CN106526182B - A kind of structure of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection - Google Patents
A kind of structure of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection Download PDFInfo
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- CN106526182B CN106526182B CN201610966066.5A CN201610966066A CN106526182B CN 106526182 B CN106526182 B CN 106526182B CN 201610966066 A CN201610966066 A CN 201610966066A CN 106526182 B CN106526182 B CN 106526182B
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- fibrin ferment
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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"
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses the structures of the paper substrate aptamer fluorescent optical sensor detected for fibrin ferment.The hydrophobic wax print pattern of the micro-fluidic paper chip of Adobe illustrator CS4 Software for Design is utilized on computers;Golden platinum bimetallic is grown in paper chip working region reduces background fluorescence signal;Modify aptamers 1 on gold-palladium bimetallic be used for specific recognition object;By specific binding of the aptamers 2 to fibrin ferment, fluorescent marker is introduced in working region, and then realizes the detection to fibrin ferment by luminoscope.
Description
Technical field
The present invention relates to refill chip technology, detection technique of fluorescences, are more specifically based on spy of the aptamers to object
Different in nature binding characteristic constructs a simple, sensitive paper substrate fluorescent optical sensor for fibrin ferment detection.
Background technology
Fibrin ferment is a kind of serine protease, in the diagnosis equimolecular of hemostasis, angiogenesis and growth and metastasis of tumours
Important role in biology.Since it is in the importance of biology, the detection and analysis of fibrin ferment include various technologies such as
Colorimetric, Surface enhanced Raman spectroscopy and surface plasma body resonant vibration etc..It is various based on electrochemistry, it is photic electricity, electrochemical luminescence and
The aptamer sensor of other analyses is widely used to the detection of various biomolecule.Compared with traditional molecular recognition system,
Oligonucleotide aptamer is simple because synthesizing, convenient for mark, with very strong competing the advantages that stronger stability, wide applicability
Strive power.
In order to improve the sensitivity of fibrin ferment detection, noble metal composite nano materials are because of its large specific surface area, bio-compatible
Property the advantages such as good very big concern is caused in science and technology field.Paper chip with this noble metal composite nano materials,
The specific surface area of paper chip is not only increased, and effectively reduces the background fluorescence of paper, thus is widely used in various types
Biosensor in.In recent years, quantum dot because its preparation process it is simple, good biocompatibility, excitation spectrum is wider, symmetry
High emission spectrum, has many advantages, such as bleach-resistant.It is made to be widely used as biological labled material.It is replaced with quantum dot organic
Fluorescence labeling material can greatly reduce cost, improve detection ground sensitivity.Select biocompatibility very well porous simultaneously
For zinc oxide as carrier, macroporous structure substantially increases the load capacity of quantum dot in the system, so as to further improve detection spirit
Sensitivity.
Since two thousand seven, paper substrate sensor is because of its low cost, bigger serface, foldable, processing, shaping, biofacies
The features such as capacitive is good is applied to the research of clinical diagnosis.We are converted into preferable conformation using the foldability of paper, and
And the capillarity of paper fiber is utilized, the device that the introducing that water can avoid pump with neous flow is played truly is simple
Change.By the rational design and cutting to paper chip, with reference to aptamers and fibrin ferment specific binding construct one it is simple,
Sensitive paper substrate fluorescent optical sensor.
The content of the invention
The purpose of the present invention is make full use of the excellent of the foldability of paper and capillarity and AuPt alloy nano particles
It is benign to prepare a kind of new paper substrate fluorescence analysis device, and the specific recognition function realization for combining aptamers is convenient and efficient
The highly sensitive detection fibrin ferment in ground.
In order to solve the above-mentioned technical problem, the present invention is realized by following measures:
(1)Paper chip hydrophobic wax print pattern as shown in Figure 1 is designed on computers;
(2)By step(1)The print pattern of middle design is printed upon the chromatographic paper for being cut to A4 sizes by wax printer
On, the A4 chromatographic papers with wax pattern are then heated into 2 min by being heated to the panel heater of 125oC, wax is made to melt simultaneously
The thickness of entire paper is impregnated with, forms hydrophobic wall;
(3)In step(2)In the obtained working region of chromatographic paper carry out functionalization, fitted after growing AuPt bimetallics
50 μ L fibrin ferments to be detected are added dropwise in the modification of ligand 1, pure with ox blood after being cleaned after 45 min of reaction with phosphate buffer solution
Albumen blocks nonactive site;
(4)In step(2)In the obtained hatching region of chromatographic paper drip the synthetic QDs ZnO fluorescent markers of 40 μ L and
1 μM of aptamers 2 of 60 μ L carry out the reaction of fluorescent marker and aptamers 2;
(5)Paper chip along fold line is folded, is connected with hairs of the fluorescent marker QDs@ZnO in paper fiber of aptamers 2
Spy is firmly lower to reach working region by fluid, is washed after reacting 30 min with phosphate buffer solution:
(6)By step(5)Middle paper chip workspace is put into fluorescence equipment, and fluorescence is carried out under the excitation wavelength of 340 nm
It measures, draws the concentration relationship of fluorescence intensity and fibrin ferment, realize the accurate detection to fibrin ferment.
