CN106248931A - A kind of antibiotic and antibacterial flash chromatography based on this antibiotic detection card - Google Patents
A kind of antibiotic and antibacterial flash chromatography based on this antibiotic detection card Download PDFInfo
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- CN106248931A CN106248931A CN201610642106.0A CN201610642106A CN106248931A CN 106248931 A CN106248931 A CN 106248931A CN 201610642106 A CN201610642106 A CN 201610642106A CN 106248931 A CN106248931 A CN 106248931A
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- antibiotic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The present invention relates to a kind of utilize can combination Non-specific with bacteria cell wall antibiotic and utilize this antibiotic as labelling molecule make flash chromatography detection card.Described antibiotic is molecule or the nano material labelling with certain optical properties, described flash chromatography detection card is many empty membrane materials, it is fixed with the antibody of specific bacterial on film, changes antibody and can form sandwich structure with the nano material of antibacterial and antibiotic marker, it is achieved detection.This advantage utilizing low cost little molecule antibiotic, it is achieved many sites of single antibacterial combine, and improve detection sensitivity, save the use of another one antibody simultaneously, greatly reduce testing cost.
Description
Technical field
The invention belongs to field of biochemistry detection, relate to a kind of antibiotic and antibacterial flash chromatography based on this antibiotic
Detection card.
Background technology
Malignant bacteria can invade host, including being adsorbed in body surface, invades tissue or cell, growth and breeding, produces toxin, be
Spread and resist the series of defence function of host to diffusion, cause body injury, therefore quick to common causative antibacterial
Detection seems particularly necessary.Traditional its morphological characteristic of the pathogenic microorganism examination Main Basis and physiological character, need to carry out carefully
Bacterium separates, cultivates and a series of biochemical reaction, and with duration, program is loaded down with trivial details, and some antibacterial tends not to be given preferably qualification
As a result, practice also encounters many problems.In recent years, the immunofluorescence technique of development, Enzyme-multiplied immune technique,
The immunological methods such as radioimmunoassay technique are the most progressively applied to the detection of pathogenic bacterium, but still suffer from complex operation, and sensitivity is on the low side
Etc. problem.And needed for comparing traditional method, instrument and equipment requirement is high, greatly limit the application of these methods and pushes away
Extensively, its practical value is greatly reduced.Fast-bacteria-detection card is due to the multiple antibody sandwich of needs at present, it is impossible to realize multiple inspection
Survey, and the use of multiple antibody also improves testing cost.Therefore, development multiple rapid detection card more at a low price will be that property causes
The important research direction of pathogenic bacteria Fast Detection Technique, breaks out effectively prevention and control pathogenic bacterium, alleviates pathogenic bacterium to the mankind
The harm caused plays a significant role.
Summary of the invention
For the problems referred to above, the invention provides a kind of antibacterial flash chromatography based on antibiotic detection card.This antibiotic
Can be specific binding with various bacteria surface molecular, by colour developing molecular marker, make each antibacterial with numerous labelling
Molecule, and owing to the labelling of antibacterial uses general antibiotic, as long as therefore by using difference anti-on T line on detection card
Body, can be achieved with Multiple detection, reduces testing cost while improving detection sensitivity.
The present invention is realized by following technical scheme:
A kind of can the antibiotic of combination Non-specific with bacteria cell wall, described antibiotic is can be with bacteria cell wall specificity
In conjunction with antibiotic, including vancomycin, norvancomycin, ciprofloxacin and modifier thereof, penicillin and modifier thereof.
Preferably, described antibiotic comprises molecule or the nano material label with certain optical properties.
Preferably, described label include on common fluorescent molecular, fluorescence shifting molecular and fluorescent molecule and
Being the fluorescent nano particle building basis by it, described label also includes gold colloidal, coloured microsphere.
Preferably, the particle diameter of described label is 1 nm-100 nm.
Present invention also offers antibacterial flash chromatography based on above-mentioned antibiotic detection card, described detection card is by base plate, sample
Product pad, gold mark pad, nitrocellulose filter and absorbent paper assemble；Described base plate has viscosity, sample pad, gold mark pad, nitric acid
Cellulose membrane and absorbent paper are all pasted onto on base plate；Described sample pad be blank glass fiber or through phosphate buffer or albumen or
High polymer and combinations thereof processes and dried glass fibre, has 2-3mm between described sample pad and described nitrocellulose filter
Overlap；Described gold mark pad connects sample pad and nitrocellulose filter, and gold mark pad one end is pasted onto below sample pad one end at nitre
Above acid cellulose, and have that 2-3mm's is overlapping between the two, gold mark pad sprays colour development material；Described colour development material is antibiosis
The molecule with certain optical properties of element labelling or nano material；Secure on described nitrocellulose filter and identify antibacterial
Antibody, as detection line, secures bacterial polysaccharides molecule as nature controlling line；Absorbent paper is pasted onto the another of described nitrocellulose filter
One end, has between described absorbent paper with described nitrocellulose filter that 2-3mm's is overlapping.
