CN106526182A - Construction of paper-based aptamer fluorescence sensor used for thrombin detection - Google Patents
Construction of paper-based aptamer fluorescence sensor used for thrombin detection Download PDFInfo
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- CN106526182A CN106526182A CN201610966066.5A CN201610966066A CN106526182A CN 106526182 A CN106526182 A CN 106526182A CN 201610966066 A CN201610966066 A CN 201610966066A CN 106526182 A CN106526182 A CN 106526182A
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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"
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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"
- 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
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Abstract
The invention discloses construction of a paper-based aptamer fluorescence sensor used for thrombin detection. According to the invention, Adobe illustrator CS4 software is used to design a hydrophobic wax printing pattern of a micro fluidic paper chip; growing gold platinum duplex metals in a work area of a paper chip to reduce a background fluorescence signal; modifying an aptamer 1 on the duplex metals for specific recognition of a target; specifically binding the thrombin through an aptamer 2, introducing a fluorescence indicator to the work area, and detecting thrombin through an fluorescence instrument.
Description
Technical field
The present invention relates to refill chip technology, detection technique of fluorescence, are more specifically based on spy of the aptamers to object
Anisogamy characteristic constructs a simple, sensitive paper substrate fluorescent optical sensor for thrombin detection.
Background technology
Thrombin is a kind of serine protease, in the diagnosis equimolecular of hemostasis, angiogenesis and growth and metastasis of tumours
Important role in biology.As which is in the importance of biology, the detection and analysis of thrombin includes various technologies such as
Colorimetric, surface enhanced raman spectroscopy and surface plasma body resonant vibration etc..It is various sent a telegraph based on electrochemistry, light, electrochemiluminescence 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 of synthesis, with very strong competing the advantages of be easy to labelling, stronger stability, wide applicability
Strive power.
In order to improve the sensitivity of thrombin detection, noble metal composite nano materials are because its specific surface area is big, bio-compatible
Property the advantage such as good cause very big concern in science and technology field.Length has the paper chip of this noble metal composite nano materials,
Not only increase the specific surface area of paper chip, and effectively reduce the background fluorescence of paper, thus be widely used in all kinds
Biosensor in.In recent years, quantum dot because its preparation process it is simple, good biocompatibility, excitation spectrum are wider, symmetry
High emission spectrum, has the advantages that bleach-resistant.Which is made to be widely used as biological labled material.Replace organic with quantum dot
Fluorescence labeling material, can greatly reduce cost, improve detection ground sensitivity.Biocompatibility very well porous is selected simultaneously
Used as carrier, macroporous structure substantially increases the load capacity of quantum dot in the system to Zinc Oxide, so as to further improve detection spirit
Sensitivity.
Since two thousand seven, paper substrate sensor is because of its low cost, bigger serface, foldable, process, molding, biofacies
The features such as capacitive is good is applied to the research of clinical diagnosises.We are converted into preferable conformation using the foldability of paper, and
And using the capillarity of paper fiber, device letter truly is played in the introducing that water can avoid pump with neous flow
Change.By the appropriate design to paper chip and cutting, with reference to aptamers and thrombin specific binding construct one it is simple,
Sensitive paper substrate fluorescent optical sensor.
The content of the invention
The purpose of the present invention be make full use of paper foldability and capillarity and AuPt alloy nano particles it is excellent
It is benign to prepare a kind of new paper substrate fluorescence analysiss device and convenient and swift with reference to the specific recognition functional realiey of aptamers
The highly sensitive detection thrombin in ground.
In order to solve 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 being cut on the chromatographic paper of A4 sizes, so by wax printer
The A4 chromatographic papers with wax pattern are heated into 2 min by being heated to the panel heater of 125 C afterwards, wax is melted and is impregnated with whole
The thickness of individual paper, forms hydrophobic wall；
（3）In step（2）In the working region of chromatographic paper that obtains carry out functionalization, carry out aptamers after growth AuPt bimetallics
1 modification, thrombin 50 μ L of Deca to be detected use bovine serum albumin after being cleaned with phosphate buffer solution after 45 min of reaction
Block nonactive site；
（4）In step（2）In the hatching region of chromatographic paper that obtains drip the synthetic QDs ZnO fluorescent markers of 40 μ L and 60 μ
The 1 M aptamers 2 of L, carry out the reaction of fluorescent labeling and aptamers 2；
（5）Paper chip is folded along fold line, the fluorescent marker QDS@ZnO for being connected with aptamers 2 make in the capillary of paper fiber
It is firmly lower that working region is reached by fluid, washed with phosphate buffer solution after 30 min of reaction：
（6）By step（5）Middle paper chip working area is put in fluorescence equipment, carries out fluorescence survey under the excitation wavelength of 340 nm
It is fixed, the concentration relationship of fluorescence intensity and thrombin is drawn, the accurate detection to thrombin is realized.
