CN106353287A - Preparation of hydrogen peroxide detection colorimetry/fluorescent paper chip in cancer cell - Google Patents
Preparation of hydrogen peroxide detection colorimetry/fluorescent paper chip in cancer cell Download PDFInfo
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- CN106353287A CN106353287A CN201610721320.5A CN201610721320A CN106353287A CN 106353287 A CN106353287 A CN 106353287A CN 201610721320 A CN201610721320 A CN 201610721320A CN 106353287 A CN106353287 A CN 106353287A
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- paper chip
- hydrogen peroxide
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- cancerous cell
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a preparation method of a colorimetry/fluorescent paper chip for detecting hydrogen peroxide in a cancer cell. The method comprises the following steps: using a wax printing technology to prepare a hydrophobic area, a hydrophillic area and a hollow passageway on the paper chip, and cutting the hollow passageway through a laser cutting machine; growing cerium dioxide on a working area 2, wherein cerium dioxide with the enzyme-like property become saffron yellow in the presence of hydrogen peroxide, thus realizing visual colorimetric detection. Because hydrogen peroxide can replace DNA absorbed on cerium dioxide, high sensitive fluoremetry is realized through the 'off-on' of a fluorescence signal.
Description
Technical field
The present invention relates to low cost, be easy to carry about with one and visual cancerous cell hydrogen peroxide technical field of analysis and detection,
In particular it is a kind of preparation of the colorimetric/fluorescence paper chip that can be used in the detection of cancerous cell hydrogen peroxide.
Background technology
Hydrogen peroxide, is best one kind of stability in reactive oxygen specieses, and it is important intracellular signal molecule, main regulation
Dna damages, and protein synthesis and cell wither.Under low concentration, hydrogen peroxide can as the intracellular second message,second messenger of generally existing
Promote the propagation of cell, differentiation and migration with activation signal bridge, cell is played with regulation protective effect.However, working as hydrogen peroxide
Concentration overexpression when, the modification of dna base will be caused, such as the oxidation of purine and pyrimidine and chain break etc., thus drawing
The variation playing cell even produces canceration.Therefore intracellular hydrogen peroxide is highly sensitive, high credibility detection is particularly important.
Except some traditional methods, create some new method for sensing, nano enzyme recently in terms of nano enzyme research
It is the nanoparticle with class enzymatic property, and nano cerium is a class with fine class enzymatic activity.When nano cerium and hydrogen peroxide
During mixing, its color is become for crocus by white.Therefore, the direct detection of the hydrogen peroxide based on color change is reported
Road.However, colorimetric bio sensor be merely able to measured object is carried out intuitively, visualization measure, because its sensitivity is low, Zhi Nengjin
Row sxemiquantitative or qualitative detection.And fluoroscopic examination, as a kind of full-fledged detection method, because of its sensitivity height, operates letter
Single, single-minded the advantages of, is widely used in biological detection and clinical medicine.Hydrogen peroxide can substitute absorption on nano cerium surface
Dna, thus producing " on/off " of fluorescence signal.The combination of two methods do not only reached highly sensitive, intuitively detect, also
The credibility making testing result is greatly improved.
And micro-fluidic paper chip, because of its low consumption, portable, the features such as controlled, it is widely used in various bio-sensings
In device.And hollow channel paper chip is because, the problems such as which obviating sample sample volatilization is serious in the channel, circulation is slow, causing
Vast researcher is more and more paid close attention to.Hollow channel makes fluorescence and the method for colorimetric effectively be combined together, and not only solves
Sensitivity and the problem of intuitive detection, and be expected to be widely used in scarcity of resources or remote districts.
Content of the invention
It is an object of the invention to provide a kind of length has the micro-fluidic paper chip of the hollow channel of ceria, by colorimetric/glimmering
The method of light detection, thus realize quick, the sensitive and Visual retrieval of low content hydrogen peroxide in cancerous cell.
