CN106771231B - 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
- CN106771231B CN106771231B CN201611046680.6A CN201611046680A CN106771231B CN 106771231 B CN106771231 B CN 106771231B CN 201611046680 A CN201611046680 A CN 201611046680A CN 106771231 B CN106771231 B CN 106771231B
- Authority
- CN
- China
- Prior art keywords
- salmonella
- chip
- carbohydrate chip
- carbohydrate
- detection
- 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.)
- Active
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 coupled to obtain by the solid phase carrier of chemical modification with mannose derivative.Can be used for quickly detecting salmonella and screen the high throughput method of anti-adhesive treatment drug the present invention provides a kind of, carbohydrate chip prepared by the present invention can the different Salmonella strains of specific detection, detection method is simple, quickly, and detection effect is good.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of carbohydrate chip in detection salmonella or screening salmonella
Application in antagonist.
Background technique
Main food-borne causal agent of the salmonella (salmonella) as zoonosis, caused disease throughout
World seriously endangers Community health and economic development.In order to effectively control salmonella, detection rapidly and efficiently is needed
Method.Currently, there are mainly three types of Detection of Salmonella: traditional conventional detection technology, immunology detection technology and molecule are raw
Object detection technique, is shown in Table 1:
The common Salmeterol fluticasone propionate technology of table 1
As seen from the table, above a variety of detection means are still each defective, therefore there is an urgent need to develop a kind of quick, sensitivity
Height, high specificity, favorable reproducibility detection salmonella methods and techniques.
Carbohydrate chip (Carbohydrate microarray) technology is emerging after genetic chip, protein-chip
The large-scale Measurement for Biotechnique of the third generation.As the important analysis technology of Study of functional sugar, advantage is embodied in dosage
Less, flux height and high specificity etc. can be widely used for the interaction between research sugar-large biological molecule.Carbohydrate chip is former
Reason is similar with other biochips, is based primarily upon sugar-protein specific effect and prepares, is by lot of trace, different knots
The saccharide compound of structure is fixed on substrate tablet made of prepared by certain organic or inorganic material in a manner of covalently or non-covalently
On, make stromal surface that there is biological activity, and then to the means that biological sample to be measured is tested and analyzed；Have with sugared probe
The sample to be tested of specific effect can be adsorbed in matrix, and the agent that is washed without specific effect washes away.It is fixed to obtain
The process that detecting probe information changes on chip mainly includes label and unmarked two kinds of detection methods: having labelling method usually right
Probe or sample to be tested are marked, such as fluorescent marker, electrochemical label, isotope labelling etc.；And unmarked rule is
It is directly changed the change of the natures such as weight, dielectric property, optical property, often combines mass spectrum, surface plasma body resonant vibration etc.
Instrument.
At present there are many for making carbohydrate chip, for analyzing sugar and the method for protein-interacting, these methods
Achieve the achievement much attracted attention.But still there are many difficulties, predominantly following two points for the development of current carbohydrate chip: first how closing
Guarantee the specificity of carbohydrate chip at target sugar compounds；Secondly how sugar compounds to be fixed in matrix and guarantees its biology
Activity can normally embody.Wherein the design Yu synthesis of glucide are to construct a key factor of carbohydrate chip.In addition, at present
Carbohydrate chip study still in its infancy, be generally usually used in the detection of protein, using carbohydrate chip detection bacterium research also compared with
It is few.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide carbohydrate chips in detection salmonella or screening Salmonella
Application in bacterium antagonist.The invention discloses one kind can be used for quickly detecting salmonella and screening anti-adhesive treatment drug
High throughput method, carbohydrate chip of the invention can the different Salmonella strains of specific detection, detection method is simple, quickly,
Detection effect is good.
To achieve the above object, the invention adopts the following technical scheme:
The invention discloses application of the carbohydrate chip in detection salmonella or screening salmonella antagonist, wherein sugared core
Piece is coupled to obtain by the solid phase carrier of chemical modification with mannose derivative.
Further, mannose derivative is the mannose derivative containing amino.
Preferably, mannose derivative is 1- amino-ethyl-α-D- mannopyranose polyglycoside (Man-1), 1- to amino-
α-D- mannopyranose glucosides (Man-2) or p- [(N-2- ethylenediamine -2,3- diepoxy butyl -1- alkenyl) amino]-α-D- pyrrole
Mutter mannose glucosides (Man-3).
