CN106771231A - Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist - Google Patents
Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist Download PDFInfo
- Publication number
- CN106771231A CN106771231A CN201611046680.6A CN201611046680A CN106771231A CN 106771231 A CN106771231 A CN 106771231A CN 201611046680 A CN201611046680 A CN 201611046680A CN 106771231 A CN106771231 A CN 106771231A
- Authority
- CN
- China
- Prior art keywords
- salmonella
- chip
- carbohydrate chip
- carbohydrate
- mannose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
Abstract
The present invention relates to a kind of application of carbohydrate chip in detection salmonella or screening salmonella antagonist, carbohydrate chip is obtained by the solid phase carrier of chemical modification with mannose derivative coupling.The invention provides a kind of high throughput method that can be used for quick detection salmonella and screening anti-adhesive treatment medicine, carbohydrate chip prepared by the present invention can the different Salmonella strains of specific detection, detection method is simple, quick, and Detection results are good.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of carbohydrate chip is in detection salmonella or screening salmonella
Application in antagonist.
Background technology
Salmonella (salmonella) as zoonosis main food-borne causal agent, caused disease spreads all over
World, serious harm Community health and economic development.In order to effectively control salmonella, it is necessary to detection rapidly and efficiently
Method.At present, mainly there are three kinds of Detection of Salmonella：The life of traditional conventional detection technology, immunology detection technology and molecule
Thing detection technique, is shown in Table 1：
The common Salmeterol fluticasone propionate technology of table 1
As seen from the table, the various detection means of the above are still each defective, therefore in the urgent need to a kind of quick, sensitivity of exploitation
Height, high specificity, the methods and techniques of the detection salmonella of favorable reproducibility.
Carbohydrate chip (Carbohydrate microarray) technology is emerging after genetic chip, protein-chip
The large-scale Measurement for Biotechnique of the third generation.Used as the important analysis technology of Study of functional sugar, its advantage is embodied in consumption
Less, flux is high and the aspect such as high specificity, can be widely used for studying the interaction between sugar-large biological molecule.Carbohydrate chip is former
Reason is similar with other biochips, is based primarily upon sugar-protein specific effect and prepares, and is by lot of trace, different knots
The saccharide compound of structure is fixed on the substrate tablet prepared by certain organic or inorganic material in the way of covalently or non-covalently
On, make stromal surface that there is BA, and then to means that biological sample to be measured is tested and analyzed；Have with sugared probe
The testing sample of specific effect be can be adsorbed in matrix, and the be washed agent without specific effect is washed away.Fixed to obtain
The process of detecting probe information change, mainly includes mark and unmarked two kinds of detection methods on chip：There is labelling method generally right
Probe or testing sample are marked, such as fluorescence labeling, electrochemical label, isotope marks etc.；And unmarked rule is
Directly change the change of the natures such as weight, dielectric property, optical property, often with reference to mass spectrum, surface plasma body resonant vibration etc.
Instrument.
Various methods for making carbohydrate chip, for analyzing sugar and protein-interacting existing at present, these methods
Achieve many achievements attracted attention.But the development of current carbohydrate chip still has many difficulties, predominantly following 2 points：First how close
Into target sugar compounds ensureing the specificity of carbohydrate chip；Secondly how sugar compounds are fixed in matrix and ensure that it is biological
Activity can normally embody.The wherein design of glucide and synthesis is a key factor for building carbohydrate chip.Additionally, at present
Carbohydrate chip research still be in the starting stage, be typically usually used in the detection of protein, using carbohydrate chip detection bacterium research also compared with
It is few.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide carbohydrate chip in detection salmonella or screening Salmonella
Application in bacterium antagonist.Can be used for quick detection salmonella and screening anti-adhesive treatment medicine the invention discloses one kind
High throughput method, carbohydrate chip of the invention can the different Salmonella strains of specific detection, detection method is simple, quick,
Detection results are good.
To achieve the above object, present invention employs following technical scheme：
The invention discloses application of the carbohydrate chip in detection salmonella or screening salmonella antagonist, wherein sugared core
Piece is obtained by the solid phase carrier of chemical modification with mannose derivative coupling.
Further, mannose derivative is the mannose derivative containing amino.
Preferably, mannose derivative be 1- amino-ethyl-α-D- mannopyranoses polyglycoside (Man-1), 1- to amino-
α-D- mannopyranoses glucosides (Man-2) or p- [(N-2- ethylenediamine -2,3- diepoxy butyl -1- alkenyls) amino]-α-D- pyrroles
Mutter mannose glucosides (Man-3).
Further, the surface of solid phase carriers of chemical modification is modified with epoxy radicals, amino and N-hydroxy-succinamide base
In one or more.
Further, solid phase carrier is slide or silicon chip.