In order to solve above-mentioned technical problem, the present invention is realized by following measures: a kind of new hollow channel
The preparation of colorimetric/fluorescence paper chip, is characterized in that comprising the following steps:
(1) the hydrophobic wax print area of fluorescence/colorimetric paper chip as shown in Figure 1 and hydrophilic working area are designed on computers
Domain；
(2) pass through wax printer and the paper chip of design in step (1) printed upper hydrophobic pattern, subsequently by the paper chip obtaining in
Heat 1-2 min under 120-150 c, so that wax is melted, form hydrophobic layer；
(3) utilize laser cutting machine, the paper chip obtaining is cut, gray area is cut away in step (2), prepare hole
Hole；
(4) ceria is grown on the hydrophilic region 1 of gained paper chip in step (3)；
(5) dna, aptamers chain and cancerous cell that the fluorescent material of good biocompatibility is modified are fixed to step (4) gained
In paper chip, subsequently block avtive spot with bovine serum albumin；
(6) hide storing solution on the paper chip hydrophilic region 2 of step (5) gained；
(7) paper chip of step (6) gained is folded in the direction of arrows, carry out visualizing colorimetric detection；It is subsequently placed into fluorescence to set
In standby, carry out accurate fluoremetry under certain excitation wavelength and launch wavelength.
The concrete size of the hollow channel paper chip designed by the present invention as shown in Figure 2, the hydrophilic area of right side paper chip
Domain 1 and a diameter of 6 mm in sample application zone, a diameter of 4 mm of hydrophilic region 2, left side paper chip gray area is and right side paper chip
The completely corresponding hole of hydrophilic region, connect two circular cavities be a length of 4 mm, the hydrophilic channel of a width of 2 mm.
Ceria is grown, concrete steps are such as on the hydrophilic region 1 of the paper chip of gained in step (3) of the present invention
Under:
After the sodium borohydride solution of the chloroplatinic acid of 20.0 mm of same volume and 10.0 mm is mixed, rapid absorption 20.0 μ l
It is added drop-wise to and grows 10 min under the working region of paper chip, room temperature, use secondary water cleaning down, be dried at room temperature for；Next
0.374 g cerous nitrate, 0.09 g hexamethylenetetramine and 0.4 g Polyvinylpyrrolidone are dissolved in 16 ml distilled water
In, mix, mixture is transferred in 50 ml autoclaves, afterwards, the paper chip that growth is had pt nanoparticle layers immerses high pressure
In kettle, and keep 100 min under 180 c, be then cooled to room temperature, after paper chip is taken out, thorough with ethanol and secondary water
Cleaning, last 60 c vacuum drying.
The fluorescent material of good biocompatibility of the present invention is: quantum dot: graphene quantum dot, the Graphene of doping
Quantum dot, carbon point, N doping, nitrogen sulfur doping carbon point；Metal cluster: golden cluster, silver-colored cluster, copper cluster, gold silver cluster.
The storing solution hidden on paper chip hydrophilic region 2 of the present invention is the corresponding stimulus object of 10 l cancerous cell
Vascular endothelial cell growth factor phorbol exters.
The determination step of sample of the present invention, the paper chip of step (6) gained is folded in the direction of arrows, carries out sample
The mensure of product, draws the standard curve of gray scale and cancerous cell concentration, realizes visualization colorimetric determination；It is subsequently placed into fluorescence equipment
In, carry out the mensure of sample under certain excitation wavelength and launch wavelength, draw the standard of fluorescence intensity and cancerous cell concentration
Curve, realizes accurate fluoremetry.
Beneficial effects of the present invention:
(1) on paper growth ceria it is achieved that the combination of colorimetric/fluorescent dual module formula, it is achieved thereby that low content in cancerous cell
Sensitive, the quick and Visual retrieval of hydrogen peroxide.
(2) pass through fine to design hollow channel paper chip, it is to avoid the Long Term Contact of stimulus object and cancerous cell.
Brief description
The hydrophobic wax print pattern of Fig. 1: paper chip；
The size of Fig. 2: paper chip and folding mode.
