and base sequence is incubated for as shown in nucleotides sequence list；It is immediately after 2 μM by 50 μ L concentration
H2 chain is defined as chain 4, and base sequence is added drop-wise to the surface of the working region of modification, then exists as shown in nucleotides sequence list
2 h are incubated under 37 °C；Then, by the S1@CuCo-CeO of 50 μ L2Solution is introduced into modified paper base working electrode, and use is ultrapure
After water rinses, the electrode of modification is incubated for 2 h under 4 °C；Later, by the electrode ultrapure water of modified to remove not
In conjunction with chain structure；
(5) it is modified in reaction zone circle hydrophilic region, specific steps are as follows: 10 μ L pH are added in round hydrophilic region
It is 7.0, concentration is the phosphate buffered saline solution of 0.1M；Then the H for being 5 mM by 200 μ L concentration2O2Solution is added drop-wise to circle
Hydrophilic region, then in 3,3', 5, the 5'- tetramethyl benzidine developing solutions that the pre-buried 20 μ L concentration of color development area is 20 mM；
(6) paper chip is simply folded, completes the detection to miRNA-155, the specific steps are as follows: pass through folding first
It will test region, reference electrode and to electrode zone, hollow region and color development area overlapping, auxiliary area and cleaning area difference
The two sides up and down of color development area are placed in, after the modification layer by layer for completing electrode, working region is cleaned, detection zone and electrode are made
Label overlapping, is fixed, pattern such as attached drawing 2 is connect with electrochemical workstation, and 60 μ L are contained 5.0 mM [Fe with clip
(CN)6] 3-/4 -PH is 7.0, and concentration is that the phosphate buffered saline solution of 0.1M is added drop-wise to detection zone, is measured and records；
(7) carry out the color developing detection of miRNA-155, the specific steps are as follows: after the completion of electric signal measurement process, by assisted tag and
Cleaning label is taken out, hydrogen peroxide and signal probe flow through hollow region and with 3,3', 5, the 5'- tetramethyls that are embedded in color development area
Base benzidine developing solution reaction, to realize Visual retrieval.
The design of paper chip of the present invention, it is characterised in that: cleaning region is half hydrophilic half hydrophobic, having a size of 12 mm
× 12 mm, working electrode area diameter are 10 mm, and modified regions diameter is 8 mm, and hollow region label is the mm of 30 mm × 30
Square structure, reference electrode and to electrode print regional diameter be 10 mm, color development area diameter be 10 mm, assisted tag
Having a size of 15mm × 35mm, cleaning label is upper 15 mm of bottom, and go to the bottom 26 mm, and a height of 65 mm's is trapezoidal.
Beneficial effects of the present invention:
(1) a kind of integrated paper base dual-mode biological sensor can be realized to miRNA-155 Sensitive Detection, reduce experimental cost.
(2) introducing of signal probe greatly improves the selectivity and sensitivity of detection.
(3) use of gold nano grain increases specific surface area and effectively reduces the background fluorescence of paper, improves detection
Sensitivity.
(4) paper base sensor flexible is flexible, easy to carry, can cut out, be bent, folding and is plastic, and post-processing is simple,
It not can cause environmental pollution.
(5) compared to traditional glass-carbon electrode and glass electrode, paper base material abundant raw material, light weight, cheap, foldable,
It is degradable.
(6) integrated paper base biosensor realizes the self-cleaning process of electrode, simplifies operating procedure, reduces cost.
Detailed description of the invention:
Present invention is further described in detail with specific embodiment with reference to the accompanying drawing:
Fig. 1 is screen printing work electrode on hydrophobic wax print pattern, reference electrode, to electrode, carbon line.
Fig. 2 is that integrated paper base equipment electrochemistry mode detection object folds schematic diagram.
Fig. 3 is that integrated paper base equipment colour developing mode detection object folds schematic diagram.