Further, salmonella is the salmonella strain that can be specifically bound with mannose, specially salmonella
ATCC9184, salmonella ATCC31685, salmonella S.typhimurium LB5010, salmonella S.typhimurium
One or more in AJB3, salmonella ATCC 14028 and salmonella S.typhimurium SL1344.
Further, the least concentration of the salmonella that carbohydrate chip can be detected is 106cells/mL。
In one embodiment, the preparation of carbohydrate chip is comprised the following steps：
Mixed solution is obtained in organic solvent by mannose derivative is molten, then the consolidating in chemical modification by mixed solution
Point sample on phase carrier, the solid phase carrier after point sample is incubated 8-12h at 15-35 DEG C under 50%~60% humidity, after taking-up
Fixed 2-4h is dried at 45-65 DEG C.
Preferably, the step of preparation of carbohydrate chip carries out protein labeling after being additionally included in and being incubated.
In one embodiment, the step of protein labeling is as follows：Using Cy3 labelled protein Con A, hydroxylamine hydrochloride, miscellaneous
Hand over buffer solution and the mixed solution of aqua sterilisa to be marked carbohydrate chip, mixed solution and carbohydrate chip are added into chip hybridization box
In, under 4-10r/min rotating speeds, 20-30 DEG C, humidity be 20-35% under the conditions of hybridize 3h.
Further, the concentration of mannose derivative is 10 μm of ol/L-20mmol/L in mixed solution.
Further, organic solvent be selected from absolute ethyl alcohol, glacial acetic acid and DMF (DMF) in one kind or
It is several.
The structural formula of Man-1 is as follows：
In one embodiment, the synthetic route of Man-1 is as follows：
The structural formula of Man-2 is as follows：
In one embodiment, the synthetic route of Man-2 is as follows：
The structural formula of Man-3 is as follows：
In one embodiment, the synthetic route of Man-3 is as follows：
Carbohydrate chip of the present invention detects that the principle of salmonella is：
Because salmonella has I type pili, the FimH albumen on I type pili is a kind of carbohydrate-binding protein, can and sweet dew
Sugar specificity is combined.Based on specific binding effect, using carbohydrate chip (Carbohydrate microarray) technology, structure
Different mannose carbohydrate chips are built, the detection of salmonella and the screening of anti-adhesive treatment agent is applied to.This research is by sweet dew
Sugar linking arm be designed, be prepared for three kinds of mannose derivatives, the mannose with different linking arms with carry do not assimilate
Learn group solid phase carrier connection, on the activation slide for using herein carry leaving group, can from the sweet of different linking arms
Dew sugar forms amido link, prepared carbohydrate chip can direct detection bacterium solution, without extracting albumen or nucleic acid etc., and required bacterium solution amount
Seldom；By mannose with linking arm and the connection of different solid phase carriers can cause mannose large space influence chip surface it is sweet
Dew sugar, so that the binding ability of mannose and albumen is influenceed, thus carbohydrate chip prepared by different mannose derivatives is to Salmonella
The detectability of bacterium is had any different.
By such scheme, the present invention at least has advantages below：
The invention discloses application of the carbohydrate chip in terms of Bacteria Detection and screening anti-adhesive treatment agent, prepared by the present invention
Carbohydrate chip can the different Salmonella strains of specific detection, detection method is simple, quick, and Detection results are good, while can apply
In the screening of anti-adhesive treatment agent；Carbohydrate chip prepared by the present invention can detect salmonella under low sugar concn, save glycogen
The consumption of material；The present invention can build carbohydrate chip using auto sample applicator, and thousands of samples can be put on same solid phase carrier,
Realize real high flux.Therefore, greater number of parallel laboratory test can be set up so as to detect a large amount of samples by carbohydrate chip technology
Product, compare with traditional method of detecting bacterium, save plenty of time and raw material.
Brief description of the drawings
Fig. 1 illustrates the comparing result of difference carbohydrate chip of the invention and salmonella binding ability；
Fig. 2 illustrates carbohydrate chip of the present invention and salmonella binding specificity；
Fig. 3 illustrates the result of the carbohydrate chip and salmonella binding ability prepared by the sugar using various concentrations；
Fig. 4 illustrates the bacterial solution of various concentrations and the fluorometric investigation result of carbohydrate chip hybridization；
Fig. 5 illustrates the concentration value result of each antagonist when carbohydrate chip is screened to different Anti-adhesive treatment agent.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiment of the invention is described in further detail.Hereinafter implement
Example is not limited to the scope of the present invention for illustrating the present invention.