The antibiotic provided in the present invention can be specific binding with various bacteria surface molecular, by colour developing molecular marker,
Make each antibacterial with numerous labelling molecule, and owing to the labelling of antibacterial uses general antibiotic, if therefore logical
Cross and the T line on detection card is used different antibodies, can be achieved with Multiple detection, while improving detection sensitivity, reduce inspection
Survey expense.Antibacterial flash chromatography based on the above-mentioned antibiotic detection card provided in invention, simple in construction, it is possible to achieve multiple inspection
Survey the advantage high with sensitivity, the expense of detection can be effectively reduced, and improve the efficiency of detection.Owing to resisting only with one
Body, therefore testing cost can be greatly lowered, and the specific binding little molecule of antibacterial also can improve detection specificity.Additionally,
Each antibacterial can in conjunction with the antibody that combines merely of little molecular proportion many, detection sensitivity also can improve much accordingly.
Accompanying drawing explanation
Fig. 1 is that the method for the invention is to staphylococcus aureus testing result；
Fig. 2 is that the method for the invention is to E. Coli results figure.
Detailed description of the invention
Below by specific embodiment, technical scheme is described in detail, but the scope of the present invention
It is not restricted by the embodiments.
Embodiment 1
The present embodiment illustrates as a example by the vancomycin detection staphylococcus aureus of fluorescent nano material converted modification:
(1) up-conversion fluorescence nanoparticle (UCNPs) labelling and gold mark pad preparation
Particle diameter is the EDC/NHS side of Y2O3:Yb, the E r up-conversion fluorescence nanoparticle employing standard of 50 nm Surface coating carboxyls
Method coupling vancomycin.EDC (4 is added in 2 mL contain the MES buffer (10 mmol/L, pH5.5) of 5 mg UCNPs
Mmol/L), NHS (10 mmol/L), to activate the carboxyl on UCNPs surface；In 30 ° of C concussion reaction 30min；Centrifugal, use 10 mM
Phosphate buffer (PBS, pH 7. 5) washs three times, is centrifuged the granule got off and is dispersed in 2 mL to contain 0.5 mg people the most mould
In the PBS (10 mmol/L, pH 7. 5) of element, on shaking table, 30 ° of C react 2 h, add 15 mg BSA (Sanguis Bovis seu Bubali
Albumin) close unreacted NHS；Wash 2 times with PBS, centrifugation, surface must be arrived and be connected with the UCNPs of vancomycin, dispersion
Contain 0.1 % BSA at 1 mL, 10% sucrose PBS in, be then sprayed on blank glass fiber make gold mark
Pad.
(2) detection line and nature controlling line
Detection line (T line) is prepared with 1cm/s speed setting-out with the Mus monoclonal antibody (1mg/mL) for staphylococcus aureus, nature controlling line
(C line) is prepared with 1cm/s speed setting-out with staphylococcus aureus lysate (1mg/mL).
(3) prepared by sample pad
Dissolve the BSA of 1% with the borate buffer of the 50mM that PH is 8.5, soak with glass fibre after drying standby.
(4) test strips assembles
Sample pad, gold mark pad, nitrocellulose membrane, absorbent paper are pasted on end liner successively according to 2-3mm overlap, and with cutting cutter and
For 3mm strip, after packaging, it is dried room temperature preservation.
(5) sample detection
Sample to be tested 1mLPBS is mixed, takes thereafter 100-150 μ L and be added in the sample pad of detection card detect.After loading
Within 5 minutes, detect with fluorescence chart scanner, as it is shown in figure 1, staphylococcus aureus negative (red) and positive (green) testing result,
Toilet seat peak is T line position, and peak, the right is C line position.
Embodiment 2
The present embodiment illustrates as a example by the ciprofloxacin detection escherichia coli that gold colloidal is modified:
(1) colloid gold label and gold mark pad preparation
The EDC/NHS method coupling ciprofloxacin of employing standard.MES buffer (10 mmol/ of 5 mg BSA are contained at 2 mL
L, pH5.5) the middle EDC (4 mmol/L) that adds, NHS (10 mmol/L), ciprofloxacin 10 mg, in 30 ° of C concussion reaction 1 h；
20 KD bag filters, 10 mM phosphate buffers (PBS, pH 7. 5) are dialysed 5 times, obtain the BSA containing ciprofloxacin labelling.
Taking 1mL colloidal gold solution to be marked and regulate PH to 7-8 with the K2CO3 of 0.1M, adding 10 μ L concentration thereafter is 1
The BSA of the ciprofloxacin labelling of mg/mL is marked, and after room temperature 30 min, the bovine serum albumin solution of addition 10% is dense to it
Degree is 1%.Room temperature stands 12000g after 1 h, 4 DEG C of centrifugal 1 h and takes precipitation, wash with the 10mM PBS containing 1%BSA, 10% sucrose
It is sprayed on blank glass fiber after washing 3 times and makes gold mark pad.