Specific embodiment
Embodiment 1(comes from human serum)
A kind of design is reasonable, low in cost, the construction method of integrated paper base dual mode device easy to operate and high sensitivity,
It is characterized in that the following steps are included:
(1) CuCo-CeO is synthesized using hydro-thermal method2Nano material, and by itself and S1 chain link, it is defined as chain 1, base sequence
As shown in nucleotides sequence list, as signal probe, it to be used for H2O2Catalysis oxidation；Firstly, by 500 mg Ce(NO3)3·6H2O
It is dissolved in 14 mL ethylene glycol with 200 mg PVP, and 30 min is stirred at room temperature；Then, by 20 mg of 0.5 mL/
The CuCl of mL2·2H220 mg of O and 0.5 mL/mL CoCl2·6H2O solution is added to above-mentioned solution；Next, will mix
It closes solution to be transferred in the autoclave for the teflon lined that capacity is 20 mL, and heats 8 h under 160 °C；It has reacted
Autoclave cooled to room temperature is collected product and is washed with absolute alcohol difference three times with distilled water by Cheng Hou；Finally, will
Product is dried overnight under 60 °C, then calcines 1 h under 300 °C with the speed of 1 °C/min, and it is double to obtain product copper cobalt
Doping cerium dioxide nano material；Then the S1 chain that 300 μ L concentration are 2 μM is added drop-wise to CuCo-CeO2Solution in, and 4
12 h are stirred under °C；Then, unbonded S1 chain is removed by being centrifuged and washing；Finally, by the S1@CuCo-CeO of acquisition2Point
It is dispersed in 2 mL ultrapure waters, and saved under 4 °C；
(2) one hydrophobic wax print pattern of Adobe Illustrator CS6 software design is utilized on computers and utilizes spray
It is then melted on the A4 size filter paper of its bulk print to cutting to wax and is permeated whole in heater plate by wax printer
The thickness of a paper forms hydrophobic region, which includes working electrode label, hollow region, reference and to electrode label, colour developing
Region, assisted tag and cleaning label；
(3) method for using silk-screen printing, is printed onto I region for carbon working electrode, Ag/AgCl reference electrode and carbon are to electrode
It is printed onto III region, carbon electrode is printed onto reaction zone, and the round hydrophilic region that hydrophobic wax in hollow region surrounds is carried out between two parties
Punching；The solid line of color development area is cut, then folds these paper chips, forms a stereochemical structure；
(4) functionalization is carried out to the hydrophilic working region of circle of detection zone, gold nano is grown by seed solution growth method first
Particle, specific growth step are as follows: 80 mL secondary waters are poured into three-necked flask be heated to 90 DEG C first, and 800 μ L matter are added
The chlorauric acid solution that score is 1% is measured, continues to be heated to 96 DEG C of 1 min of holding, being eventually adding 2.8 mL mass fractions is 1%
Sodium citrate continues to heat 15 min, and natural cooling obtains gold seeds solution, and the gold seeds solution for taking 60 μ L to obtain is added dropwise
Working region, standing are dried, and in triplicate, are rinsed 3 times with secondary water, will are then 2 μM of H1 chain by 50 μ L concentration, determine
Justice is chain 2, and base sequence is added drop-wise to the working region of gold nanoparticle modification as shown in nucleotides sequence list, and in room temperature
18 h of lower incubation；Later, the electrode of preparation is washed with distilled water, and with 50 μ L, concentration is the 6- sulfydryl -1- of 1.0 mM
Hexanol closes 1 h, to remove non-specific adsorption；Then, by the miRNA- of the working region modified and 50 μ L various concentrations
155 chains are defined as chain 3, and base sequence is incubated for as shown in nucleotides sequence list；It is immediately after 2 μM by 50 μ L concentration
H2 chain is defined as chain 4, and base sequence is added drop-wise to the surface of the working region of modification, then exists as shown in nucleotides sequence list
2 h are incubated under 37 °C；Then, by the S1@CuCo-CeO of 50 μ L2Solution is introduced into modified paper base working electrode, and use is ultrapure
After water rinses, the electrode of modification is incubated for 2 h under 4 °C；Later, by the electrode ultrapure water of modified to remove not
In conjunction with chain structure；