Further, the surface of solid phase carriers of chemical modification is modified with epoxy group, amino and n-hydroxysuccinimide base
One or more of.
Further, solid phase carrier is slide or silicon wafer.
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 of AJB3, salmonella ATCC 14028 and salmonella S.typhimurium SL1344.
Further, the least concentration of carbohydrate chip salmonella that can be detected is 106cells/mL。
In one embodiment, carbohydrate chip preparation the following steps are included:
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 are incubated for 8-12h at 15-35 DEG C under 50%~60% humidity, after taking-up
Dry fixed 2-4h at 45-65 DEG C.
Preferably, the preparation of carbohydrate chip further includes the steps that carrying out protein labeling after incubation.
In one embodiment, the step of protein labeling is as follows: using Cy3 labelled protein Con A, hydroxylamine hydrochloride, miscellaneous
It hands over the mixed solution of buffer and aqua sterilisa that carbohydrate chip is marked, chip hybridization box is added in mixed solution and carbohydrate chip
In, under 4-10r/min revolving speed, 20-30 DEG C, humidity hybridizes 3h under the conditions of being 20-35%.
Further, the concentration of mannose derivative is 10 μm of ol/L-20mmol/L in mixed solution.
Further, organic solvent be selected from one of dehydrated alcohol, glacial acetic acid and n,N-Dimethylformamide (DMF) 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:
The principle of carbohydrate chip detection salmonella of the present invention are as follows:
Since salmonella has I type pili, the FimH albumen on I type pili is a kind of carbohydrate-binding protein, energy and sweet dew
Sugar specificity combines.It is acted on based on the specific binding, utilizes carbohydrate chip (Carbohydrate microarray) technology, structure
Different mannose carbohydrate chips are built, applied to the detection of salmonella and the screening of anti-adhesive treatment agent.This research passes through to sweet dew
Sugar linking arm be designed, be prepared for three kinds of mannose derivatives, the mannose with different linking arms with have do not assimilate
The solid phase carrier connection for learning group, leaving group is had on the activation slide used herein, can from it is sweet with different linking arms
Dew sugar forms amido bond, and prepared carbohydrate chip can directly detect bacterium solution, without extracting albumen or nucleic acid etc., and required bacterium solution amount
Seldom；Since mannose institute band connection arm and the connection of different solid phase carriers will lead to mannose large space, to influence chip surface sweet
Dew sugar, so that the binding ability of mannose and albumen is influenced, thus the carbohydrate chip of different mannose derivative preparations is to Salmonella
The detectability of bacterium is had any different.
According to the above aspect of the present invention, the present invention has at least the following advantages:
It is prepared by the present invention the invention discloses application of the carbohydrate chip in terms of Bacteria Detection and screening anti-adhesive treatment agent
Carbohydrate chip can the different Salmonella strains of specific detection, detection method is simple, quickly, and detection effect is good, while can apply
In the screening of anti-adhesive treatment agent；Salmonella can be detected in carbohydrate chip prepared by the present invention under low sugar concn, saves glycogen
The consumption of material；The present invention can construct carbohydrate chip using auto sample applicator, and thousands of a samples can be put on the same solid phase carrier,
Realize real high throughput.Therefore, greater number of parallel laboratory test can be established to detect a large amount of samples by carbohydrate chip technology
Product are compared with traditional method of detecting bacterium, save plenty of time and raw material.
