CN110470719A - A kind of preparation method of portable thermosensitive paper base bio-sensing chip - Google Patents
A kind of preparation method of portable thermosensitive paper base bio-sensing chip Download PDFInfo
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- CN110470719A CN110470719A CN201910920716.6A CN201910920716A CN110470719A CN 110470719 A CN110470719 A CN 110470719A CN 201910920716 A CN201910920716 A CN 201910920716A CN 110470719 A CN110470719 A CN 110470719A
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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
- G01N27/28—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
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- 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
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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
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Abstract
The invention discloses a kind of preparation methods of portable thermosensitive paper base bio-sensing chip, comprising the following steps: prepares paper electrode-synthesis MnO of polyphenyl amino-functionalization using electro-deposition techniques using chromatographic paper as paper substrates-2The composite heat sensitive material of nanoparticle and Ag nanoparticle-building temperature-sensitive paper base bio-sensing chip.The method have the characteristics that selecting the paper electrode of polyphenyl amino-functionalization as sensor base, MnO is used in combination2The composite heat sensitive material of nanoparticle and Ag nanoparticle, realizes the important breakthrough that portable, the highly sensitive detection of target antigen can be completed without large-scale instrument and equipment, and the research and development for novel sensing chip provide new direction.
Description
Technical field
The present invention relates to a kind of preparation methods of paper base bio-sensing chip, more specifically a kind of to have temperature-sensitive response
Portable paper base bio-sensing chip carcinomebryonic antigen detection in application.
Background technique
In recent years, the people not only propose requirements at the higher level to material and cultural life, while tracking self health status need to
Ask continuous improvement.However, high-incidence state one of of the China as cancer, newly-increased cases of cancer and cancer mortality case are in every year
Therefore the global umber one, compatriots' health status are also seriously threatened.Therefore, the early diagnosis of cancer becomes research hotspot instantly
One of problem.
The diagnosis of cancer at present, mainly by the influence equipment of hospital, such as ultrasound diagnosis, magnetic resonance imaging, endoscopy
It looks into etc. and to complete.These detection methods detection speed is fast, high sensitivity, however, being required to complete by large-scale instrument and equipment, examines
Higher cost is surveyed, the remote districts for being not suitable for falling behind relatively use.Therefore, develop a kind of simple, portable, highly sensitive swell
Tumor markers detection chip is the problem that researcher emphasis is captured.
Thermometer is common a kind of simple, cheap measuring tool in daily life, it is that the heat expansion based on liquid is cold
Made of contracting property, the temperature change for accurately judging and measuring solid, liquid, gas etc. can be used to.Paper base bio-sensing
Chip is that one kind is immobilized using paper substrates as biomolecule, identifies that substrate is borrowed using biological sensitive materials as recognition component
Energy converter appropriate is helped, realizes the portable device of object content detection.With paper base bio-sensing chip surface, temperature-sensitive is introduced
Effect material is realized tumor markers Sensitive Detection, is expected to as its people by simply measuring the variation of paper chip surface temperature
A kind of cancer detection platform of household formula is provided, provides important scientific basis for the early warning of cancer.
Summary of the invention
The technical problem to be solved by the present invention is to make a kind of portable thermosensitive paper base bio-sensing chip, to realize cancer embryo
The highly sensitive detection of antigen.
