CN102175666A - Nano-biosensor for fast detecting enterohemorrhagic escherichia coli enteritis - Google Patents
Nano-biosensor for fast detecting enterohemorrhagic escherichia coli enteritis Download PDFInfo
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- CN102175666A CN102175666A CN2011100287703A CN201110028770A CN102175666A CN 102175666 A CN102175666 A CN 102175666A CN 2011100287703 A CN2011100287703 A CN 2011100287703A CN 201110028770 A CN201110028770 A CN 201110028770A CN 102175666 A CN102175666 A CN 102175666A
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Abstract
The invention relates to a photochemical biosensor based on an adaptor and used for detecting enterohemorrhagic escherichia coli enteritis, in particular relating to a polydiacetylene structural organized molecular assembly photochemical biosensor for distinguishing a target molecule (enterohemorrhagic escherichia coli enteritis) lipopolysaccharide by utilizing oligonucleotide adaptors; and the polydiacetylene nano-vesica photochemical biosensor is used for detecting the enterohemorrhagic escherichia coli enteritis. In the photochemical biosensor related by the invention, the adaptor of the specifically combined enterohemorrhagic escherichia coli enteritis is combined at the surface of the polydiacetylene structural organized molecular assembly, so that the specific enterohemorrhagic escherichia coli enteritis is detected/distinguished by virtue of the changed ultraviolet-visual light absorbance or fluorescence intensity of the polydiacetylene nano-vesica when the enterohemorrhagic escherichia coli enteritis is exposed at the corresponding adaptor.
Description
Technical field
The invention belongs to field of biosensors, relate to a kind of novel optical chemical biosensor, and this optical chemical biosensor is used to detect EHEC based on aptamers.
Background technology
The lipoid that contains the diacetylene structure after the arrangement closely, can polymerization form blue poly-diacetylene structure forming high-sequential under the UV-irradiation of 254nm.This poly-diacetylene structure is along with the change of temperature, solvent, the pH of environment of living in and the mechanical stress that is subjected to thereof, and corresponding variation can take place its color.Based on this special optical property, make lipoid become the ideal material of the signal transport element of preparation biology sensor with poly-diacetylene structure.
People such as D.H.Charych had delivered one piece about utilizing functional poly diacetylene optically variable films can discern the article of influenza virus on Science in 1993, had caused people's common concern.They utilize 10, and 12-pentacosyl diacetylenic acid (PCDA) is basic fat, and sialylated PCDA is an acceptor molecule, preparation functionalization LB film (Langmuir-Blodgett Films), and this film presents blueness.When sialic acid runs into the information source common cold virus, cause the electronic state of poly-diacetylene structure and the change of weight, finally cause the color of information processing part (poly-diacetylene structure) red by blue stain, this phenomenon naked eyes obviously as seen, thereby started the beginning that poly-diacetylene prepares biology sensor.
Aptamers be a class have high affinity and can high degree of specificity the oligonucleotide sequence of identification combining target molecule, its target molecule scope is wide, from small-molecule drug and dyestuff, to complicated biomacromolecule such as enzyme molecule, polypeptide and protein.This artificial part is by a kind of new in-vitro screening technology-SELEX (evolution of index concentration Fas lignand system), and screening obtains from single strain oligonucleotide library at random.Because aptamers has identical with antibody or is better than the selectivity and the affinity of antibody, carry out protein molecule identification and become a kind of trend so in detection system, replace antibody with aptamers.In addition, compare with antibody, aptamers also has several advantages, as thermal stability increase, the anti-value of pH widely and salinity and synthetic, modification and curing be easy.The more important thing is that aptamers can lose the ability that combines with the target material by the nucleotide sex change, utilize this specific character can make reusable biology sensor.
At present, people have been developed the whole bag of tricks and aptamers are solidificated in solid surface and are applied to albumen and catch and separate and the preparation of biology sensor.Yet, utilize aptamers to be prepared the aptamers biology sensor now as molecular recognition elements, often modification is too complicated, and it is loaded down with trivial details to detect the target molecule method, is not suitable for the requirement of fast detecting target molecule.
Summary of the invention
The object of the invention provides the simple a kind of novel optical chemical biosensor based on aptamers of modification, and utilizes described biology sensor fast detecting EHEC.
This novel optical chemical biosensor is characterised in that with chemistry, physics or biological method modifies poly-diacetylene structural molecule organized assembles with aptamers, is built into the optical chemical biosensor with specific recognition EHEC function.