Detailed description of the invention
Fig. 1 illustrates the comparing results of different carbohydrate chips and salmonella binding ability of the invention；
Fig. 2 illustrates carbohydrate chip of the present invention and salmonella binding specificity；
Fig. 3 illustrates the result of carbohydrate chip prepared by the sugar using various concentration Yu salmonella binding ability；
The fluorometric investigation result that Fig. 4 illustrates the bacterial solution of various concentration and carbohydrate chip hybridizes；
Fig. 5 illustrates the concentration value result of each antagonist when carbohydrate chip screens different Anti-adhesive treatment agent.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
The preparation of embodiment 1 1- amino-ethyl-α-D- mannopyranose polyglycoside (Man-1) compound
The synthetic route of Man-1 compound is as follows:
The specific steps of which are as follows:
Under conditions of ice bath, 135mg DMPA (dihydromethyl propionic acid) and 72mL Py are added in the eggplant-shape bottle of 500mL
After being slowly added to 40mL acetic anhydride after mixing evenly, 5g mannose is added in (pyridine), is stirred to react 8h, and TLC detects raw material reaction
Completely.It is successively molten using deionized water (3 × 300mL), saturated potassium carbonate after the methylene chloride of 200mL is added in reaction solution
It until liquid (3 × 300mL) cleans organic phase to bubble-free, then is cleaned with saturated sodium chloride solution (1 × 300mL), is finally used
Anhydrous sodium sulfate is dry.Organic phase is collected, is evaporated under reduced pressure to remove organic phase, full acetylated mannan sugar (compound 1 in figure) is obtained,
It is put in -20 DEG C of refrigerators and saves.
The full acetylated mannan sugar of 1.5g is dissolved in 10mL methylene chloride, the molecule after 0.84g bromoethanol and activation is added
Sieve (high-temperature activation vacuumizes, respectively three times), TMSOTf (trifluoro methylsulphur is added under the protection of nitrogen in ice bath stirring 0.5h
Sour trimethylsilyl group) 0.84mL.After mixing evenly, ice bath is removed, is then stirred to react for 24 hours, TLC detects fully reacting.Reaction
Liquid is terminated with same volume saturated potassium carbonate to react, then is extracted with saturated sodium bicarbonate (3 × 30mL) and saturated salt solution (3 × 30mL)
It takes, merges organic phase, and dry with anhydrous sodium sulfate.It filters, after filtrate is spin-dried for, gained crude product silica gel column chromatography purifies (stone
Oily ether: ethyl acetate=10:1-4:1) after obtain white powder (in figure compound 2,0.91g, 60.3%).
0.12g sodium azide is added after taking 0.5g compound 2 to be dissolved in methylene chloride, is stirred to react 12h, TLC detection reaction
Completely.The ethyl acetate of equal volume is added in reaction solution, after deionized water (3 × 30mL) extraction, collects organic phase, is used in combination
Anhydrous sodium sulfate is dry.Filter, after filtrate is spin-dried for, gained crude product silica gel column chromatography purify (petroleum ether: ethyl acetate=10:
1-4:1) obtain white powder (in figure compound 3,0.42g, 83.0%).
It takes 0.4g compound 3 to be dissolved in methanol solution, sodium methoxide-carbinol mixture of 0.04g is added, after stirring 2h, TLC inspection
Fully reacting is surveyed, cation exchange resin is added and adjusts pH to 5-6,10min is stirred at room temperature.It filters, after revolving removes solvent, gained
Crude product purified with silica gel column chromatography obtain after (methylene chloride: methanol=50:1-10:1) white powder (compound 4 in figure,
0.2g, 99.0%).
It takes 0.2g compound 4 to be dissolved in the methanol solution of 2mL, 10% Pd/C of 10mg is added, in the Hydrogen Vapor Pressure of 4atm
Under, reaction is stirred at room temperature.After TLC detects reaction raw materials fully reacting, filtered with diatomite, after rotating away solvent, by product
It is put in -20 DEG C of refrigerators and saves, product is Man-1 (0.15g, 83.0%).
Preparation of 2 1- of embodiment to amino-α-D- mannopyranose glucosides (Man-2) compound
The synthetic route of Man-2 compound is as follows:
Specific step is as follows:
It takes the full acetylated mannan of 2.0g sugared (compound 1) to be dissolved in 10mL methylene chloride, the anhydrous p-nitrophenol of 1.8g is added
And activated molecular sieve, after ice bath stirring 0.5h, under the protection of nitrogen, TMSOTf 1.12mL is added.It stirs evenly
Afterwards, ice bath is removed, is stirred to react, after TLC detects fully reacting, is used again after terminating reaction with isometric unsaturated carbonate potassium solution
Saturated sodium bicarbonate (3 × 30mL) and saturated salt solution (3 × 30mL) extract, dry with anhydrous sodium sulfate after merging organic phase.