(2) detection line and nature controlling line
Detection line (T line) is prepared with 1cm/s speed setting-out with for colibacillary Mus monoclonal antibody (1mg/mL), nature controlling line (C line)
Prepare with 1cm/s speed setting-out with E. coli lysate (1mg/mL).
(3) prepared by sample pad
Dissolve the BSA of 1% with the borate buffer of the 50mM that PH is 8.5, soak with glass fibre after drying standby.
(4) test strips assembles
Sample pad, gold mark pad, nitrocellulose membrane, absorbent paper are pasted on end liner successively according to 2-3mm overlap, and with cutting cutter and
For 3mm strip, after packaging, it is dried room temperature preservation.
(5) sample detection
Sample to be tested 1mLPBS is mixed, takes thereafter 100-150 μ L and be added in the sample pad of detection card detect, after loading
The detection of 5 minutes naked eyes, result is as in figure 2 it is shown, escherichia coli negative (left) and the positive (right) testing result.
It is pointed out that above two embodiment is explanation of the invention, be not the restriction to invention,
In the case of the spirit of the present invention, the present invention can make any type of amendment, and such as simple heating system is more
Change or the change of reactor all should be within technical solution of the present invention protection domain.
Claims (5)
1. one kind can the antibiotic of combination Non-specific with bacteria cell wall, it is characterised in that: described antibiotic be can and antibacterial
The antibiotic that cell wall is specific binding, including vancomycin, norvancomycin, ciprofloxacin and modifier thereof, penicillin
And modifier.
The most as claimed in claim 1 can the antibiotic of combination Non-specific with bacteria cell wall, it is characterised in that described antibiotic
Comprise molecule or the nano material label with certain optical properties.
The most as claimed in claim 2 can the antibiotic of combination Non-specific with bacteria cell wall, it is characterised in that described label
It is the fluorescence nano building basis including shifting molecular on common fluorescent molecular, fluorescence and fluorescent molecule and by it
Grain, described label also includes gold colloidal, coloured microsphere.
The most as claimed in claim 4 can the antibiotic of combination Non-specific with bacteria cell wall, it is characterised in that described label
Particle diameter be 1 nm-100 nm.
5. an antibacterial flash chromatography based on the arbitrary described antibiotic of claim 1-4 detection card, described detection card by base plate,
Sample pad, gold mark pad, nitrocellulose filter and absorbent paper assemble；Described base plate has viscosity, sample pad, gold mark pad, nitre
Acid cellulose film and absorbent paper are all pasted onto on base plate；Described sample pad is blank glass fiber or through phosphate buffer or albumen
Or high polymer and combinations thereof processes and dried glass fibre, between described sample pad and described nitrocellulose filter, there is 2-
The overlap of 3mm；Described gold mark pad connects sample pad and nitrocellulose filter, and gold mark pad one end is pasted onto one end below sample pad and exists
Above celluloid, and have that 2-3mm's is overlapping between the two, gold mark pad sprays colour development material；Described colour development material is anti-
The molecule with certain optical properties of raw element labelling or nano material；Identification antibacterial is secured on described nitrocellulose filter
Antibody as detection line, secure bacterial polysaccharides molecule as nature controlling line；Absorbent paper is pasted onto described nitrocellulose filter
The other end, has between described absorbent paper with described nitrocellulose filter that 2-3mm's is overlapping.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610642106.0A CN106248931A (en) | 2016-08-08 | 2016-08-08 | A kind of antibiotic and antibacterial flash chromatography based on this antibiotic detection card |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610642106.0A CN106248931A (en) | 2016-08-08 | 2016-08-08 | A kind of antibiotic and antibacterial flash chromatography based on this antibiotic detection card |
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| CN201610642106.0A Pending CN106248931A (en) | 2016-08-08 | 2016-08-08 | A kind of antibiotic and antibacterial flash chromatography based on this antibiotic detection card |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107478833A (en) * | 2017-08-22 | 2017-12-15 | 西北农林科技大学 | A kind of sensitive probe and preparation method and the method using its detection Bacterium enteritidis |
| CN111505278A (en) * | 2020-04-15 | 2020-08-07 | 西北农林科技大学 | Staphylococcus aureus detection test strip, detection method and application |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107478833A (en) * | 2017-08-22 | 2017-12-15 | 西北农林科技大学 | A kind of sensitive probe and preparation method and the method using its detection Bacterium enteritidis |
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| CN111505278A (en) * | 2020-04-15 | 2020-08-07 | 西北农林科技大学 | Staphylococcus aureus detection test strip, detection method and application |
| CN113281507A (en) * | 2021-05-23 | 2021-08-20 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
| CN113281507B (en) * | 2021-05-23 | 2022-08-16 | 吉林大学 | Rapid detection method and kit for staphylococcus aureus |
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Application publication date: 20161221 |