The step of hydrophilic region functionalization to paper chip of the present invention is：Work(is carried out to the working region of chromatographic paper
Concretely comprise the following steps to energyization：100 μ L golden nanometer particles are measured with liquid-transfering gun to drip in hydrophilic working region, then at room temperature
Spontaneously dry, be repeated 5 times, secondary water washing, be dried under room temperature and stand；By 1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL
1% platinum acid chloride solution is quickly mixed, and liquid-transfering gun measures 60 μ L mixed liquors and is added drop-wise to working region, is then measured with liquid-transfering gun rapidly
Take the freshly prepared 1wt% sodium citrate aqueous solutions of 40 μ L and drip to working region, rinsed with secondary water after 30 min of reaction, then
Measured 100 μ L, 1 M aptamers 1 and dripped with liquid-transfering gun and hatch 1 h in working region, be that 7.4 phosphate buffer solutions are clear with pH
Wash three times nonactive site is blocked with 1% bovine serum albumin solution afterwards.
The preparation process of QDs@ZnO of the present invention is：Weigh 0.3 g white carbon blacks to be dissolved in 60 mL, 6 M nitric acid, surpass
Flow back after 2 h of sound under the conditions of 120 °C 24 h, above-mentioned solution is dropped to and be centrifuged after room temperature and be dried, product is dissolved in two then
Dialyse three days after secondary water, obtain graphene quantum dot;Weigh 3.5 g soluble starches to be dissolved in 80 mL boiling water, under 85 C
6 mmol zinc nitrate hexahydrates are added after stirring 5 min, are adjusted to continue after 8-9 by pH value of solution by being added dropwise over sodium hydroxide
30 min are heated, then by the pelleting centrifugation for obtaining, washing 2 h of the roasting acquisitions porous zinc bloom under 500 C, 0.6 is weighed
The porous zinc bloom that g is obtained stirs 4 h in being dissolved in the p-Mercaptoaniline solution of 0.1 mM；It is synthetic that liquid-transfering gun measures 500 μ L
Quantum dot mix with the Zinc Oxide of 2 mL functionalization after in 10 mg mL-11- (3- dimethylamino-propyls) -3- ethyls carbon two
Inferior amine salt hydrochlorate and 20 mg mL-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 fluorescent probe is simple, good biocompatibility.
（2）Using adaptation body technique, it is to avoid traditional antigen-antibody recognition methodss.
（3）Method testing cost of the present invention is cheap, simple to operate, sensitivity is high.
Accompanying drawing 1：The size and dimension of paper chip.
Specific embodiment
For a better understanding of the present invention, present disclosure is further elucidated with reference to embodiment, but the present invention
Content is not limited solely to following enforcement.