Filter, after filtrate is spin-dried for, gained crude product purified with silica gel column chromatography obtain after (petroleum ether: ethyl acetate=10:1-4:1) it is yellowish
Color powder (in figure compound 5,1.12g, 59.3%).
It takes 1g compound 5 to be dissolved in 10mL methanol, 10% Pd/C of 100mg, under the Hydrogen Vapor Pressure of 4atm, room temperature is added
It is stirred to react.After TLC detects reaction raw materials fully reacting, filtered with diatomite, after concentrated by rotary evaporation, gained crude product silica gel column layer
Pale yellow powder (in figure compound 6,0.88g, 88.9%) is obtained after analysis purifying (petroleum ether: ethyl acetate=10:1-1:1).
It takes 0.8g compound 6 to be dissolved in 8mL methanol solution, sodium methoxide-carbinol mixture of 0.08g is added, after stirring 2h,
TLC detects fully reacting, and cation exchange resin is added and adjusts pH to 5-6,10min is stirred at room temperature.It filters, revolving is except after solvent
Pale yellow powder is obtained, finally product is put in -20 DEG C of refrigerators and is saved, product is Man-2 (0.48g, 99.0%).
3 p- of embodiment [(N-2- ethylenediamine -2,3- diepoxy butyl -1- alkenyl) amino]-α-D- mannopyranose glucosides
(Man-3) preparation of compound
The synthetic route of Man-3 compound is as follows:
Specific step is as follows:
1g compound 5 is taken, fragrant acid diesters 0.63mL is added after being dissolved in 10mL methylene chloride, is stirred to react, TLC detection
Fully reacting, revolving remove solvent, and gained crude product is purified with silica gel column chromatography to be obtained after (petroleum ether: ethyl acetate=10:1-1:2)
Yellow powder (in figure compound 7,0.73g, 67.5%).
0.5g compound 7 is taken, 0.05g sodium methoxide-carbinol mixture is added after being dissolved in 5mL methanol solution, after stirring 2h,
TLC detects fully reacting, and cation exchange resin is added and adjusts pH to 5-6,10min is stirred at room temperature.It filtering, revolving removes solvent,
Gained crude product purified with silica gel column chromatography obtain after (methylene chloride: methanol=50:1-10:1) yellow powder (compound 8 in figure,
0.28g, 95.3%).
0.20g compound 8 is taken, after being dissolved in 2mL methanol solution, the stirring of 50mg ethylenediamine solution, TLC detection reaction is added
Terminate.Revolving removes solvent, and products obtained therefrom is put in -20 DEG C of refrigerators and saves, and product is Man-3 (0.22g, 98.0%).
The preparation of 4 epoxy slide of embodiment
The reaction route of epoxy slide is as follows:
By GPTS (propyl trimethoxy silicane) approach, coupling agent silane coupling agent (2,3- the third oxygen of epoxy) propyl is selected
Trimethoxy silane) it is used as arm molecule, alkoxy can be reacted with carrier surface of glass slide hydroxyl, to form silanol
Key, silane molecule are orderly coupled at surface of glass slide, obtain epoxy slide.
Concrete operations are as follows:
Slide (20) is shaken in ultrapure water respectively and is washed 4 times, shakes and washes 2 times in dehydrated alcohol, be every time 5-10min,
After centrifugal drying 10min (600r/min) at normal temperature；Slide after centrifugal drying is placed in 5%HCl solution, with 40
DEG C, it is slowly uniformly shaken 4 hours under conditions of 60r/min；Slide is placed in Piranha solution Piranha solution again
(the wherein proportion 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 1:1), 85 DEG C of water-bath 2h；Slide after immersion shakes in ultrapure water to be washed 4 times, in dehydrated alcohol
It shakes and washes 2 times, each 5-10min, centrifuge dripping (600r/min) at normal temperature after cleaning；It is dry to centrifugation using 12%NaOH solution
Slide after dry is handled, and temperature is 40 DEG C, and revolving speed is uniform shaken over night at a slow speed under conditions of 60rpm/min；After overnight
Slide is taken out, slide is immersed in ultrapure water to shake and is washed 4 times, shakes and washes 2 times in dehydrated alcohol, each 5-10min, in room temperature after cleaning
Lower centrifuge dripping (600r/min)；Slide after drying 5%GPTS (configuration method: 12.5mL GPTS, 360 μ L glacial acetic acid,
240mL dehydrated alcohol) in solution, it is protected from light, 40 DEG C are uniformly shaken 6 hours at a slow speed, and shaking speed is 60r/min；After taking out slide, use
Dehydrated alcohol, which shakes, to be washed 3 times (10min/ times), centrifugal drying 10min (600r/min)；It is finally placed under the conditions of 37 DEG C, in vacuum
In drying box after dry 2h, it is stored in spare in 4 DEG C of driers.