The preparation of embodiment 1 1- amino-ethyl-α-D- mannopyranoses polyglycoside (Man-1) compound
The synthetic route of Man-1 compounds is as follows：
It is comprised the following steps that：
Under conditions of ice bath, 135mg DMPA (dihydromethyl propionic acid) and 72mL Py is added in the eggplant-shape bottle of 500mL
(pyridine), after 40mL acetic anhydride is slowly added to after stirring, adds 5g mannoses, stirring reaction 8h, TLC detection raw material reaction
Completely.It is molten using deionized water (3 × 300mL), saturated potassium carbonate successively after the dichloromethane for adding 200mL in reaction solution
Liquid (3 × 300mL) cleaning organic phase is cleaned to bubble-free, then with saturated nacl aqueous solution (1 × 300mL), is finally used
Anhydrous sodium sulfate drying.Organic phase is collected, vacuum distillation obtains full acetylated mannan sugar (compound 1 in figure) to remove organic phase,
It is put in preservation in -20 DEG C of refrigerators.
The full acetylated mannan sugar of 1.5g is dissolved in 10mL dichloromethane, the molecule after 0.84g bromoethanols and activation is added
Sieve (high-temperature activation, vacuumizes, three times respectively), ice bath stirring 0.5h under the protection of nitrogen, adds TMSOTf (fluoroform sulphurs
Sour trimethylsilyl group) 0.84mL.After stirring, ice bath is removed, then stirring reaction 24h, TLC detection reactions are complete.Reaction
Liquid same volume saturated potassium carbonate terminating reaction, then extracted with saturated sodium bicarbonate (3 × 30mL) and saturated aqueous common salt (3 × 30mL)
Take, merge organic phase, and use anhydrous sodium sulfate drying.Suction filtration, after filtrate is spin-dried for, gained crude product silica gel column chromatography purifies (stone
Oily ether:Ethyl acetate=10:1-4:1) white powder (compound 2,0.91g, 60.3% in figure) is obtained after.
Take after 0.5g compounds 2 are dissolved in dichloromethane and add 0.12g sodium azide, stirring reaction 12h, TLC detection reaction
Completely.Add the ethyl acetate of equal volume, after being extracted with deionized water (3 × 30mL), collect organic phase in reaction solution, be used in combination
Anhydrous sodium sulfate drying.Suction filtration, after filtrate is spin-dried for, gained crude product silica gel column chromatography purifies (petroleum ether:Ethyl acetate=10:
1-4:1) white powder (compound 3,0.42g, 83.0% in figure) is obtained.
Take 0.4g compounds 3 and be dissolved in methanol solution, add the sodium methoxide-carbinol mixture of 0.04g, after stirring 2h, TLC inspections
Survey reaction complete, add cationic ion-exchange resin to adjust pH to 5-6,10min is stirred at room temperature.Suction filtration, after revolving is except solvent, gained
Crude product silica gel column chromatography purifies (dichloromethane:Methyl alcohol=50:1-10:1) obtained after white powder (compound 4 in figure,
0.2g, 99.0%).
The methanol solution that 0.2g compounds 4 are dissolved in 2mL is taken, the 10% of 10mg Pd/C is added, in the Hydrogen Vapor Pressure of 4atm
Under, reaction is stirred at room temperature.After TLC detection reaction raw materials reactions completely, filtered with diatomite, after rotating away solvent, by product
Preservation in -20 DEG C of refrigerators is put in, product is Man-1 (0.15g, 83.0%).
Preparations of the 1- of embodiment 2 to amino-α-D- mannopyranoses glucosides (Man-2) compound
The synthetic route of Man-2 compounds is as follows：
Comprise the following steps that：
Take full acetylated mannans sugar (compound 1) of 2.0g to be dissolved in 10mL dichloromethane, add the anhydrous p-nitrophenols of 1.8g
And the molecular sieve for having activated, after ice bath stirring 0.5h, under the protection of nitrogen, add TMSOTf 1.12mL.Stir
Afterwards, ice bath is removed, stirring reaction, after TLC detection reactions completely, with being used again after isometric unsaturated carbonate potassium solution terminating reaction
Saturated sodium bicarbonate (3 × 30mL) and saturated aqueous common salt (3 × 30mL) are extracted, and anhydrous sodium sulfate drying is used after merging organic phase.
Suction filtration, after filtrate is spin-dried for, gained crude product silica gel column chromatography purifies (petroleum ether:Ethyl acetate=10:1-4:1) obtained after yellowish
Color powder (compound 5,1.12g, 59.3% in figure).
Take 1g compounds 5 and be dissolved in 10mL methyl alcohol, add the 10% of 100mg Pd/C, under the Hydrogen Vapor Pressure of 4atm, room temperature
Stirring reaction.After TLC detection reaction raw materials reactions completely, filtered with diatomite, after concentrated by rotary evaporation, gained crude product silica gel column layer
Analysis purifying (petroleum ether:Ethyl acetate=10:1-1:1) pale yellow powder (compound 6,0.88g, 88.9% in figure) is obtained after.