The amphipathic monomer molecule that contains the diacetylene structure, in aqueous solution, its hydrophilic radical is very stable in water, and hydrophobic grouping is extremely unstable, on the whole, is Unstable Systems on the energy.Amphipathic molecule will be in low-yield on thermodynamics, at first is its hydrophobic chain to be climbed up on top of the water go up the sensing air, reduces the immiscible property of molecule in water.When molecule can't enter the surface in the water when being paved with amphipathic molecule on the water surface, then these molecules were ganged up in water, made the hydrophilic group of molecule point to aqueous medium, in the hope of stablizing on the energy.The amphipathic molecule of air-water interface tiling is passed through the LB technology, be transferred on the solid support, promptly make poly-diacetylene structure LB film (Langmuir-Blodgett Films).And the amphiphile, amphiphilic molecule in the water is carried out sonicated, can be prepared into the vesica structure.Poly-diacetylene LB film has similar structure with poly-diacetylene vesica, just two kinds of multi-form molecular assembly assemblys.In actual applications, because poly-diacetylene vesica has space three-dimensional structure, its surperficial recognition component is easier to be combined with target molecule, and sensitivity is higher.We can will gather the diacetylene vesica by suitable function key and modify on the solid support, promptly make the curing vesica.
Chemical method of the present invention is a carbonization diamines method, and promptly the free carboxyl of effect activation PCDA by N-hydroxy-succinamide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide salt (EDC) forms active ester.Active ester and the amino reaction of aptamers 5 ' end form amido link, thereby the EHEC aptamers is modified poly-diacetylene structural molecule organized assembles, be prepared into optical chemical biosensor with specific recognition EHEC function.
The present invention is a kind of optical chemical biosensor based on aptamers, comprises molecular recognition elements and signal conduction device, be used for molecular recognition aptamers its be modified at poly-diacetylene structural molecule organized assembles surface.
Above-mentioned molecular recognition elements is the aptamers of energy specific recognition in conjunction with EHEC.
Above-mentioned signal conduction device is poly-diacetylene structural molecule organized assembles.
The above-mentioned mode that aptamers is modified at poly-diacetylene structural molecule organized assembles surface is modified for the covalency function key
The invention provides the method that detects the target material based on the optical chemical biosensor of aptamers, comprising:
1. the poly-diacetylene structural molecule organized assembles of preparation, wherein the aptamers that will be used to detect is modified at the surface of above-mentioned poly-diacetylene nano vesicle, can discern structure in conjunction with EHEC so that above-mentioned aptamers to be provided;
2. at the poly-diacetylene structural molecule organized assembles of modifying aptamers, be exposed under the situation of EHEC environment, measure the ultraviolet-visible optical absorbance of poly-diacetylene nano vesicle and the variation of fluorescence intensity;
3. based on the change-detection/evaluation EHEC of absorbance and fluorescence intensity.
The utility model is compared with the biological identification element of assembling in the past, modification is simple, can carry out fast detecting, therefore the utility model detects EHEC based on the optical chemical biosensor of aptamers and with it, can be widely used in aspects such as biochemical research, biological anti-terrorism, epidemic disease generaI investigation and clinical examination diagnosis.
Description of drawings
Fig. 1 is the synoptic diagram that carbonization diamines method mediation amination aptamers is modified poly-diacetylene vesica surface;
Fig. 2 is a characteristic absorption spectrum of modifying the poly-diacetylene vesica of aptamers;
Fig. 3 is an aptamers vesica colorimetric detection EHEC synoptic diagram;
Vesica changed CR% value change curve in time after Fig. 4 represented to add EHEC;
Fig. 5 represents that the variable concentrations EHEC causes that the CR% value of poly-diacetylene vesica changes and the detection range of linearity;
Symbol description
X is the characteristic absorption spectrum of the poly-diacetylene vesica after aptamers is modified; Y is the characteristic absorption spectrum of the poly-diacetylene vesica do not modified with aptamers; Z is the microwell plate blank.
Embodiment
The utility model is described in further detail below in conjunction with specific embodiments and the drawings, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1
1. the preparation of nano biological sensor
(1) EHEC specificity aptamers is synthetic and amido modified
The EHEC specificity aptamers that 5 ' end amination is modified is responsible for synthetic by Bai Ye trade (Shanghai) Co., Ltd. and is finished amido modified.Utilize the Maldi-Tof mass spectrophotometry to carry out quality control.