The preparation of 5 NHS slide of embodiment
The preparation route of NHS slide is as follows:
By APTS (aminopropyl triethoxysilane) approach, amination slide is prepared first, and wherein coupling agent is amino
Coupling agent, the coupling agent have the silane group with amino, can be reacted with the hydroxyl of carrier surface of glass slide, form silanol
Key, so that amino molecule is orderly coupled at surface of glass slide.Route is as follows:
Above-mentioned amination slide further is modified with TGDD (two succinimide base disuccinic acid of tetraethylene glycol), is obtained
NHS slide, route are as follows:
Specific step is as follows for the preparation method of NHS slide:
1) take 10 above-mentioned amination slides be based on hand basket in, put figure containing TGDD DMF solution (TGDD:10mM, DIPA:
100mM, this solution is through 0.22 μm of organic membrane filter) in, fast 60-70rmp reaction is shaken under the conditions of 40 DEG C overnight.
2) after reaction, ultrasonic 15min, with washes of absolute alcohol 3 times, each 10min, recyclable third time, which is shaken, is washed
First time cleaning of the dehydrated alcohol for modification next time chip base.Shake fast 110-120rmp；Piece after cleaning is based on room temperature 1000rmp
Centrifuge dripping obtains NHS slide, and it is spare to put it into room temperature preservation in drier.
The preparation and verifying of 6 NHS carbohydrate chip of embodiment
By taking Man-1, Man-2 and NHS slide as an example, specific preparation process is as follows for carbohydrate chip:
(1) by 20M solution Man-1 (weigh 1.8mg Man-1, be dissolved in 25% DMF aqueous solution, DMF: aqua sterilisa=
100 μ L:300 μ L) and Man-2 (2.2mg Man-2 is weighed, is dissolved in 25% DMF aqueous solution, DMF: aqua sterilisa=100 μ L:
300 μ L) solution and blank control group 25% DMF aqueous solution, with point sample instrument by 3 groups of solution point on NHS slide respectively
Sample, repetition point sample is multiple, each sampling point accommodates 1nL solution；Slide after point sample is placed on as early as possible (50% in constant temperature humidity cabinet
~60% humidity) it is incubated overnight.
(2) after carbohydrate chip is incubated overnight, the fixed 2h of vacuum in the vacuum desiccator that temperature is 60 DEG C.
(3) it after the carbohydrate chip after fixation being taken out cooling, is placed in the chromatography ware equipped with 1 × PBST of 100mL and vibrates clearly
It washes 2 times (5min/ times)；After 1 × PBST solution once cleans, change second of cleaning again again of chip upper and lower position；Twice 1
It after × PBST is cleaned, then is cleaned 2 times with 1 × PBS, each 5min.Vibrating the oscillator used is the multi-purpose vibration of HY-2 speed regulation
Swing device, chip placement direction should be parallel with orientation of oscillation when oscillation, chip uniform force when guaranteeing oscillation cleaning.Ensure chip
It after cleaning up, is dried using miniature slide dryer, revolving speed controls within 75r/min, guarantees two sides drying.
(4) chip after drying is placed in Block buffer (1 × PBS containing 1%BSA, 0.1% polysorbas20 of about 600 μ L
Buffer) in, and form chip upper interior space solution can with uniform rotation and without multiple minute bubbles, to guarantee its flowing
Property；Finally being shelved on temperature is 25 DEG C, and humidity closes 1h under conditions of being 25%, will not be tied on slide by the closed process
Close the position closing of glycosyl group.
(5) chip that closing terminates is taken out, the cleaning process of (3) is repeated.