Take 0.8g compounds 6 and be dissolved in 8mL methanol solutions, add the sodium methoxide-carbinol mixture of 0.08g, after stirring 2h,
TLC detection reactions are complete, add cationic ion-exchange resin to adjust pH to 5-6, and 10min is stirred at room temperature.Suction filtration, after revolving is except solvent
Pale yellow powder is obtained, finally product is put in -20 DEG C of refrigerators and is preserved, product is Man-2 (0.48g, 99.0%).
The p- of embodiment 3 [(N-2- ethylenediamine -2,3- diepoxy butyl -1- alkenyls) amino]-α-D- mannopyranose glucosides
(Man-3) preparation of compound
The synthetic route of Man-3 compounds is as follows：
Comprise the following steps that：
1g compounds 5 are taken, fragrant acid diesters 0.63mL, stirring reaction, TLC detections are added after being dissolved in 10mL dichloromethane
Reaction is complete, and revolving removes solvent, and gained crude product silica gel column chromatography purifies (petroleum ether:Ethyl acetate=10:1-1:2) obtained after
Yellow powder (compound 7,0.73g, 67.5% in figure).
0.5g compounds 7 are taken, 0.05g sodium methoxides-carbinol mixture is added after being dissolved in 5mL methanol solutions, after stirring 2h,
TLC detection reactions are complete, add cationic ion-exchange resin to adjust pH to 5-6, and 10min is stirred at room temperature.Suction filtration, revolving removes solvent,
Gained crude product silica gel column chromatography purifies (dichloromethane:Methyl alcohol=50:1-10:1) obtained after yellow powder (compound 8 in figure,
0.28g, 95.3%).
0.20g compounds 8 are taken, after being dissolved in 2mL methanol solutions, the stirring of 50mg ethylenediamine solutions, TLC detection reactions is added
Terminate.Revolving removes solvent, and products obtained therefrom is preserved in being put in -20 DEG C of refrigerators, and product is Man-3 (0.22g, 98.0%).
The preparation of the epoxy slide of embodiment 4
The reaction scheme of epoxy slide is as follows：
By GPTS (propyl trimethoxy silicane) approach, coupling agent silane coupler (2,3- the third oxygen of epoxy) propyl group is selected
Trimethoxy silane) used as arm molecule, its alkoxy can be reacted with carrier surface of glass slide hydroxyl, so as to form silanol
Key, silane molecule is coupled at surface of glass slide in order, obtains epoxy slide.
Concrete operations are as follows：
Slide (20) is shaken in ultra-pure water respectively and is washed 4 times, shaken in absolute ethyl alcohol and washed 2 times, 5-10min is every time,
Centrifugal drying 10min (600r/min) at normal temperatures after end；Slide after centrifugal drying is placed in 5%HCl solution, with 40
DEG C, slow uniform shake 4 hours under conditions of 60r/min；Slide is placed in Piranha solution Piranha solution again
(the wherein proportioning of Piranha solution：30%H2O2:98%H2SO4=1:1, under ice bath stirring, by 30%H2O2It is slow to be added dropwise to
98% dense H2SO4In, volume ratio is 1:1), 85 DEG C of water-bath 2h；Slide after immersion shakes in ultra-pure water to be washed 4 times, in absolute ethyl alcohol
Shake and wash 2 times, each 5-10min, centrifuge dripping (600r/min) at normal temperatures after cleaning；It is dry to centrifugation using 12%NaOH solution
Slide after dry is processed, and temperature is 40 DEG C, and rotating speed is uniform shaken over night at a slow speed under conditions of 60rpm/min；After overnight
Slide is taken out, slide is immersed to be shaken in ultra-pure water and is washed 4 times, shaken in absolute ethyl alcohol and washed 2 times, each 5-10min, in normal temperature after cleaning
Lower centrifuge dripping (600r/min)；Dried slide is in 5%GPTS (collocation methods：12.5mL GPTS, 360 μ L glacial acetic acid,
240mL absolute ethyl alcohols) in solution, lucifuge, speed is shaken for 60r/min in 40 DEG C of uniform shake 6 hours at a slow speed；After taking out slide, use
Absolute ethyl alcohol shakes to be washed 3 times (10min/ times), centrifugal drying 10min (600r/min)；Finally it is positioned under the conditions of 37 DEG C, in vacuum
After drying 2h in drying box, it is stored in standby in 4 DEG C of driers.
The preparation of the NHS slides of embodiment 5
The syntheti c route of NHS slides is as follows：
By APTS (aminopropyl triethoxysilane) approach, amination slide is prepared first, wherein coupling agent is amino
Coupling agent, the coupling agent has the silane group with amino, can be reacted with the hydroxyl of carrier surface of glass slide, forms silanol
Key so that amino molecule is coupled at surface of glass slide in order.Route is as follows：
Above-mentioned amination slide further is modified with TGDD (the succinimide base disuccinic acid of TEG two), is obtained
NHS slides, route is as follows：
The preparation method of NHS slides is comprised the following steps that：
1) 10 above-mentioned amination slides are taken to be based in hand basket, DMF solution (TGDD of the figure containing TGDD is put:10mM, DIPA:
100mM, this solution is through 0.22 μm of organic membrane filter) in, reacted overnight with shaking fast 60-70rmp under the conditions of 40 DEG C.