Embodiment 1：Paper substrate is fit, and fluorescent optical sensor is detected for thrombin, 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 being cut on the chromatographic paper of A4 sizes, so by wax printer
The A4 chromatographic papers with wax pattern are heated into 2 min by being heated to the panel heater of 125 C afterwards, wax is melted and is impregnated with whole
The thickness of individual paper, forms hydrophobic wall；
（3）In step（2）In the working region of chromatographic paper that obtains carry out functionalization, measure 100 μ L gold nano with liquid-transfering gun
Particle is dripped in hydrophilic working region, is then spontaneously dried at room temperature, is repeated 5 times, secondary water washing, and under room temperature, drying is quiet
Put；1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL, 1% chloroplatinic acids are quickly mixed, liquid-transfering gun measures 60 μ L mixed liquors
Working region is added drop-wise to, and then the freshly prepared 1wt% sodium citrate aqueous solutions of 40 μ L is measured rapidly with liquid-transfering gun and is dripped to work
Region, is rinsed with secondary water after 30 min of reaction, then measures 100 μ L, 1 M aptamers 1 with liquid-transfering gun and drip in working region
Hatch 1 h, with pH be 7.4 phosphate buffer solutions clean three times afterwards with 1% bovine serum albumin solution block non-active site
Point；
（4）In step（2）In the hatching region of chromatographic paper that obtains once operated；100 μ L Jenners are measured with liquid-transfering gun
Rice corpuscles are dripped in hydrophilic working region, are then spontaneously dried at room temperature, are repeated 5 times, secondary water washing, and under room temperature, drying is quiet
Put；1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL, 1% chloroplatinic acids are quickly mixed, liquid-transfering gun measures 60 μ L mixed liquors
Working region is added drop-wise to, and then the freshly prepared 1wt% sodium citrate aqueous solutions of 40 μ L is measured rapidly with liquid-transfering gun and is dripped to work
Region, is rinsed with secondary water after 30 min of reaction, then measures 100 μ L, 1 M aptamers 1 with liquid-transfering gun and drip in working region
Hatch 1 h, with pH be 7.4 phosphate buffer solutions clean three times afterwards with 1% bovine serum albumin solution block non-active site
Point；
（5）Paper chip is folded along fold line, the fluorescent marker QDS@ZnO for being connected with aptamers 2 make in the capillary of paper fiber
It is firmly lower that working region is reached by fluid, washed with phosphate buffer solution after 30 min of reaction；Concretely comprise the following steps：Weigh 0.3
G white carbon blacks are dissolved in 60 mL, 6 M nitric acid, and flow back after ultrasonic 2 h under the conditions of 120 °C 24 h, and above-mentioned solution is dropped to room temperature
After be centrifuged and be dried, then by product be dissolved in after secondary water dialyse three days, obtain graphene quantum dot;Weigh 3.5 g solubilities
Starch is dissolved in 80 mL boiling water, adds 6 mmol zinc nitrate hexahydrates, by being added dropwise under 85 C after stirring 5 min
PH value of solution is adjusted to continue 30 min of heating after 8-9 by sodium hydroxide, then by the pelleting centrifugation for obtaining, washing under 500 C
2 h of roasting obtains porous zinc bloom, and the porous zinc bloom for weighing 0.6 g acquisitions is dissolved in the p-Mercaptoaniline solution of 0.1 mM
Stir 4 h；Liquid-transfering gun is measured after the synthetic quantum dots of 500 μ L are mixed with the Zinc Oxide of 2 mL functionalization in 10 mg mL-1
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and 20 mg mL-1N- hydroxysuccinimides effect
30 min of lower reaction, centrifugation, are dissolved in after washing in 3 mL phosphate buffer solutions；
（6）By step（5）Middle paper chip working area is put in fluorescence equipment, carries out fluorescence survey under the excitation wavelength of 340 nm
It is fixed, the concentration relationship of fluorescence intensity and thrombin is drawn, the accurate detection to thrombin is realized.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>A kind of structure of the fit fluorescent optical sensor of paper substrate for thrombin detection
<130> 2016
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213>Synthetic
<400> 1
ggttggtgtg gttgg 15
<210> 2
<211> 44
<212> DNA
<213>Synthetic
<400> 2
tttttttttt tttttagtcc gtggtagggc aggttggggt gact 44
Claims (3)
1. a kind of structure of the fit fluorescent optical sensor of paper substrate for thrombin detection, is characterized in that comprising the following steps：
1.1 design paper chip hydrophobic wax print pattern as shown in Figure 1 on computers；
The print pattern designed in step 1.1 is printed upon being cut on the chromatographic paper of A4 sizes, so by 1.2 by wax printer
The A4 chromatographic papers with wax pattern are heated into 2 min by being heated to the panel heater of 125 C afterwards, wax is melted and is impregnated with whole
The thickness of individual paper, forms hydrophobic wall；
The working region of the chromatographic paper obtained in step 1.2 is carried out functionalization by 1.3, carries out aptamers after growth AuPt bimetallics
1 modification, thrombin 50 μ L of Deca to be detected are cleaned with phosphate buffer solution after 45 min of reaction, then pure with Sanguis Bovis seu Bubali
Albumen blocks nonactive site；
Drip the synthetic QDs ZnO fluorescent markers of 40 μ L and 60 μ in the hatching region of 1.4 chromatographic papers obtained in step 1.2
The 1 M aptamers 2 of L, carry out the reaction of fluorescent marker and aptamers 2；
Paper chip is folded by 1.5 along fold line, and the fluorescent marker QDS@ZnO for being connected with aptamers 2 are reached containing object
Working region, is washed with phosphate buffer solution after 30 min of reaction；
1.6 are put into paper chip working area in step 1.5 in fluorescence equipment, carry out fluorescence survey under the excitation wavelength of 340 nm
It is fixed, the concentration relationship of fluorescence intensity and thrombin is drawn, the accurate detection to thrombin is realized.