(6) take 20 μ L Cy3 labelled protein Con A, the hydroxylamine hydrochloride of 30 μ L 4M, 300 μ L 2 × hybridization buffer and
The aqua sterilisa of 250 μ L mixes, and is finally made into 600 μ L mixed solutions, is added in chip hybridization box, with made carbohydrate chip into
Row hybridization；Slowly rotated in hybridization case with 4r/min speed (temperature: 25 DEG C, humidity: 25%, hybridization time: 3h)；After hybridization
Carbohydrate chip dries after 1 × PBST and 1 × PBS cleaning.
(7) carbohydrate chip is scanned using chip scanner, laser intensity is selected spotted area after 100% prescan, accurately
Scanning.Scanning result shows that, greater than 0.5 compared with the fluorescent value before cleaning after cleaning, carbohydrate chip is made.
Embodiment 7 detects salmonella using carbohydrate chip
Epoxidation slide and NHS slide is selected to react to prepare carbohydrate chip, then with above-mentioned 3 kinds of mannose derivatives respectively
With salmonella ATCC31685 strain hybrid, fluorescence signal intensity is detected with chip scanner.
As a result as shown in Figure 1, Fig. 1 (a) is the carbohydrate chip prepared using NHS slide, the mannose wherein in 1-4 column is derivative
Object is Man-1, and 5-8 is classified as Man-2, and 9-12 is classified as Man-3, and every bore dia is about 180 μm；Difference can be detected in three kinds of carbohydrate chips
The fluorescence signal of intensity is 1830 using Man-3, uses wherein the use of the fluorescent value of the carbohydrate chip of Man-1 preparation being 3100
Man-2 is 1050.Fig. 1 (b) is the carbohydrate chip prepared using epoxy slide, and wherein the mannose derivative in 1-4 column is
Man-1,5-8 are classified as Man-2, and 9-12 is classified as Man-3, and every bore dia is about 180 μm, and the concentration of three kinds of mannose derivatives is
10mmol/L；The fluorescence signal of varying strength can be detected in three kinds of carbohydrate chips, wherein the fluorescence of the carbohydrate chip using Man-1 preparation
Value is 850, the use of Man-3 is 450, is detected using the fluorescence signal of Man-2 close to background value, it is difficult to capture.Comparison diagram 1
(a) (b) can have found, the combination effect of epoxy slide and three kinds of mannose derivatives is not so good as NHS slide；Different mannose chemical combination
Carbohydrate chip made by object is different from the binding ability of salmonella, sramana in conjunction with carbohydrate chip made by NHS slide and Man-1
Salmonella ability is most strong.
Two kinds of salmonella ATCC9184 and ATCC31685 to mannose with different affinity are chosen, bacterium is dilute
It releases in chip hybridization solution, is detected using NHS slide and carbohydrate chip made by Man-1.As a result as shown in Fig. 2, Fig. 2
(a) bacterium detected is ATCC31685, and the bacterium of Fig. 2 (b) detection is ATCC9184, is 10 in bacterial concentration9cells·mL-1
When, (Mean Fluorescence is about with fluorescence signal intensity of the lower ATCC9184 bacterial strain of mannose affinity after carbohydrate chip detects
It is 230, close to background value) far below the ATCC31685 bacterial strain 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 slide and Man-1, it, then will be after hybridization by the hybridization hatching 1h at room temperature of the chip after closing
Slide takes out, and is successively cleaned 2 times (5min/ times) with 1 × PBST and 1 × PBS buffer solution, then clean 1 (5min/ with ultrapure water
It is secondary), to wash away the cell on non-hybridized；Then shadow of the sugared carbohydrate chip prepared to the fluorescence signal of detection of various concentration is measured
It rings, the bacteria concentration of salmonella detected is 109Cells/mL, as a result as shown in Figure 3.