Specific embodiment
Embodiment 1
The preparation of mcf-7 intracellular hydrogen peroxide detection colorimetric/fluorescence paper chip:
(1) the hydrophobic wax print area of colorimetric/fluorescence paper chip as shown in Figure 1 and hydrophilic working area are designed on computers
A diameter of 6 mm in domain, the hydrophilic region 1 of right side paper chip and sample application zone, a diameter of 4 mm of hydrophilic region 2, left side paper chip ash
Zone domain is hole completely corresponding with the hydrophilic region of right side paper chip, and that connect two circular cavities is a length of 4 mm, a width of 2
The hydrophilic channel of mm；
(2) pass through wax printer and the paper chip of design in step (1) printed upper hydrophobic pattern, subsequently by the paper chip obtaining in
Heat 1-2 min under 120-150 c, so that wax is melted, form hydrophobic layer；
(3) utilize laser cutting machine, the paper chip obtaining is cut, gray area is cut away in step (2), prepare hole
Hole；
(4) in step (3) on the hydrophilic region 1 of gained paper chip grow ceria, also will same volume 20.0 mm
Chloroplatinic acid and 10.0 mm sodium borohydride solution mixing after, rapid draw the working region that 20.0 μ l are added drop-wise to paper chip,
Grow 10 min under room temperature, use secondary water cleaning down, be dried at room temperature for；Next by 0.374 g cerous nitrate, 0.09 g
Hexamethylenetetramine and 0.4 g Polyvinylpyrrolidone are dissolved in 16 ml distilled water, mix, mixture is transferred to 50
In ml autoclave, afterwards, paper chip growth being had pt nanoparticle layers immerses in autoclave, and keeps 100 under 180 c
Min, is then cooled to room temperature, after paper chip is taken out, with ethanol and secondary water thoroughly cleaning, last 60 c vacuum drying；
(5) dna of the modified by graphene quantum dot of good biocompatibility, aptamers chain and mcf-7 cell are fixed to step (4)
In the paper chip of gained, subsequently block avtive spot with bovine serum albumin；
(6) the corresponding stimulus object blood vessel endothelium hiding 10 l cancerous cell on the paper chip hydrophilic region 2 of step (5) gained is thin
Intracellular growth factor phorbol exters；
(7) paper chip of step (6) gained is folded in the direction of arrows, carry out the mensure of sample, draw gray scale dense with cancerous cell
The standard curve of degree, realizes visualization colorimetric determination；It is subsequently placed in fluorescence equipment, in certain excitation wavelength and launch wavelength
Under carry out the mensure of sample, draw the standard curve of fluorescence intensity and cancerous cell concentration, realize accurate fluoremetry.
Embodiment 2
Preparation process, with example 1, is a difference in that: cancerous cell used is a549 cell, and fluorescent material used is carbon point.
sequence listing
<110>University Of Ji'nan
<120>cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip
<130> 2016
<160> 2
<170> patentin version 3.3
<210> 1
<211> 6
<212> dna
<213>synthetic
<400> 1
aaaaaa 6
<210> 2
<211> 36
<212> dna
<213>synthetic
<400> 2
gcagttgatc ctttggatac cctggttttt tttttt 36
Claims (6)
1. cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, it is characterized in that comprising the following steps:
1.1 design the hydrophobic wax print area of colorimetric/fluorescence paper chip and hydrophilic working region on computers；
1.2 pass through wax printer prints upper hydrophobic pattern by the paper chip of design in step 1.1, subsequently by the paper chip obtaining in
Heat 1-2 min under 120-150 c, so that wax is melted, form hydrophobic layer；
1.3 utilize laser cutting machine, the paper chip obtaining is cut, gray area is cut away in step 1.2, prepare hole
Hole；
Ceria is grown on 1.4 hydrophilic regions 1 of gained paper chip in step 1.3；
1.5 dna modifying the fluorescent material of good biocompatibility, aptamers chain and cancerous cell are fixed to step 1.4 gained
In paper chip, subsequently block avtive spot with bovine serum albumin；
1.6 hide storing solution on the paper chip hydrophilic region 2 of step 1.5 gained；
The paper chip of step 1.6 gained is folded by 1.7 in the direction of arrows, carries out visualizing colorimetric detection；It is subsequently placed into fluorescence to set
In standby, carry out accurate fluoremetry under certain excitation wavelength and launch wavelength.