In order to solve the above-mentioned technical problem, the present invention is realized by following measures: a kind of portable thermosensitive paper base
The preparation method of bio-sensing chip, it is characterized in that the following steps are included:
(1) select chromatographic paper as paper substrates, using AI software, in designing close and distant water patterns, pattern such as 1 institute of attached drawing in paper substrates
Show, the scraps of paper are cut to A4 paper size；
(2) wax printer is utilized, the close and distant water patterns of paper substrates printout surface obtained by step (1), wherein black portions are hydrophobic
Area, white portion are hydrophilic areas；
The close and distant water patterns, it is characterized in that: hydrophobic pattern width is 9 mm, length is 12 mm, and hydrophilic pattern width is 6 mm, long
Degree is 9 mm；
(3) it by paper substrates obtained by step (2), is put in 120 °C of baking ovens, heats 2 min, melt paper substrates surface wax, in paper
Hydrophobic wall is formed between piece upper and lower surface；
(4) screen printing technique is used, the hydrophilic region in the paper substrates obtained by step (3) prints one layer of conductive carbon paste, preparation
Obtain carbon functionalization paper electrode；Using hand papercutter, carbon functionalization paper electrode is cut, being cut to width is 9 mm, length
For the carbon functionalization paper electrode of 12 mm；
(5) carbon functionalization paper electrode surface, electro-deposition polyaniline nano material obtained by step (4) prepare polyphenyl amino-functionalization
Paper electrode；
The preparation of the paper electrode of the polyphenyl amino-functionalization, comprising the following steps: with the resulting carbon functionalization paper electrode of step (4)
For working electrode, in the aniline mixed liquor that the sulfuric acid solution and concentration that concentration is 0.05 M are 0.1 M, under 0.8 V current potential,
3 min of electro-deposition at room temperature, then, using cyclic voltammetric technology, in -0.3 V to 1.0 V potential regions, with 0.15 V/s
Speed, the paper electrode of polyphenyl amino-functionalization is prepared in 60 s of electro-deposition polyaniline, finally, by the paper of polyphenyl amino-functionalization
Electrode is dried at room temperature；
(6) using the paper electrode of polyphenyl amino-functionalization prepared by step (5) as sensor base, temperature-sensitive paper base bio-sensing is constructed
Chip；
The building of the temperature-sensitive paper base bio-sensing chip, comprising the following steps: firstly, synthesis MnO2Nanoparticle and Ag nanometers
The composite heat sensitive material of particle, is defined as MnO2@Ag: weighing the potassium permanganate powder of 0.25 g, is dissolved in the water of 125 mL,
1 h is stirred at room temperature, and the oleic acid solutions of 2.5 mL are then added into above-mentioned solution, solution is uniformly mixed, react at room temperature
24 h, can be obtained MnO2Nanoparticle, the MnO that will be obtained2Nanoparticle centrifugation, successively cleans centrifugation with ethyl alcohol and secondary water
Clean MnO can be obtained in obtained precipitating2Nanoparticle, by MnO212 h are dried in vacuo under 60 °C of nanoparticle；Weigh 40
The MnO of mg2Nanoparticle is put in a round bottom flask, and 100 microlitres of 3- aminopropyl triethoxysilane is added into round-bottomed flask
And 20 mL toluene solution, round-bottomed flask is put in 120 °C of oil bath pans, 10 h of back flow reaction in nitrogen atmosphere, obtain
The MnO of amino functional2Nanoparticle, by the MnO of amino functional2Nanoparticle is cleaned three times with toluene solution, in 60 °C
Dry 12 h in vacuum oven；Then, the MnO of the amino functional of 20 mg is weighed2Nanoparticle is added to 100 mL circle
In the flask of bottom, the ethylene glycol solution of 15 mL is added into round-bottomed flask, solution is heated to 140 °C in the case where stirring energetically, and
Afterwards, the ethylene glycol that the polyvinylpyrrolidone of silver nitrate and 3.5 g that 15 mL contain 0.1 g is added into round-bottomed flask is molten
Liquid, meanwhile, reaction solution is warming up to 160 °C, reacts 50 min, completes MnO2The preparation of@Ag composite heat sensitive material；Secondly,
Prepare the MnO of signal antibody label2@Ag composite heat sensitive material: pipetting 20 microlitres of concentration is 20 μ g mL-1Signal antibody it is molten
Liquid is added to the MnO of 2 mL2In@Ag composite heat sensitive material, 24 h of concussion reaction, completes signal antibody label at room temperature
MnO2The preparation of@Ag composite heat sensitive material；And then, temperature-sensitive paper base bio-sensing chip is prepared: in the paper of polyphenyl amino-functionalization
Electrode surface, the glutaraldehyde solution that the volumetric concentration for being added dropwise 50 microlitres is 2.5%, reacts 40 min at room temperature, then uses secondary water
Paper electrode surface is rinsed, again dries paper electrode at room temperature, then, it is 20 μ g that 100 microlitres of concentration, which are added dropwise, to paper electrode surface
mL-1Coated antibody solution, react 2 h at room temperature, after secondary water rinses electrode surface, dry paper electrode at room temperature and then adopt
With 100 microlitres, the bovine serum albumin(BSA) that mass fraction is 1% blocks the remaining active site of electrode surface, then by 100 microlitres
The carcinomebryonic antigen of various concentration be added drop-wise to electrode surface, hatch 2 h at room temperature, subsequent secondary water rinses paper electrode surface, room
The lower drying of temperature, finally, pipetting the MnO of 100 microlitres of signal antibody label2@Ag composite heat sensitive material is added drop-wise to paper electrode surface,
After hatching 40 min at room temperature, paper electrode surface is rinsed with secondary water again, completes the building of temperature-sensitive paper base bio-sensing chip；
(7) paper electrode surface temperature obtained by determination step (6), completes the detection of carcinomebryonic antigen；
The measurement of the paper electrode surface temperature, it is characterized in that: being first 10% in the mass fraction that paper electrode surface is added 80 microlitres
Hydrogenperoxide steam generator, react 3 min after, thermometer probe is inserted into paper electrode surface solution, read and recording sheet electricity
The real time temperature of pole surface draws the response curve between carcinomebryonic antigen and temperature.