In Fig. 3,1-12 column sugared concentration successively reduce, 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 increased with the raising of sugared concentration, in 10mmol/L
When, fluorescence intensity nearly reaches maximum value, this shows that fixed mannose in carbohydrate chip has been saturated, even if it is dense to increase sugar
Degree, fluorescence intensity are not also further added by；When 7th column (white box institute marked position in Fig. 3) represent sugar concentration as 313 μm of ol/L, still may be used
Detect the fluorescence signal higher than background value.In addition, in Fig. 4, after the Man-1 sugar juice production 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 fluorescence signal detected is with bacterial concentration in figure
It reduces and reduces.When ATCC31685 bacterial strain concentration is greater than or equal to 106When cells/mL, fluorescence signal can be detected；Continue to drop
After low bacterial strain concentration, fluorescence signal is difficult to detect, it is known that the minimum Bacteria Detection of this experiment carbohydrate chip detection salmonella
Concentration is 106cells/mL。
The result shows that carbohydrate chip technology can be applied to Salmeterol fluticasone propionate, and (313 μm of ol/L) can under low sugar concn
It detects salmonella, the consumption of glycogen material can be saved.In addition, existing carbohydrate chip is configured to manual construction, since aperture is big,
More than 200 samples can only be put on same slide, therefore this kind of carbohydrate chip can only be known as " more arrays " (Multiarray) mostly, with
" microarray " (Microarray) there are also larger gaps.And the present invention can construct carbohydrate chip, each structure using auto sample applicator
The point sample aperture built is 180-200 μm, can greatly increase reticular density, and thousands of can be put on the same slide
Sample realizes real high throughput.Therefore, greater number of parallel laboratory test can be established to which detection is a large amount of by carbohydrate chip technology
Sample is compared with traditional method of detecting bacterium, saves plenty of time and raw material.
Embodiment 8 screens agglutinin FimH antagonist using carbohydrate chip
It is related that related document shows that agglutinin FimH and enteric bacteria stick epithelial cell, can cause the diseases such as urinary tract infections
Disease；A variety of mannose derivatives and mannose (under the conditions of micromole) can block bacterial adhesion (FimH is protein mediated),
It thus can be used as the therapeutic agent of related disease.
Choose three kinds it has been reported that antagonist (can be effectively suppressed 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- mannopyranose glycosides (p-NPMan) and
It is a kind of it is covalent key connection D-MANNOSE amphiphilic polymers molecule PGMA-Mannose (PGMA, polymethylacrylic acid shrink it is sweet
Grease).Chip hybridization solution is the solution combined for detection bacterium and carbohydrate chip, if antagonist can be hindered when being incubated for
Disconnected bacterium is in conjunction with chip, then this antagonist has preferable receptor combination.
Shown using result detected by carbohydrate chip of the invention (Fig. 5): the concentration of three kinds of antagonists is different, lower
Concentration represents inhibits antibacterial effect more excellent accordingly, and PGMA-Mannose (50 μm of ol/L) inhibits the effect of bacterium to be 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
Carbohydrate chip antagonist detected effect and report it is consistent: p-NPMan is a kind of better antagonist compared to mannose,
It can form stable interaction with large biological molecule, and have been demonstrated can for the mannose compound with aryl, heteroaryl or heterocycle
It is used to inhibit urinary tract infections caused by Escherichia coli 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 monosaccharide and salmonella.
Provable by above-mentioned experiment, carbohydrate chip of the invention can be used for detecting antagonist to the inhibiting effect of agglutinin,
And quantitative analysis is carried out to it, to accelerate the progress of bacterial adhesion antagonist, providing one kind for new drug development has
Effect tool.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (8)
1. application of the carbohydrate chip in detection salmonella or screening salmonella antagonist, the application is non-diagnostic method
Using the carbohydrate chip is coupled to obtain by the solid phase carrier of chemical modification with mannose derivative, and the mannose derivative is
1- amino-ethyl-α-D- mannopyranose polyglycoside, the surface of solid phase carriers of the chemical modification are modified with N- hydroxysuccinimidyl acyl
Imido grpup.
2. application according to claim 1, it is characterised in that: the solid phase carrier is slide or silicon wafer.
3. application according to claim 1, it is characterised in that: the salmonella is salmonella ATCC9184, sramana
Salmonella ATCC31685, salmonella S.typhimurium LB5010, salmonella S.typhimurium AJB3, Salmonella
One or more of bacterium ATCC 14028 and salmonella S.typhimurium SL1344.