Aptamers: 5 ' Amine-C6-ATCAAATGTGCAGATATCAAGACGATTTGTACAAGAT
Above-mentioned aptamers is dissolved with aqua sterilisa, packing ,-20 ℃ of preservations.Before the use, 95 ℃ of sex change 5min.
The preparation and the modification of (2) preparation of EHEC optical chemical biosensor-poly-diacetylene lipid nanometer vesica
PCDA is dissolved in chloroform, is made into the 1mmol/L final concentration.Get an amount of this solution under blanket of nitrogen, vacuum drying becomes thin lipid film, adds deionized water and makes the solution final concentration to 1mmol/L, makes that the total concentration of lipoid is 1mmol/L.Lucifuge, 72 ℃ of ultrasonic emulsification 15min, transparent or semitransparent until solution.Place 4 ℃ to spend the night the solution that obtains, so that vesica is closed fully.Take out closed vesica solution, treat that it returns to room temperature, with hand-held uviol lamp (254nm) irradiation 30min, until mazarine occurring, obtain poly-diacetylene vesica, the vesica for preparing places 4 ℃ of preservations.Above process all needs to carry out under the lucifuge condition before the ultraviolet irradiation polymerization.
Get the 1mmol/L PCDA vesica of 100 μ L,, behind the reaction 2h, add 10nmol aptamers aqueous solution, replenish distilled water to 1mL to wherein adding 4mmol/L NHS aqueous solution 50 μ L and 4mmol/L EDCHCl aqueous solution 50 μ L respectively.Continue room temperature reaction 8h.After reaction finishes, at D-Tube
TMFully dialyse among the Dialyzer (merck) and remove unreacted aptamers etc.
2. nano biological sensor fast detecting EHEC
(1) cultivation of bacterium and counting
Cultivate 16～18h in the LB Liquid Culture under based on 37 ℃ of conditions, with aqua sterilisa thalline solution is diluted to 8 concentration gradients successively then, be respectively charged into 8 bottles, from 101 to 108, every bottle of 1ml, label 1～8 respectively.Traditional plate count method is used in cell count, gets the surface that 50 μ l are tiled in plain agar in the solution that contains viable bacteria that branch installs, and counts behind 37 ℃ of cultivation 24h.Culture counting is finished back and the bottle that viable bacteria is housed 121 ℃ of following 30min deactivations, in 4 ℃ of refrigerators preservations.
(2) EHEC optical chemical biosensor colorimetric detection EHEC
For the change color degree of blueness-redness of the poly-diacetylene nano vesicle of quantitative test, we define:
Than colour response (colorimetric response writes a Chinese character in simplified form CR) CR%=[(PB0-PBf)/PB0] * 100%
Wherein, PB=Ablue/ (Ablue+Ared), A is that blue poly-diacetylene component is in absorption intensity about 640nm or the red poly-absorption intensity of diacetylene component about 540nm, PB0 is that poly-diacetylene absorbs shared percentage to red ripple before adding fused protein, and PBf is that the red ripple of poly-diacetylene absorbs shared percentage after adding fused protein.The CR% value is big more, shows that the color of vesica solution is red more.
The poly-diacetylene lipid nanometer vesica of the modification aptamers of above-mentioned preparation is sub-packed in the 96 hole Microtiter-UV microwell plates each 100 μ L of every hole with pipettor.To be made into a series of bacterial concentration (transferring to 108CFU/ml, 107CFU/ml, 106CFU/ml, 105CFU/ml, 104CFU/ml, 103CFU/ml respectively) after the dilution of EHEC bacterium liquid, after adding in the above-mentioned hole of containing vesica fully mixing, at 37 ℃ or incubated at room 30min.Adopt the multi-functional microplate reader of Varioskan Flash (Thermo Scientific) to carry out the full wavelength scanner of 400～700nm, the visible absorption spectra before and after test is hatched detects A640 and A540 before and after hatching simultaneously, and calculates CR% value, curve plotting.
(3) experimental result
Carbonization diamines method mediation amination aptamers is modified the synoptic diagram on poly-diacetylene vesica surface and is seen Fig. 1.
Modify the characteristic absorption spectrum of the poly-diacetylene vesica of aptamers and see Fig. 2, the X curve is the characteristic absorption spectrum of the poly-diacetylene vesica after aptamers is modified among the figure, the Y curve is the characteristic absorption spectrum of the poly-diacetylene vesica do not modified with aptamers, and the Z curve is the absorption spectrum of blank.