Beneficial effects of the present invention
(1) MnO is selected2@Ag can speed up hydrogen peroxide and decompose to give off heat as composite heat sensitive material, convenient for using temperature
Meter carries out portable quantitative analysis to target antigen.
(2) the temperature-sensitive paper base bio-sensing chip constructed by, can be not suitable for any large scale equipment, complete carcinomebryonic antigen
Sensitive Detection.
(3) biomolecule can greatly be increased in paper as sensor base using the paper electrode of polyphenyl amino-functionalization
Electrode surface load capacity improves detection sensitivity.
Detailed description of the invention
Fig. 1: paper base bottom surface hydrophobe pattern.
Specific embodiment
For a better understanding of the present invention, below by the embodiment content that the present invention is furture elucidated, but the present invention is not
It is limited only to the following examples.
Embodiment 1
A kind of preparation method of portable thermosensitive paper base bio-sensing chip, specific preparation process are as follows:
(1) it selects chromatographic paper as paper substrates, is cut to the scraps of paper in designing close and distant water patterns in paper substrates using AI software
A4 paper size；
(2) wax printer is utilized, the close and distant water patterns of paper substrates printout surface obtained by step (1), wherein black portions are hydrophobic
Area, white portion are hydrophilic areas；
The close and distant water patterns, it is characterized in that: hydrophobic pattern width is 9 mm, length is 12 mm, and hydrophilic pattern width is 6 mm, long
Degree is 9 mm；
(3) it by paper substrates obtained by step (2), is put in 120 °C of baking ovens, heats 2 min, melt paper substrates surface wax, in paper
Hydrophobic wall is formed between piece upper and lower surface；
(4) screen printing technique is used, the hydrophilic region in the paper substrates obtained by step (3) prints one layer of conductive carbon paste, preparation
Obtain carbon functionalization paper electrode；Using hand papercutter, carbon functionalization paper electrode is cut, being cut to width is 9 mm, length
For the carbon functionalization paper electrode of 12 mm；
(5) carbon functionalization paper electrode surface, electro-deposition polyaniline nano material obtained by step (4) prepare polyphenyl amino-functionalization
Paper electrode；
The preparation of the paper electrode of the polyphenyl amino-functionalization, comprising the following steps: with the resulting carbon functionalization paper electrode of step (4)
For working electrode, in the aniline mixed liquor that the sulfuric acid solution and concentration that concentration is 0.05 M are 0.1 M, under 0.8 V current potential,
3 min of electro-deposition at room temperature, then, using cyclic voltammetric technology, in -0.3 V to 1.0 V potential regions, with 0.15 V/s
Speed, the paper electrode of polyphenyl amino-functionalization is prepared in 60 s of electro-deposition polyaniline, finally, by the paper of polyphenyl amino-functionalization
Electrode is dried at room temperature；
(6) using the paper electrode of polyphenyl amino-functionalization prepared by step (5) as sensor base, temperature-sensitive paper base bio-sensing is constructed
Chip；
The building of the temperature-sensitive paper base bio-sensing chip, comprising the following steps: firstly, synthesis MnO2Nanoparticle and Ag nanometers
The composite heat sensitive material of particle, is defined as MnO2@Ag: weighing the potassium permanganate powder of 0.25 g, is dissolved in the water of 125 mL,
1 h is stirred at room temperature, and the oleic acid solutions of 2.5 mL are then added into above-mentioned solution, solution is uniformly mixed, react at room temperature
24 h, can be obtained MnO2Nanoparticle, the MnO that will be obtained2Nanoparticle centrifugation, successively cleans centrifugation with ethyl alcohol and secondary water
Clean MnO can be obtained in obtained precipitating2Nanoparticle, by MnO212 h are dried in vacuo under 60 °C of nanoparticle；Weigh 40
The MnO of mg2Nanoparticle is put in a round bottom flask, and 100 microlitres of 3- aminopropyl triethoxysilane is added into round-bottomed flask
And 20 mL toluene solution, round-bottomed flask is put in 120 °C of oil bath pans, 10 h of back flow reaction in nitrogen atmosphere, obtain
The MnO of amino functional2Nanoparticle, by the MnO of amino functional2Nanoparticle is cleaned three times with toluene solution, in 60 °C
Dry 12 h in vacuum oven；Then, the MnO of the amino functional of 20 mg is weighed2Nanoparticle is added to 100 mL circle