4. application according to claim 1, it is characterised in that: the lower limit of the carbohydrate chip salmonella that can be detected is dense
Degree is 106cells/mL。
5. application according to claim 1, which is characterized in that the preparation of the carbohydrate chip the following steps are included:
The mannose derivative is dissolved in organic solvent and obtains mixed solution, then by the mixed solution in the chemistry
Point sample on modified solid phase carrier, the solid phase carrier after point sample are incubated for 8-12h under 50%~60% humidity at 15-35 DEG C.
6. application according to claim 5, it is characterised in that: the preparation of the carbohydrate chip further includes carrying out egg after incubation
The step of white marker.
7. application according to claim 5, it is characterised in that: in the mixed solution, the concentration of mannose derivative is
10μmol/L-20mmol/L。
8. application according to claim 5, it is characterised in that: the organic solvent is selected from dehydrated alcohol, glacial acetic acid and N,
One or more of 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 CN106771231A (en) | 2017-05-31 |
| CN106771231B true 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) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114594253A (en) * | 2020-12-03 | 2022-06-07 | 中国科学院大连化学物理研究所 | Method for screening carbohydrate chip of carbohydrate binding protein competitive inhibitor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6660484B2 (en) * | 1992-11-13 | 2003-12-09 | Regents Of The University Of California | Colorimetric glycopolythiophene biosensors |
-
2016
- 2016-11-23 CN CN201611046680.6A patent/CN106771231B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| 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;王雪玉 等;《生物工程学报》;20161025;第32卷(第10期);第1362-1371页 |
| 糖芯片的研究进展;邹兰 等;《有机化学》;20091231;第29卷(第11期);第1689-1699页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106771231A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| 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 | |
| CN108802374A (en) | Excretion body nucleic acid detection technique based on magnetic enrichment electrochemical luminescence | |
| CN104597230B (en) | A kind of functional polymer film, preparation method and applications | |
| WO2008006373A1 (en) | Solid-phase detection of terminal monosaccharides cleaved from glycosylated substrates | |
| CN105699650B (en) | Carbofuran bio-barcode immune analytic reagent kit and its application | |
| CN104181221B (en) | Medicine target capturing method | |
| CN110004148B (en) | Alternariol aptamer affinity column and preparation method and application thereof | |
| CN106540473A (en) | A kind of method that aptamer modified magnetic photonic crystal microsphere is enriched with ochratoxin A | |
| CN104689806B (en) | Functional mesoporous SiO2/TiO2Composite nano-microsphere solid phase extraction column and preparation method thereof | |
| CN106771231B (en) | Application of the carbohydrate chip in detection salmonella or screening salmonella antagonist | |
| CN107698614A (en) | A kind of carborane diimide derivative and synthetic method and the sensor array based on it and preparation method and application | |
| CN105651992A (en) | Triazophos bio-barcode immunoassay determination kit and application thereof | |
| CN104897826A (en) | Method for detecting interaction of small molecular compound and target protein at cell in-situ level | |
| CN114113582B (en) | Metal organic framework nanoenzyme biological probe and ELISA kit | |
| CN106399317A (en) | DNA (deoxyribonucleic acid) aptamer of phthalate type plasticizer and screening and expression method and electro-chemical sensor thereof | |
| CN108982842A (en) | A kind of zearalenone pretreatment reagent kit using immunomagnetic bead technique | |
| RU2597768C2 (en) | Method of functionalising surfaces for detecting analytes | |
| CN102288763B (en) | Immunofluorescence method and special kit for detecting ractopamine based on quantum dots | |
| US7611888B2 (en) | Rotaxane compound, rotaxane-bonded solid substrate, and biochip using the same | |
| CN105297141A (en) | Micro-array chip detection method for 10 on-melon diseases and used chip probe | |
| CN107505419A (en) | One kind is used to be enriched with, purify, detect AFB1Modification inverse opal photonic crystal microballoon and its preparation method and application | |
| JP2021533778A (en) | Nucleic acid isolation and related methods | |
| CN104931687A (en) | Three-dimensional biological surface, preparation method thereof, three-dimensional biological chip and application of three-dimensional biological chip | |
| WO2011150542A1 (en) | Process for labeling antibody with copper oxide nanoparticle, kit and use thereof | |
| KR100761881B1 (en) | Versatile Linker Compound, Ligand and Producing Method Thereof |
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 |