The step of hydrophilic region functionalization of the present invention to paper chip is:Work(is carried out to the working region of chromatographic paper
Concretely comprise the following steps to energyization:100 μ L gold nanoparticles drop is measured in hydrophilic working region with liquid-transfering gun, then at room temperature
It spontaneously dries, is repeated 5 times, secondary water washing, it is dry at room temperature to stand;By 1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL
The quick mixing of 1wt% platinum acid chloride solutions, liquid-transfering gun measures 60 μ L mixed liquors and is added drop-wise to working region, then rapid with liquid-transfering gun
The 1wt% sodium citrate aqueous solutions for measuring 40 μ L Fresh drip to working region, are rinsed after reacting 30 min with secondary water, so
It is measured 100 μ L, 1 μM of aptamers 1 with liquid-transfering gun afterwards and is dripped and hatch 1 h in working region, be 7.4 phosphate buffer solutions with pH
Cleaning is three times afterwards with the bovine serum albumin solution block nonactive site of 1wt%.
The preparation process of QDs@ZnO of the present invention is:It weighs 0.3 g carbon blacks to be dissolved in 60 mL, 6 M nitric acid, surpass
Flow back 24 h after 2 h of sound under the conditions of 120 °C, is centrifuged after above-mentioned solution is dropped to room temperature and dry, product then is dissolved in two
It dialyses three days after secondary water, obtains graphene quantum dot;It weighs 3.5 g soluble starches to be dissolved in 80 mL boiling water, under 85 oC
6 mmol zinc nitrate hexahydrates are added in after stirring 5 min, are continued after pH value of solution is adjusted to 8-9 by the way that sodium hydroxide is added dropwise
30 min are heated, then the precipitation of acquisition is centrifuged, is washed and 2 h acquisition porous zinc blooms are roasted under 500 oC, weigh 0.6
The porous zinc bloom that g is obtained, which is dissolved in the p-Mercaptoaniline solution of 0.1 mM, stirs 4 h;It is synthetic that liquid-transfering gun measures 500 μ L
Quantum dot mixed with the zinc oxide of 2 mL functionalization after in 10 mgmL-11- (3- dimethylamino-propyls) -3- ethyls carbon two
Inferior amine salt hydrochlorate and 20 mgmL-1N- hydroxysuccinimides effect 30 min of lower reaction, centrifugation is dissolved in 3 mL after washing
In phosphate buffer solution.
Beneficial effects of the present invention
(1)The synthetic method of fluorescence probe is simple, good biocompatibility.
(2)Using body technique is adapted to, traditional antigen-antibody recognition methods is avoided.
(3)Method testing cost of the present invention is cheap, easy to operate, high sensitivity.
Attached drawing 1:The size and shape of paper chip.
Specific embodiment
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to following implementation.