In the flask of bottom, the ethylene glycol solution of 15 mL is added into round-bottomed flask, solution is heated to 140 °C in the case where stirring energetically, and
Afterwards, the ethylene glycol that the polyvinylpyrrolidone of silver nitrate and 3.5 g that 15 mL contain 0.1 g is added into round-bottomed flask is molten
Liquid, meanwhile, reaction solution is warming up to 160 °C, reacts 50 min, completes MnO2The preparation of@Ag composite heat sensitive material；Secondly,
Prepare the MnO of signal antibody label2@Ag composite heat sensitive material: pipetting 20 microlitres of concentration is 20 μ g mL-1Signal antibody it is molten
Liquid is added to the MnO of 2 mL2In@Ag composite heat sensitive material, 24 h of concussion reaction, completes signal antibody label at room temperature
MnO2The preparation of@Ag composite heat sensitive material；And then, temperature-sensitive paper base bio-sensing chip is prepared: in the paper of polyphenyl amino-functionalization
Electrode surface, the glutaraldehyde solution that the volumetric concentration for being added dropwise 50 microlitres is 2.5%, reacts 40 min at room temperature, then uses secondary water
Paper electrode surface is rinsed, again dries paper electrode at room temperature, then, it is 20 μ g that 100 microlitres of concentration, which are added dropwise, to paper electrode surface
mL-1Coated antibody solution, react 2 h at room temperature, after secondary water rinses electrode surface, dry paper electrode at room temperature and then adopt
With 100 microlitres, the bovine serum albumin(BSA) that mass fraction is 1% blocks the remaining active site of electrode surface, then by 100 microlitres
The carcinomebryonic antigen of various concentration be added drop-wise to electrode surface, hatch 2 h at room temperature, subsequent secondary water rinses paper electrode surface, room
The lower drying of temperature, finally, pipetting the MnO of 100 microlitres of signal antibody label2@Ag composite heat sensitive material is added drop-wise to paper electrode surface,
After hatching 40 min at room temperature, paper electrode surface is rinsed with secondary water again, completes the building of temperature-sensitive paper base bio-sensing chip；
(7) paper electrode surface temperature obtained by determination step (6), completes the detection of carcinomebryonic antigen；
The measurement of the paper electrode surface temperature, it is characterized in that: being first 10% in the mass fraction that paper electrode surface is added 80 microlitres
Hydrogenperoxide steam generator, react 3 min after, thermometer probe is inserted into paper electrode surface solution, read and recording sheet electricity
The real time temperature of pole surface draws the response curve between carcinomebryonic antigen and temperature.
Claims (1)
1. a kind of preparation method of portable thermosensitive paper base bio-sensing chip, it is characterised in that the preparation step of this method is such as
Under:
(1) it selects chromatographic paper as paper substrates, is cut to the scraps of paper in designing close and distant water patterns in paper substrates using AI software
A4 paper size；
(2) wax printer is utilized, the close and distant water patterns of paper substrates printout surface obtained by step (1), wherein black portions are hydrophobic
Area, white portion are hydrophilic areas；
The close and distant water patterns, it is characterized in that: hydrophobic pattern width is 9 mm, length is 12 mm, and hydrophilic pattern width is 6 mm, long
Degree is 9 mm；
(3) it by paper substrates obtained by step (2), is put in 120 °C of baking ovens, heats 2 min, melt paper substrates surface wax, in paper
Hydrophobic wall is formed between piece upper and lower surface；
(4) screen printing technique is used, the hydrophilic region in the paper substrates obtained by step (3) prints one layer of conductive carbon paste, preparation
Obtain carbon functionalization paper electrode；Using hand papercutter, carbon functionalization paper electrode is cut, being cut to width is 9 mm, length
For the carbon functionalization paper electrode of 12 mm；
(5) carbon functionalization paper electrode surface, electro-deposition polyaniline nano material obtained by step (4) prepare polyphenyl amino-functionalization
Paper electrode；
The preparation of the paper electrode of the polyphenyl amino-functionalization, comprising the following steps: with the resulting carbon functionalization paper electrode of step (4)
For working electrode, in the aniline mixed liquor that the sulfuric acid solution and concentration that concentration is 0.05 M are 0.1 M, under 0.8 V current potential,
3 min of electro-deposition at room temperature, then, using cyclic voltammetric technology, in -0.3 V to 1.0 V potential regions, with 0.15 V/s