2) after reaction terminates, ultrasonic 15min, with washes of absolute alcohol 3 times, each 10min, recyclable third time shakes what is washed
Absolute ethyl alcohol is used for the first time cleaning of modification next time chip base.Shake fast 110-120rmp；Piece after cleaning is based on room temperature 1000rmp
Centrifuge dripping, obtains NHS slides, and room temperature preservation is standby in putting it into drier.
The preparation of the NHS carbohydrate chips of embodiment 6 and checking
By taking Man-1, Man-2 and NHS slide as an example, the specific preparation process of carbohydrate chip is as follows：
(1) 20M solution Man-1 (is weighed 1.8mg Man-1, is dissolved in the 25% DMF aqueous solution, DMF:Aqua sterilisa=
100μL:300 μ L) and Man-2 (weigh 2.2mg Man-2, be dissolved in the 25% DMF aqueous solution, DMF:Aqua sterilisa=100 μ L:
300 μ L) solution, and blank control group 25% the DMF aqueous solution, with point sample instrument by 3 groups of solution point on NHS slides respectively
Sample, repeats point sample repeatedly, and each sampling point accommodates 1nL solution；Slide after point sample is placed on (50% in constant temperature humidity cabinet as early as possible
~60% humidity) overnight incubation.
(2) after carbohydrate chip overnight incubation, vacuum fixes 2h in the vacuum desiccator that temperature is 60 DEG C.
(3) after the carbohydrate chip after fixation being taken out into cooling, it is positioned in the chromatography ware equipped with 1 × PBST of 100mL and vibrates clearly
Wash 2 times (5min/ times)；After 1 × PBST solution is once cleaned, change second of cleaning again again of chip upper-lower position；Twice 1
After × PBST cleanings terminate, then with 1 × PBS 2 times, each 5min.The oscillator that vibration is used shakes for HY-2 speed governing is multiplex
Device is swung, chip placement direction should be parallel with orientation of oscillation during vibration, to ensure chip uniform force during oscillation cleaning.Ensure chip
After cleaning up, dried using miniature slide drier, rotating speed is controlled within 75r/min, it is ensured that two sides dries.
(4) dried chip is placed in the Block buffer (1 × PBS containing 1%BSA, 0.1% polysorbas20 of about 600 μ L
Buffer solution) in, and chip upper interior space solution is formed with uniform rotation and without multiple minute bubbles, to ensure that it flows
Property；Temperature is finally shelved on for 25 DEG C, humidity is to close 1h under conditions of 25%, will be on slide without knot by the closed process
Close the position closing of glycosyl group.
(5) chip that closing terminates is taken out, repeats the cleaning process of (3).
(6) take 20 μ L Cy3 labelled protein Con A, the hydroxylamine hydrochloride of 30 μ L 4M, the 2 × hybridization buffer of 300 μ L and
The aqua sterilisa mixing of 250 μ L, is finally made into 600 μ L mixed solutions, adds in chip hybridization box, enters with made carbohydrate chip
Row hybridization；(temperature is slowly rotated with 4r/min speed in hybridization case：25 DEG C, humidity：25%, hybridization time：3h)；After hybridization
Carbohydrate chip is dried after 1 × PBST and 1 × PBS.
(7) using chip scanner scan carbohydrate chip, laser intensity be 100% prescan after, select spotted area, accurately
Scanning.Scanning result shows that compare more than 0.5 with the fluorescent value before cleaning after cleaning, carbohydrate chip is made.
Embodiment 7 detects salmonella using carbohydrate chip
From epoxidation slide and NHS slides respectively with the reaction of above-mentioned 3 kinds of mannose derivatives to prepare carbohydrate chip, then
With salmonella ATCC31685 strain hybrids, fluorescence signal intensity is detected with chip scanner.