Embodiment 1:Paper substrate aptamer fluorescent optical sensor detects for fibrin ferment, it is characterized in that comprising the following steps:
(1)Paper chip hydrophobic wax print pattern as shown in Figure 1 is designed on computers;
(2)By step(1)The print pattern of middle design is printed upon the chromatographic paper for being cut to A4 sizes by wax printer
On, the A4 chromatographic papers with wax pattern are then heated into 2 min by being heated to the panel heater of 125oC, wax is made to melt simultaneously
The thickness of entire paper is impregnated with, forms hydrophobic wall;
(3)To step(2)In the obtained working region of chromatographic paper carry out functionalization, measure 100 μ L gold with liquid-transfering gun
Nano-particle is dripped in hydrophilic working region, is then spontaneously dried, is repeated 5 times at room temperature, secondary water washing is dried at room temperature
It stands;By 1.0 mL 1wt% aqueous solution of chloraurate and the 2.5 quick mixings of mL 1wt% chloroplatinic acids, liquid-transfering gun measures 60 μ L and mixes
It closes drop and is added to working region, the 1wt% sodium citrate aqueous solutions for then measuring 40 μ L Fresh rapidly with liquid-transfering gun drip to
Working region is rinsed after reacting 30 min with secondary water, and then measuring 100 μ L, 1 μM of aptamers 1 with liquid-transfering gun drips in work
1 h is hatched in region, is cleaned with pH for 7.4 phosphate buffer solutions and is used the bovine serum albumin solution block of 1wt% non-afterwards three times
Active site;
(4)To step(2)In the obtained hatching region of chromatographic paper operated:It weighs 0.3 g carbon blacks and is dissolved in 60 mL
In 6 M nitric acid, flow back 24 h after 2 h of ultrasound under the conditions of 120 °C, is centrifuged after above-mentioned solution is dropped to room temperature and dry, then
It dialyses three days after product is dissolved in secondary water, obtains graphene quantum dot;It weighs 3.5 g soluble starches and is dissolved in 80 mL boiling water
In, 6 mmol zinc nitrate hexahydrates are added in after 5 min are stirred under 85 oC, by the way that sodium hydroxide is added dropwise by pH value of solution tune
Continue to heat 30 min after to 8-9, then the precipitation of acquisition is centrifuged, is washed and the porous oxygen of 2 h acquisitions is roasted under 500 oC
Change zinc, the porous zinc bloom for weighing 0.6 g acquisitions is dissolved in the p-Mercaptoaniline solution of 0.1 mM and stirs 4 h;Liquid-transfering gun measures
Quantum dot synthetic 500 μ L mixed with the zinc oxide of 2 mL functionalization after in 10 mgmL-11- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides and 20 mgmL-1N- hydroxysuccinimides effect 30 min of lower reaction, centrifugation,
It is dissolved in after washing in 3 mL phosphate buffer solutions, measure the synthetic QDs@ZnO fluorescent markers of 40 μ L and 60 μ L 1 μM is suitable
Ligand 2 carries out the reaction of fluorescent marker and aptamers 2 in hatching region;
(5)Paper chip along fold line is folded, is connected with hairs of the fluorescent marker QDs@ZnO in paper fiber of aptamers 2
Spy is firmly lower to reach working region by fluid, is washed after reacting 30 min with phosphate buffer solution;
(6)By step(5)Middle paper chip workspace is put into fluorescence equipment, and fluorescence is carried out under the excitation wavelength of 340 nm
It measures, draws the concentration relationship of fluorescence intensity and fibrin ferment, realize the accurate detection to fibrin ferment.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>A kind of structure of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection
<130> 2016
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>It is artificial synthesized
<400> 1
ggttggtgtg gttgg 15
<210> 2
<211> 44
<212> DNA
<213>It is artificial synthesized
<400> 2
tttttttttt tttttagtcc gtggtagggc aggttggggt gact 44
Claims (3)
1. a kind of construction method of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection, it is characterized in that comprising the following steps:
1.1 design paper chip hydrophobic wax print pattern on computers, and paper chip hydrophobic wax print pattern is by two length of sides
The square composition of 20mm, two squares present side by side and the intermediate folding region there are 1mm wide, the upper left corner of left side square
It is the hydrophilic hatching region and working region of circle of a diameter of 8mm respectively with the lower right corner, the upper right corner and lower-left of right side square
Angle is the circular hole area of two a diameter of 8mm respectively, just be overlapped with left side border circular areas after being folded at folding region;
It has been cut to 1.2 the print pattern designed in step 1.1 is printed upon by wax printer on the chromatographic paper of A4 sizes, so
The A4 chromatographic papers with wax pattern are heated into 2 min by being heated to the panel heater of 125oC afterwards, melts wax and is impregnated with whole
The thickness of a paper forms hydrophobic wall, obtains for fluorimetric working region and for fluorescent marker and adaptation precursor reactant
Hatching region;
The working region of the chromatographic paper obtained in step 1.2 is carried out functionalization by 1.3, and aptamers are carried out after growing AuPt bimetallics
1 modification is added dropwise 50 μ L fibrin ferments to be detected, is cleaned after reacting 45 min with phosphate buffer solution, then pure with ox blood
Albumen blocks nonactive site;
Drip the synthetic QDs ZnO fluorescent markers and 60 of 40 μ L in the hatching region of 1.4 chromatographic papers obtained in step 1.2
1 μM of aptamers 2 of μ L carry out the reaction of fluorescent marker and aptamers 2;
1.5 fold paper chip along fold line, and the fluorescent marker QDs@ZnO for being connected with aptamers 2 are reached containing object
Working region is washed after reacting 30 min with phosphate buffer solution;
1.6 are put into paper chip working region in step 1.5 in fluorescence equipment, and fluorescence survey is carried out under the excitation wavelength of 340 nm
It is fixed, the concentration relationship of fluorescence intensity and fibrin ferment is drawn, realizes the accurate detection to fibrin ferment.