Speed, the paper electrode of polyphenyl amino-functionalization is prepared in 60 s of electro-deposition polyaniline, finally, by the paper of polyphenyl amino-functionalization
Electrode is dried at room temperature；
(6) using the paper electrode of polyphenyl amino-functionalization prepared by step (5) as sensor base, temperature-sensitive paper base bio-sensing is constructed
Chip；
The building of the temperature-sensitive paper base bio-sensing chip, comprising the following steps: firstly, synthesis MnO2Nanoparticle and Ag nanometers
The composite heat sensitive material of particle, is defined as MnO2@Ag: weighing the potassium permanganate powder of 0.25 g, is dissolved in the water of 125 mL,
1 h is stirred at room temperature, and the oleic acid solutions of 2.5 mL are then added into above-mentioned solution, solution is uniformly mixed, react at room temperature
24 h, can be obtained MnO2Nanoparticle, the MnO that will be obtained2Nanoparticle centrifugation, successively cleans centrifugation with ethyl alcohol and secondary water
Clean MnO can be obtained in obtained precipitating2Nanoparticle, by MnO212 h are dried in vacuo under 60 °C of nanoparticle；Weigh 40
The MnO of mg2Nanoparticle is put in a round bottom flask, and 100 microlitres of 3- aminopropyl triethoxysilane is added into round-bottomed flask
And 20 mL toluene solution, round-bottomed flask is put in 120 °C of oil bath pans, 10 h of back flow reaction in nitrogen atmosphere, obtain
The MnO of amino functional2Nanoparticle, by the MnO of amino functional2Nanoparticle is cleaned three times with toluene solution, in 60 °C
Dry 12 h in vacuum oven；Then, the MnO of the amino functional of 20 mg is weighed2Nanoparticle is added to 100 mL circle
In the flask of bottom, the ethylene glycol solution of 15 mL is added into round-bottomed flask, solution is heated to 140 °C in the case where stirring energetically, and
Afterwards, the ethylene glycol that the polyvinylpyrrolidone of silver nitrate and 3.5 g that 15 mL contain 0.1 g is added into round-bottomed flask is molten
Liquid, meanwhile, reaction solution is warming up to 160 °C, reacts 50 min, completes MnO2The preparation of@Ag composite heat sensitive material；Secondly,
Prepare the MnO of signal antibody label2@Ag composite heat sensitive material: pipetting 20 microlitres of concentration is 20 μ g mL-1Signal antibody it is molten
Liquid is added to the MnO of 2 mL2In@Ag composite heat sensitive material, 24 h of concussion reaction, completes signal antibody label at room temperature
MnO2The preparation of@Ag composite heat sensitive material；And then, temperature-sensitive paper base bio-sensing chip is prepared: in the paper of polyphenyl amino-functionalization
Electrode surface, the glutaraldehyde solution that the volumetric concentration for being added dropwise 50 microlitres is 2.5%, reacts 40 min at room temperature, then uses secondary water
Paper electrode surface is rinsed, again dries paper electrode at room temperature, then, it is 20 μ g that 100 microlitres of concentration, which are added dropwise, to paper electrode surface
mL-1Coated antibody solution, react 2 h at room temperature, after secondary water rinses electrode surface, dry paper electrode at room temperature and then adopt
With 100 microlitres, the bovine serum albumin(BSA) that mass fraction is 1% blocks the remaining active site of electrode surface, then by 100 microlitres
The carcinomebryonic antigen of various concentration be added drop-wise to electrode surface, hatch 2 h at room temperature, subsequent secondary water rinses paper electrode surface, room
The lower drying of temperature, finally, pipetting the MnO of 100 microlitres of signal antibody label2@Ag composite heat sensitive material is added drop-wise to paper electrode surface,
After hatching 40 min at room temperature, paper electrode surface is rinsed with secondary water again, completes the building of temperature-sensitive paper base bio-sensing chip；
(7) paper electrode surface temperature obtained by determination step (6), completes the detection of carcinomebryonic antigen；
The measurement of the paper electrode surface temperature, it is characterized in that: being first 10% in the mass fraction that paper electrode surface is added 80 microlitres
Hydrogenperoxide steam generator, react 3 min after, thermometer probe is inserted into paper electrode surface solution, read and recording sheet electricity
The real time temperature of pole surface draws the response curve between carcinomebryonic antigen and temperature.
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Application publication date: 20191119 |