As shown in figure 1, Fig. 1 (a) is the carbohydrate chip prepared using NHS slides, the mannose in wherein 1-4 row derives result
Thing is Man-1, and 5-8 is classified as Man-2, and 9-12 is classified as Man-3, and 180 μm are about per bore dia；Three kinds of detectable differences of carbohydrate chip
The fluorescence signal of intensity, wherein the fluorescent value of the carbohydrate chip prepared using Man-1 is 3100, the use of Man-3 is 1830, use
Man-2's is 1050.Fig. 1 (b) is the carbohydrate chip prepared using epoxy slide, and the mannose derivative in wherein 1-4 row is
Man-1,5-8 are classified as Man-2, and 9-12 is classified as Man-3, and 180 μm are about per bore dia, and the concentration of three kinds of mannose derivatives is
10mmol/L；Three kinds of fluorescence signals of the detectable varying strength of carbohydrate chip, wherein the fluorescence of the carbohydrate chip prepared using Man-1
It is 850 to be worth, and the use of Man-3 is 450, is detected close to background value using the fluorescence signal of Man-2, it is difficult to catch.Comparison diagram 1
A () (b) can have found, epoxy slide and three kinds of combination effects of mannose derivative are not so good as NHS slides；Different mannose chemical combination
Carbohydrate chip made by thing is different from the binding ability of salmonella, NHS slides and the carbohydrate chip combination sramana made by Man-1
Salmonella ability is most strong.
Two kinds of salmonella ATCC9184 and ATCC31685 to mannose with different affinity are chosen, bacterium is dilute
Release in chip hybridization solution, detected with the carbohydrate chip made by Man-1 using NHS slides.Result is as shown in Fig. 2 Fig. 2
A the bacterium of () detection is ATCC31685, the bacterium of Fig. 2 (b) detections is ATCC9184, is 10 in bacterial concentration9cells·mL-1
When, (Mean Fluorescence is about through the fluorescence signal intensity after carbohydrate chip detection for the ATCC9184 bacterial strain relatively low with mannose affinity
It is 230, close to background value) far below the ATCC31685 bacterial strains with mannose with high-affinity, (average fluorescence signal is about
3000)。
Condition when prepared by carbohydrate chip makes following change：Change the concentration of Man-1 in 10 μm of ol/L-20mmol/L,
Carbohydrate chip is prepared using NHS slides and Man-1, by the hybridization hatching 1h at room temperature of the chip after closing, then by after hybridization
Slide takes out, and is successively cleaned 2 times (5min/ times) with 1 × PBST and 1 × PBS, then 1 (5min/ is cleaned with ultra-pure water
It is secondary), to wash away the cell on non-hybridized；Then the shadow of fluorescence signal of the sugared carbohydrate chip for preparing of various concentrations to detecting is determined
Ring, the bacteria concentration of the salmonella for being detected is 109Cells/mL, as a result as shown in Figure 3.
In Fig. 3,1-12 row sugared concentration reduce successively, concentration value be respectively 20mmol/L, 10mmol/L, 5mmol/L,
2.5mmol/L、1.25mmol/L、625μmol/L、313μmol/L、156μmol/L、78μmol/L、39μmol/L、20μmol/
L、10μmol/L.As can be seen from Figure 3, detected fluorescence signal is raised with the rising of sugared concentration, in 10mmol/L
When, fluorescence intensity nearly reaches maximum, and this shows the saturation of fixed mannose in carbohydrate chip, even if it is dense to increase sugar
Degree, fluorescence intensity is not also further added by；When 7th row (white box institute marked position in Fig. 3) represent sugared concentration as 313 μm of ol/L, still may be used
Detect the fluorescence signal higher than background value.Additionally, in Fig. 4, with after the Man-1 sugar juices making carbohydrate chip of 10mmol/L and not
Bacterial solution with concentration hybridizes, and from left to right, bacterial solution concentration is respectively 108cells/mL、107cells/mL、
106cells/mL、105Cells/mL and 104Cells/mL, it can be seen that the fluorescence signal detected in figure is with bacterial concentration
Reduce and reduce.When ATCC31685 bacterial strains concentration is more than or equal to 106During cells/mL, fluorescence signal is can detect；Continue to drop
After low bacterial strain concentration, fluorescence signal is difficult to detect, so understanding that this experiment carbohydrate chip detects the minimum Bacteria Detection of salmonella
Concentration is 106cells/mL。
Result shows that carbohydrate chip technology can be applied to Salmeterol fluticasone propionate, and (313 μm of ol/L) can under low sugar concn
Salmonella is detected, the consumption of glycogen material can be saved.In addition, existing carbohydrate chip is configured to manual construction, because aperture is big,
The sample of more than 200 can only be put on same slide, therefore this kind of carbohydrate chip can only be referred to as " many arrays " (Multiarray) mostly, with
" microarray " (Microarray) also has larger gap.And the present invention can build carbohydrate chip, each structure using auto sample applicator
The point sample aperture built is 180-200 μm, can greatly increase reticular density, you can thousands of can be put on same slide
Sample, realizes real high flux.Therefore, greater number of parallel laboratory test can be set up so as to detect largely by carbohydrate chip technology
Sample, compares with traditional method of detecting bacterium, saves plenty of time and raw material.