From the result as can be seen, the poly-diacetylene vesica of modifying aptamers absorption peak occurred at the 260nm place, and this absorption peak characteristic peak of nucleotide just, we can see that the poly-diacetylene vesica of not modifying with aptamers does not but have absorption peak in the relevant position simultaneously, can think that aptamers successfully has been modified at poly-diacetylene vesica surface.
Aptamers vesica colorimetric detection EHEC synoptic diagram is seen Fig. 3.
Colorimetric detection EHEC in microcentrifugal tube, add EHEC and can cause the metachromasia that gathers the diacetylene vesica, Fig. 4 is along with the prolongation that adds the EHEC time, and blue-red variable color increases, and the CR% value increases gradually.And along with the EHEC concentration that adds increases, blue-red variable color increases, the characteristic absorption spectrum of poly-diacetylene vesica is also along with the adding of EHEC, maximum absorption band is blue shifted to the 540nm place from 640nm, and the amount of the EHEC that adds is big more, the absorbance at 640nm place descends many more, and absorbance rising in 540nm place is big more.Fig. 5 has reflected the degree of change color quantitatively with CR%, and along with the increase of EHEC concentration, the CR% value increases gradually, 10
4～10
8In the CFU/mL scope, CR% value and bacterial concentration are good linear relationship, and linear equation is Y=10.89X-45.05, and related coefficient=0.9991 is detected and is limited to 10
4CFU/mL (S/N=3).
Claims (6)
1. the optical chemical biosensor based on aptamers comprises molecular recognition elements and signal conduction device, it is characterized in that: the aptamers that is used for molecular recognition is modified at poly-diacetylene nano vesicle surface.
2. the optical chemical biosensor based on aptamers according to claim 1 is characterized in that: described molecular recognition elements is the aptamers of energy specific recognition in conjunction with EHEC.
3. the optical chemical biosensor based on aptamers according to claim 1 is characterized in that: described signal conduction device is poly-diacetylene structural molecule organized assembles.
4. according to a kind of optical chemical biosensor based on aptamers of claim 3, wherein said poly-diacetylene structural molecule organized assembles is a vesica.
5. the optical chemical biosensor based on aptamers according to claim 1 is characterized in that: the mode that aptamers is modified at poly-diacetylene structural molecule organized assembles surface is modified for the covalency function key.
6. one kind is detected target material according to each described optical chemical biosensor based on aptamers of claim 1～4, comprising:
1. preparation gathers diacetylene structural molecule organized assembles, and the EHEC aptamers that wherein will be used to detect is modified at the surface of described poly-diacetylene structural molecule organized assembles, so that EHEC aptamers sequence to be provided;
2. at the poly-diacetylene structural molecule organized assembles of modifying aptamers, be exposed under the situation of EHEC environment, measure the ultraviolet-visible optical absorbance of poly-diacetylene nano vesicle and the variation of fluorescence intensity;
3. based on the change-detection/evaluation EHEC of absorbance and fluorescence intensity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087555A (en) * | 2014-07-16 | 2014-10-08 | 江苏东博生物医药有限公司 | Folic acid targeting magnetic color-developing nanoparticles and preparation method thereof |
| CN110726768A (en) * | 2019-10-15 | 2020-01-24 | 复旦大学 | Ultra-sensitive bacterium identification method based on plasmon gold nano vesicles |
| CN113702308A (en) * | 2021-08-25 | 2021-11-26 | 青岛大学 | Aptamer nano colorimetric biosensor, application thereof, product and detection method of escherichia coli |
-
2011
- 2011-01-21 CN CN2011100287703A patent/CN102175666A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104087555A (en) * | 2014-07-16 | 2014-10-08 | 江苏东博生物医药有限公司 | Folic acid targeting magnetic color-developing nanoparticles and preparation method thereof |
| CN110726768A (en) * | 2019-10-15 | 2020-01-24 | 复旦大学 | Ultra-sensitive bacterium identification method based on plasmon gold nano vesicles |
| CN110726768B (en) * | 2019-10-15 | 2022-04-12 | 复旦大学 | Ultra-sensitive bacterium identification method based on plasmon gold nano vesicles |
| CN113702308A (en) * | 2021-08-25 | 2021-11-26 | 青岛大学 | Aptamer nano colorimetric biosensor, application thereof, product and detection method of escherichia coli |
| CN113702308B (en) * | 2021-08-25 | 2024-01-30 | 青岛大学 | Aptamer nano colorimetric biosensor for escherichia coli detection and application |
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Application publication date: 20110907 |