(5) it is modified in reaction zone circle hydrophilic region, specific steps are as follows: 10 μ L pH are added in round hydrophilic region
It is 7.0, concentration is the phosphate buffered saline solution of 0.1M；Then the H for being 5 mM by 200 μ L concentration2O2Solution is added drop-wise to circle
Hydrophilic region, then in 3,3', 5, the 5'- tetramethyl benzidine developing solutions that the pre-buried 20 μ L concentration of color development area is 20 mM；
(6) paper chip is simply folded, completes the detection to miRNA-155, the specific steps are as follows: pass through folding first
It will test region, reference electrode and to electrode zone, hollow region and color development area overlapping, auxiliary area and cleaning area difference
The two sides up and down of color development area are placed in, after the modification layer by layer for completing electrode, working region is cleaned, detection zone and electrode are made
Label overlapping, is fixed, pattern such as attached drawing 2 is connect with electrochemical workstation, and 60 μ L are contained 5.0 mM [Fe with clip
(CN)6] 3-/4 -PH is 7.0, and concentration is that the phosphate buffered saline solution of 0.1M is added drop-wise to detection zone, is measured and records；
(7) carry out the color developing detection of miRNA-155, the specific steps are as follows: after the completion of electric signal measurement process, by assisted tag and
Cleaning label is taken out, hydrogen peroxide and signal probe flow through hollow region and with 3,3', 5, the 5'- tetramethyls that are embedded in color development area
Base benzidine developing solution reaction, to realize Visual retrieval.
Sequence table
<110>University Of Ji'nan
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taatcgtgat aggggtatgg acatggaacc cctatcacga ttagcattaa agash 55
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atggacatgg ataatcgtga taggggttcc atgtccatac ccctatgaag gagcgact 58
Claims (2)
1. a kind of method of integrated paper base dual-mode biological sensor detection miRNA-155, it is characterized in that the following steps are included:
(1) CuCo-CeO is synthesized using hydro-thermal method2Nano material, and by itself and S1 chain link, it is defined as chain 1, base sequence
As shown in nucleotides sequence list, as signal probe, it to be used for H2O2Catalysis oxidation；Firstly, by 500 mg Ce(NO3)3·6H2O
It is dissolved in 14 mL ethylene glycol with 200 mg PVP, and 30 min is stirred at room temperature；Then, by 20 mg of 0.5 mL/
The CuCl of mL2·2H220 mg of O and 0.5 mL/mL CoCl2·6H2O solution is added to above-mentioned solution；Next, will mix
It closes solution to be transferred in the autoclave for the teflon lined that capacity is 20 mL, and heats 8 h under 160 °C；It has reacted
Autoclave cooled to room temperature is collected product and is washed with absolute alcohol difference three times with distilled water by Cheng Hou；Finally, will
Product is dried overnight under 60 °C, then calcines 1 h under 300 °C with the speed of 1 °C/min, and it is double to obtain product copper cobalt
Doping cerium dioxide nano material；Then the S1 chain that 300 μ L concentration are 2 μM is added drop-wise to CuCo-CeO2Solution in, and 4
12 h are stirred under °C；Then, unbonded S1 chain is removed by being centrifuged and washing；Finally, by the S1@CuCo-CeO of acquisition2Point
It is dispersed in 2 mL ultrapure waters, and saved under 4 °C；
(2) one hydrophobic wax print pattern of Adobe Illustrator CS6 software design is utilized on computers and utilizes spray
It is then melted on the A4 size filter paper of its bulk print to cutting to wax and is permeated whole in heater plate by wax printer
The thickness of a paper, formed hydrophobic region, the device include working electrode label, hollow region, reference and to electrode label, colour developing
Region, assisted tag and cleaning label；
(3) method for using silk-screen printing, is printed onto I region for carbon working electrode, Ag/AgCl reference electrode and carbon are to electrode
It is printed onto III region, carbon electrode is printed onto reaction zone, and the round hydrophilic region that hydrophobic wax in hollow region surrounds is carried out between two parties
Punching；The solid line of color development area is cut, then folds these paper chips, forms a stereochemical structure；
(4) functionalization is carried out to the hydrophilic working region of circle of detection zone, gold nano is grown by seed solution growth method first