2. the construction method of a kind of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection according to claim 1, special
Sign is, the working region of the chromatographic paper obtained in step 1.2 is carried out functionalization described in step 1.3, and step is as follows:With
Liquid-transfering gun measures 100 μ L gold nanoparticles and drips in hydrophilic working region, then spontaneously dries, is repeated 5 times at room temperature, and two
Secondary water washing, it is dry at room temperature to stand;1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL 1wt% platinum acid chloride solutions are fast
Fast mixing, liquid-transfering gun measure 60 μ L mixed liquors and are added drop-wise to working region, then measure 40 μ L Fresh rapidly with liquid-transfering gun
1wt% sodium citrate aqueous solutions drip to working region, rinsed after reacting 30 min with secondary water, then measure 100 with liquid-transfering gun
1 μM of aptamers 1 of μ L are dripped hatches 1 h in working region, is that 7.4 phosphate buffer solutions are cleaned three times afterwards with 1wt%'s with pH
Bovine serum albumin solution blocks nonactive site.
3. the construction method of a kind of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection according to claim 1, special
Sign is that the preparation process of the QDs@ZnO described in step 1.4 is:It weighs 0.3 g carbon blacks and is dissolved in 60 mL, 6 M nitric acid
In, flow back 24 h after 2 h of ultrasound under the conditions of 120 °C, is centrifuged after above-mentioned solution is dropped to room temperature and dry, then by product
It dialyses three days after being dissolved in secondary water, obtains graphene quantum dot;It weighs 3.5 g soluble starches to be dissolved in 80 mL boiling water, 85
6 mmol zinc nitrate hexahydrates are added in after 5 min are stirred under oC, it is follow-up to be adjusted to 8-9 by sodium hydroxide being added dropwise by pH value of solution
Then the precipitation of acquisition is centrifuged, washed and 2 h acquisition porous zinc blooms are roasted under 500 oC, weighed by 30 min of continuous heating
The porous zinc bloom that 0.6 g is obtained, which is dissolved in the p-Mercaptoaniline solution of 0.1 mM, stirs 4 h;Liquid-transfering gun measures 500 μ L synthesis
Good quantum dot mixed with the zinc oxide of 2 mL functionalization after in 10 mgmL-11- (3- dimethylamino-propyls) -3- ethyl carbon
Diimmonium salt hydrochlorate and 20 mgmL-1N- hydroxysuccinimides effect 30 min of lower reaction, centrifugation is dissolved in 3 after washing
In mL phosphate buffer solutions.
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CN108918872A (en) * | 2018-07-25 | 2018-11-30 | 济南大学 | A kind of construction method for the photic electrochemical immunosensor of paper base detecting fibrin ferment |
CN109709182B (en) * | 2019-03-04 | 2021-03-26 | 济南大学 | Based on g-C3N4-MnO2Method for ultrasensitive detection of glutathione by photo-electrochemical method of nano composite material |
CN110702665B (en) * | 2019-11-14 | 2022-03-22 | 济南大学 | Preparation of paper-based coupling enhanced Raman sensor and application of paper-based coupling enhanced Raman sensor in okadaic acid detection |
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CN101587071A (en) * | 2009-07-01 | 2009-11-25 | 东南大学 | Fluorescence immunoassay method of using zinc oxide quantum dots to mark antibody |
CN104122393A (en) * | 2014-07-31 | 2014-10-29 | 济南大学 | Preparation of three-dimensional photoelectrochemical paper chip and application of three-dimensional photoelectrochemical paper chip in tumor detection |
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CN101587071A (en) * | 2009-07-01 | 2009-11-25 | 东南大学 | Fluorescence immunoassay method of using zinc oxide quantum dots to mark antibody |
CN104122393A (en) * | 2014-07-31 | 2014-10-29 | 济南大学 | Preparation of three-dimensional photoelectrochemical paper chip and application of three-dimensional photoelectrochemical paper chip in tumor detection |
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