Embodiment 8 screens agglutinin FimH antagonists using carbohydrate chip
Relevant document shows, it is related that agglutinin FimH and enteric bacteria stick epithelial cell, can cause the diseases such as urinary tract infections
Disease；Various mannose derivatives and mannose (under the conditions of micromole) just can block bacterial adhesion (FimH is protein mediated),
Thus can be used as the therapeutic agent of relevant disease.
Choose three kinds it has been reported that antagonist (can effectively suppress agglutinin FimH effect), addition contains ATCC31685
The chip hybridization solution of cell (contains 1mmol/L CaCl2, 1mmol/L MnCl2And hydroxylamine hydrochloride) in, and prepared with the present invention
Carbohydrate chip hatching.Selected antagonist be respectively D-MANNOSE, p- nitrobenzophenone-α-D- mannopyranoses glycosides (p-NPMan) and
(PGMA, polymethylacrylic acid shrinks sweet a kind of amphiphilic polymers molecule PGMA-Mannose of covalent bond connection D-MANNOSE
Grease).Chip hybridization solution is the solution combined for detection bacterium and carbohydrate chip, if antagonist can hinder when being incubated
Disconnected bacterium is combined with chip, then this antagonist has preferable acceptor combination.
Shown using the result (Fig. 5) detected by carbohydrate chip of the invention：The concentration of three kinds of antagonists differs, relatively low
The corresponding suppression antibacterial effect of concentration representative is more excellent, and the effect that PGMA-Mannose (50 μm of ol/L) suppresses bacterium is substantially better than ratio
P-NPMan (1000 μm of ol/L), and p-NPMan (1000 μm of ol/L) is better than mannose (50000 μm of ol/L).Use the present invention
The effect of antagonist that is detected of carbohydrate chip it is consistent with report：P-NPMan is a kind of more preferable antagonist compared to mannose,
It can form stabilization and interact with large biological molecule, and the mannose compound with aryl, heteroaryl or heterocyclic radical have been demonstrated can
It is used to suppress the urinary tract infections that Escherichia coli cause as the antagonist for preventing or controlling inflammatory bowel disease；And PGMA-Mannose and sand
The combination of door Salmonella is much better than the combination of monose and salmonella.
Provable by above-mentioned experiment, carbohydrate chip of the invention can be used to detect inhibitory action of the antagonist to agglutinin,
And quantitative analysis is carried out to it, so as to accelerate the progress of bacterial adhesion antagonist, have for new drug development provides one kind
Effect instrument.
The above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, on the premise of the technology of the present invention principle is not departed from, can also make it is some improvement and
Modification, these are improved and modification also should be regarded as protection scope of the present invention.
Claims (11)
1. application of the carbohydrate chip in detection salmonella or screening salmonella antagonist, the carbohydrate chip is by chemical modification
Solid phase carrier is obtained with mannose derivative coupling.
2. application according to claim 1, it is characterised in that：The mannose derivative is that the mannose containing amino spreads out
It is biological.
3. application according to claim 2, it is characterised in that：The mannose derivative is 1- amino-ethyl-α-D- pyrroles
Mannose polyglycoside, 1- mutter to amino-α-D- mannopyranoses glucosides or p- [(N-2- ethylenediamine -2,3- diepoxy butyl -1-
Alkenyl) amino]-α-D- mannopyranose glucosides.
4. the application according to any one of claim 1-3, it is characterised in that：The surface of solid phase carriers of the chemical modification
It is modified with one or more in epoxy radicals, amino and N-hydroxy-succinamide base.
5. application according to claim 1, it is characterised in that：The solid phase carrier is slide or silicon chip.
6. the application according to any one of claim 1-3, it is characterised in that：The salmonella is salmonella
ATCC9184, salmonella ATCC31685, salmonella S.typhimurium LB5010, salmonella S.typhimurium
One or more in AJB3, salmonella ATCC 14028 and salmonella S.typhimurium SL1344.
7. the application according to any one of claim 1-3, it is characterised in that：The Salmonella that the carbohydrate chip can be detected
The least concentration of bacterium is 106cells/mL。
8. application according to claim 1, it is characterised in that the preparation of the carbohydrate chip is comprised the following steps：
The mannose derivative is dissolved in mixed solution is obtained in organic solvent, then by the mixed solution in the chemistry
Point sample on modified solid phase carrier, the solid phase carrier after point sample is incubated 8-12h at 15-35 DEG C under 50%~60% humidity.
9. application according to claim 8, it is characterised in that：The preparation of the carbohydrate chip carries out egg after being additionally included in incubation
The step of white marker.