Particle, specific growth step are as follows: 80 mL secondary waters are poured into three-necked flask be heated to 90 DEG C first, and 800 μ L matter are added
The chlorauric acid solution that score is 1% is measured, continues to be heated to 96 DEG C of 1 min of holding, being eventually adding 2.8 mL mass fractions is 1%
Sodium citrate continues to heat 15 min, and natural cooling obtains gold seeds solution, and the gold seeds solution for taking 60 μ L to obtain is added dropwise
Working region, standing are dried, and in triplicate, are rinsed 3 times with secondary water, will are then 2 μM of H1 chain by 50 μ L concentration, determine
Justice is chain 2, and base sequence is added drop-wise to the working region of gold nanoparticle modification as shown in nucleotides sequence list, and in room temperature
18 h of lower incubation；Later, the electrode of preparation is washed with distilled water, and with 50 μ L, concentration is the 6- sulfydryl -1- of 1.0 mM
Hexanol closes 1 h, to remove non-specific adsorption；Then, by the miRNA- of the working region modified and 50 μ L various concentrations
155 chains are defined as chain 3, and base sequence is incubated for as shown in nucleotides sequence list；It is immediately after 2 μM by 50 μ L concentration
H2 chain is defined as chain 4, and base sequence is added drop-wise to the surface of the working region of modification, then exists as shown in nucleotides sequence list
2 h are incubated under 37 °C；Then, by the S1@CuCo-CeO of 50 μ L2Solution is introduced into modified paper base working electrode, and use is ultrapure
After water rinses, the electrode of modification is incubated for 2 h under 4 °C；Later, by the electrode ultrapure water of modified to remove not
In conjunction with chain structure；
(5) it is modified in reaction zone circle hydrophilic region, specific steps are as follows: 10 μ L pH are added in round hydrophilic region
It is 7.0, concentration is the phosphate buffered saline solution of 0.1M；Then the H for being 5 mM by 200 μ L concentration2O2Solution is added drop-wise to circle
Hydrophilic region, then in 3,3', 5, the 5'- tetramethyl benzidine developing solutions that the pre-buried 20 μ L concentration of color development area is 20 mM；
(6) paper chip is simply folded, completes the detection to miRNA-155, the specific steps are as follows: pass through folding first
It will test region, reference electrode and to electrode zone, hollow region and color development area overlapping, auxiliary area and cleaning area difference
The two sides up and down of color development area are placed in, after the modification layer by layer for completing electrode, working region is cleaned, detection zone and electrode are made
Label overlapping, is fixed, pattern such as attached drawing 2 is connect with electrochemical workstation, and 60 μ L are contained 5.0 mM [Fe with clip
(CN)6] 3-/4 -PH is 7.0, and concentration is that the phosphate buffered saline solution of 0.1M is added drop-wise to detection zone, is measured and records；
(7) carry out the color developing detection of miRNA-155, the specific steps are as follows: after the completion of electric signal measurement process, by assisted tag and
Cleaning label is taken out, hydrogen peroxide and signal probe flow through hollow region and with 3,3', 5, the 5'- tetramethyls that are embedded in color development area
Base benzidine developing solution reaction, to realize Visual retrieval.
2. a kind of method of paper base bimodulus electrochemical sensor detection adenosine triphyosphate according to claim 1,
It is characterized in that cleaning region is half hydrophilic half hydrophobic, having a size of the mm of 12 mm × 12, working electrode area diameter is 10 mm, modification
Regional diameter is 8 mm, and hollow region label is the square structure of the mm of 30 mm × 30, reference electrode and to electrode print area
Domain diameter is 10 mm, and color development area diameter is 10 mm, and for assisted tag having a size of 15mm × 35mm, cleaning label is upper bottom 15
Mm, go to the bottom 26 mm, and a height of 65 mm's is trapezoidal.
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| CN111732121A (en) * | 2020-07-13 | 2020-10-02 | 济南大学 | CuInS2@CuCo-CeO2Composite nano probe and preparation method thereof |
| CN112547094A (en) * | 2021-01-04 | 2021-03-26 | 济南大学 | Preparation method of palladium in-situ modified copper-cobalt doped cerium dioxide nanospheres |
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