2. the structure of the fit fluorescent optical sensor of paper substrate of thrombin detection is used for according to claims 1, it is characterised in that
The working region of chromatographic paper is concretely comprised the following steps with carrying out functionalization described in step 1.3：100 μ L are measured with liquid-transfering gun golden
Nanoparticle is dripped in hydrophilic working region, is then spontaneously dried at room temperature, is repeated 5 times, secondary water washing, is dried under room temperature
Stand；1.0 mL 1wt% aqueous solution of chloraurate and 2.5 mL, 1% platinum acid chloride solutions are quickly mixed, liquid-transfering gun measures 60 μ L
Mixed liquor is added drop-wise to working region, then measures rapidly the freshly prepared 1wt% sodium citrate aqueous solutions drops of 40 μ L with liquid-transfering gun
To working region, rinsed with secondary water after 30 min of reaction, then 100 μ L, 1 M aptamers 1 are measured with liquid-transfering gun and dripped in work
Make region hatch 1 h, with pH be 7.4 phosphate buffer solutions clean three times afterwards with 1% bovine serum albumin solution block it is non-
Avtive spot.
3. the structure of the fit fluorescent optical sensor of paper substrate of thrombin detection is used for according to claims 1, it is characterised in that
The preparation process of the QDs@ZnO described in step 1.4 is：Weigh 0.3 g white carbon blacks to be dissolved in 60 mL, 6 M nitric acid, ultrasound 2
Flow back after h under the conditions of 120 °C 24 h, above-mentioned solution is dropped to and be centrifuged after room temperature and be dried, product is dissolved in secondary water then
Dialyse three days afterwards, obtain graphene quantum dot;Weigh 3.5 g soluble starches to be dissolved in 80 mL boiling water, stir under 85 C
6 mmol zinc nitrate hexahydrates are added after 5 min, are adjusted to continue heating after 8-9 by pH value of solution by being added dropwise over sodium hydroxide
30 min, then by the pelleting centrifugation for obtaining, washing 2 h of the roasting acquisitions porous zinc bloom under 500 C, weigh 0.6 g and obtain
The porous zinc bloom for obtaining stirs 4 h in being dissolved in the p-Mercaptoaniline solution of 0.1 mM；Liquid-transfering gun measures the synthetic amounts of 500 μ L
Son is put after being mixed with the Zinc Oxide of 2 mL functionalization in 10 mg mL-11- (3- dimethylamino-propyls) -3- ethyls carbon two it is sub-
Amine hydrochlorate and 20 mg mL-1N- hydroxysuccinimides effect 30 min of lower reaction, centrifugation is dissolved in 3 mL phosphorus after washing
In acid buffering solution.
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| CN201610966066.5A CN106526182B (en) | 2016-11-02 | 2016-11-02 | A kind of structure of paper substrate aptamer fluorescent optical sensor for fibrin ferment detection |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN109709182A (en) * | 2019-03-04 | 2019-05-03 | 济南大学 | A kind of g-C3N4-MnO2The photic electrochemical process super sensitivity detection glutathione of nanocomposite |
| CN110702665A (en) * | 2019-11-14 | 2020-01-17 | 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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| MENG ZHANG 等: "A disposable electrochemiluminescence device for ultrasensitive monitoring of K562 leukemia cells based on aptamers and ZnO@carbon quantum dots", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 陈慧娟 等: "葡萄糖氧化酶在石墨烯-纳米氧化锌修饰玻碳电极上的直接电化学及对葡萄糖的生物传感", 《分析化学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109709182A (en) * | 2019-03-04 | 2019-05-03 | 济南大学 | A kind of g-C3N4-MnO2The photic electrochemical process super sensitivity detection glutathione of nanocomposite |
| CN110702665A (en) * | 2019-11-14 | 2020-01-17 | 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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