2. according to claims 1, cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, and its feature exists
In, the hydrophilic region 1 of right side paper chip and a diameter of 6 mm in sample application zone, a diameter of 4 mm of hydrophilic region 2, left side paper chip ash
Zone domain is hole completely corresponding with the hydrophilic region of right side paper chip, and that connect two circular cavities is a length of 4 mm, a width of 2
The hydrophilic channel of mm.
3. according to claims 1, cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, and its feature exists
In on the hydrophilic region 1 of gained paper chip in step 1.3, growth ceria, specifically comprises the following steps that
After the sodium borohydride solution of the chloroplatinic acid of 20.0 mm of same volume and 10.0 mm is mixed, rapid absorption 20.0 μ l
It is added drop-wise to and grows 10 min under the working region of paper chip, room temperature, use secondary water cleaning down, be dried at room temperature for；Next
0.374 g cerous nitrate, 0.09 g hexamethylenetetramine and 0.4 g Polyvinylpyrrolidone are dissolved in 16 ml distilled water
In, mix, mixture is transferred in 50 ml autoclaves, afterwards, the paper chip that growth is had pt nanoparticle layers immerses high pressure
In kettle, and keep 100 min under 180 c, be then cooled to room temperature, after paper chip is taken out, thorough with ethanol and secondary water
Cleaning, last 60 c vacuum drying.
4. according to claims 1, cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, and its feature exists
In the fluorescent material of the good biocompatibility described in step 1.5 is: quantum dot: graphene quantum dot, the Graphene amount of doping
Sub-, carbon point, N doping, nitrogen sulfur doping carbon point；Metal cluster: golden cluster, silver-colored cluster, copper cluster, gold silver cluster.
5. according to claims 1, cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, and its feature exists
In the storing solution hidden on paper chip hydrophilic region 2 is the corresponding stimulus object vascular endothelial cell growth factor of 10 l cancerous cell
Phorbol exters.
6. according to claims 1, cancerous cell hydrogen peroxide detects the preparation of colorimetric/fluorescence paper chip, and its feature exists
In the paper chip of step 1.6 gained being folded in the direction of arrows, carries out the mensure of sample, draw gray scale and cancerous cell concentration
Standard curve, realizes visualization colorimetric determination；It is subsequently placed in fluorescence equipment, enter under certain excitation wavelength and launch wavelength
The mensure of row sample, draws the standard curve of fluorescence intensity and cancerous cell concentration, realizes accurate fluoremetry.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107356588A (en) * | 2017-06-30 | 2017-11-17 | 济南大学 | The superoxide radical and hydrogen peroxide paper substrate detection method of a kind of simple and sensitive |
| CN109628557A (en) * | 2019-01-07 | 2019-04-16 | 济南大学 | Dual signal enhances application of the paper base biosensor in miRNA detection |
| CN110205236A (en) * | 2019-06-12 | 2019-09-06 | 中国检验检疫科学研究院 | A kind of paper micro-fluidic chip quickly detecting nucleic acid based on RPA technology |
| CN110411990A (en) * | 2018-04-27 | 2019-11-05 | 中国科学院福建物质结构研究所 | A method of hydrogen peroxide and related objective object are detected based on nano-probe |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107356588A (en) * | 2017-06-30 | 2017-11-17 | 济南大学 | The superoxide radical and hydrogen peroxide paper substrate detection method of a kind of simple and sensitive |
| CN110411990A (en) * | 2018-04-27 | 2019-11-05 | 中国科学院福建物质结构研究所 | A method of hydrogen peroxide and related objective object are detected based on nano-probe |
| CN110411990B (en) * | 2018-04-27 | 2020-11-20 | 中国科学院福建物质结构研究所 | Method for detecting hydrogen peroxide and related target object based on nano probe |
| CN109628557A (en) * | 2019-01-07 | 2019-04-16 | 济南大学 | Dual signal enhances application of the paper base biosensor in miRNA detection |
| CN110205236A (en) * | 2019-06-12 | 2019-09-06 | 中国检验检疫科学研究院 | A kind of paper micro-fluidic chip quickly detecting nucleic acid based on RPA technology |
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