10. application according to claim 8, it is characterised in that：In the mixed solution, the concentration of mannose derivative is
10μmol/L-20mmol/L。
11. applications according to claim 8, it is characterised in that：The organic solvent is selected from absolute ethyl alcohol, glacial acetic acid and N,
One or more in dinethylformamide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611046680.6A CN106771231B (en) | 2016-11-23 | 2016-11-23 | Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611046680.6A CN106771231B (en) | 2016-11-23 | 2016-11-23 | Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106771231A true CN106771231A (en) | 2017-05-31 |
| CN106771231B CN106771231B (en) | 2019-02-12 |
Family
ID=58974376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611046680.6A Active CN106771231B (en) | 2016-11-23 | 2016-11-23 | Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106771231B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010026915A1 (en) * | 1992-11-13 | 2001-10-04 | The Regents Of The University Of California | Colorimetric glycopolythiophene biosensors |
| CN102740859A (en) * | 2009-12-01 | 2012-10-17 | 艾克塞拉医疗有限责任公司 | Method for removing cytokines from blood with surface immobilized polysaccharides |
| CN103842815A (en) * | 2011-05-17 | 2014-06-04 | 萨班吉大学 | Whole-cell bacterial bio-capacitor chip and a method for detecting cellular stress induced by toxic chemicals by use of the chip |
-
2016
- 2016-11-23 CN CN201611046680.6A patent/CN106771231B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010026915A1 (en) * | 1992-11-13 | 2001-10-04 | The Regents Of The University Of California | Colorimetric glycopolythiophene biosensors |
| CN102740859A (en) * | 2009-12-01 | 2012-10-17 | 艾克塞拉医疗有限责任公司 | Method for removing cytokines from blood with surface immobilized polysaccharides |
| CN103842815A (en) * | 2011-05-17 | 2014-06-04 | 萨班吉大学 | Whole-cell bacterial bio-capacitor chip and a method for detecting cellular stress induced by toxic chemicals by use of the chip |
Non-Patent Citations (2)
| Title |
|---|
| 王雪玉 等: "利用糖芯片技术检测氨基糖苷类抗生素与RNAs和蛋白质之间的相互作用", 《生物工程学报》 * |
| 邹兰 等: "糖芯片的研究进展", 《有机化学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106771231B (en) | 2019-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105683761B (en) | The device and method of substance is tested and analyzed according to the movement of particle | |
| CN110204735A (en) | A kind of preparation method and application of the hollow porous type molecularly imprinted polymer satellite assembly of the magnetic core-of macrolide antibiotics | |
| CN102012358B (en) | Study and application of quantum dot molecular imprinting microsphere quartz fluorescent sensor for detecting trace multicomponent food additives quickly on site | |
| CN108802374A (en) | Excretion body nucleic acid detection technique based on magnetic enrichment electrochemical luminescence | |
| CN105651999B (en) | Molybdenum disulfide-based sensor and preparation method and application thereof | |
| CN105699650B (en) | Carbofuran bio-barcode immune analytic reagent kit and its application | |
| CN107698614B (en) | A kind of carborane-diimide derivative and synthetic method and sensor array and preparation method and application based on it | |
| CN105651992B (en) | Hostathion bio-barcode immune analytic reagent kit and its application | |
| CN102416313B (en) | Bisphenol A dummy template molecularly imprinted stir bar and preparation method thereof | |
| WO2023202034A1 (en) | Oligosaccharide fluorescent marker, preparation method therefor, and use thereof | |
| CN104897826A (en) | Method for detecting interaction of small molecular compound and target protein at cell in-situ level | |
| CN106399317A (en) | DNA (deoxyribonucleic acid) aptamer of phthalate type plasticizer and screening and expression method and electro-chemical sensor thereof | |
| CN104829687B (en) | A kind of method for a variety of enzymes and albumen for extracting earthworm | |
| CN106366075A (en) | Pyrrolidine compounds, salts thereof, applications of compounds or salts thereof in nuclear magnetic probes and medicines, reagent, and medicine | |
| CN110004148B (en) | Alternariol aptamer affinity column and preparation method and application thereof | |
| Hu et al. | A novel solid fluorescence method for the fast determination of quercetin in biological samples based on the quercetin–Al (III) complex imprinted polymer | |
| CN106771231B (en) | Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist | |
| CN114113582A (en) | Metal organic framework nanoenzyme biological probe and ELISA kit | |
| CN102507671A (en) | Porous silicon biochip and preparation method thereof | |
| CN106831534A (en) | Phenolphthalein haptens, artificial antigen and preparation method thereof | |
| CN105518452B (en) | Mark the modulator approach of sugar chain sample | |
| CN101832996A (en) | Substrate of traditional Chinese medicine biochip as well as preparation method and use thereof | |
| CN107505419A (en) | One kind is used to be enriched with, purify, detect AFB1Modification inverse opal photonic crystal microballoon and its preparation method and application | |
| CN103558178A (en) | Chlorpyrifos detecting method and device by terahertz wave spectrum combining with biosensing technique | |
| CN104931687A (en) | Three-dimensional biological surface, preparation method thereof, three-dimensional biological chip and application of three-dimensional biological chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |