CN107632156A - Detect Escherichia coli O 157:H7 upper conversion immuno-chromatographic test paper strip and detection method - Google Patents
Detect Escherichia coli O 157:H7 upper conversion immuno-chromatographic test paper strip and detection method Download PDFInfo
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Abstract
The invention provides one kind to detect Escherichia coli O 157:H7 upper conversion immuno-chromatographic test paper strip and its detection method, the test strips include sample pad, pad, analyzing film, adsorptive pads, the sample pad and pad are glass fibre element film, the analyzing film is nitrocellulose filter, the adsorptive pads are cellulose membrane, the analyzing film is provided with detection line and nature controlling line, and the sample pad, pad, adsorptive pads are pasted onto in PVC board successively, and up-conversion luminescence nano-particle Escherichia coli O 157 is fixed with the pad:H7 monoclonal antibody IgG conjugates, the detection line on the analyzing film are fixed with Escherichia coli O 157:H7 antigens, the nature controlling line are fixed with MAbF1.The sensitivity of Immunofluorescence test paper strip of the present invention based on up-conversion nano material is low, and detection range is wider, signal stabilization, and no photobleaching, background is low, and signal is sensitive, as a result accurately and reliably.
Description
Technical field
The invention belongs to pathogenic microorganisms detection technique field, and Escherichia coli O 157 is detected more particularly, to one kind:H7's
Upper conversion-immuno-chromatographic test paper strip and detection method.
Background technology
Escherichia coli O 157:H7 is a kind of pathogenic microorganism of important infecting both domestic animals and human, people's ehec infection O157:H7
The severe complications such as diarrhoea, hemorrhagic colitis, uremia can be suffered from, severe patient can cause death.O157:H7 breaks out stream because having
Row trend, the features such as strong pathogenic and lethal and antibiosis extract for treating may aggravate one's illness, it has also become global
Public health problem.
ICA be used to detect human chorionic gonadotropin (HCG) in 1980, show specific good, sensitivity
The advantages of high.From then on, researcher constantly explores to ICA technologies, existing ICA can be widely applied to biomolecule, infect because
Detection in terms of son and chemical pollutant.Classical immuno-sandwich method in ICA[64]It is wherein to apply most biology sample detections
Method.Immuno-sandwich method is divided into double antigens sandwich and double-antibody sandwich both of which.Double antigens sandwich pattern can be used for detecting
Antibody, double-antibody sandwich pattern is then used for the macromolecular antigen that detection at least has two antigenic determinants, therefore the latter is more suitable
For the situation of clinically detection machine body-sensing infectious pathogen, such as HBsAg, HBeAg, AFP.It is often used as ICA signal detection
Thing has nanogold, quantum dot and up-conversion nanoparticles.ICA based on nanogold is called colloidal gold immunity chromatography, it is not necessary to
Instrument can observes phenomenon, but it is normally only used for quantitative and semi-quantitative detection, it is difficult to accomplishes to quantify, this is to a certain degree
On limit the application of colloidal gold immunity chromatography.With the development of nanometer technology, the gradual quilt of up-conversion nano material (UCPs)
Applied in terms of ICA.ICA based on up-conversion nano material, because UCPs is excited in NIR region, the inanimate object sample at NIR
Product autofluorescence, no light scattering phenomenon, therefore background is low, signal is sensitive, as a result accurately.UCPs chemical property itself is stable, unglazed
Bleaching, so signal stabilization, these features are provided theories integration.Up-conversion nanoparticles can pass through a series of group
Dress and modification, material that obtain functionalization, that biocompatibility is good, this is conversion-immuno-chromatographic test paper strip on structure
(UCPs-ICA) Rapid detection test strip provides technical support associated with.Escherichia coli O 157 at present:H7 detection method is more
Colloidal gold strip method is limited as, this method can not meet quantitative, highly sensitive detection needs.
The content of the invention
In view of this, the present invention is directed to propose a kind of detection Escherichia coli O 157:H7 upper conversion-immuno-chromatographic test paper strip
And detection method, to improve Escherichia coli O 157:H7 detection sensitivity, detection range is wider, signal stabilization.
To reach above-mentioned purpose, the technical proposal of the invention is realized in this way：
One kind detection Escherichia coli O 157:H7 upper conversion-immuno-chromatographic test paper strip, the test strips include sample pad,
Pad, analyzing film, adsorptive pads, the sample pad and pad are glass fibre element film, and the analyzing film is nitrocellulose
Film, the adsorptive pads are cellulose membrane, and the analyzing film is provided with detection line and nature controlling line, the sample pad, pad, water suction
Pad is pasted onto in PVC board successively；
Up-conversion luminescence nano-particle-Escherichia coli O 157 is fixed with the pad:H7 monoclonal antibody IgG conjugates；
Detection line on the analyzing film is fixed with Escherichia coli O 157:H7 antigens, the nature controlling line are fixed with MAbF1.
Further, the sample pad, pad, analyzing film, adsorptive pads are successively with overlapping 1~2mm spacing alternation of bed
Folded to paste in PVC board, the width of the test strips is 4mm.
Described detection Escherichia coli O 157:The preparation method of H7 upper conversion-immuno-chromatographic test paper strip includes following step
Suddenly：
(1) NaYF of green light core shell structure4:Yb,Er/NaYF4The preparation of up-conversion nanoparticles；
S1：2~4mL oleic acid is added into round-bottomed flask, adds 2~5mmol rare earth ion chlorides, rare earth element
Molar ratio is Y:Yb:Tm=(50~70%):(5~15%):(0.5~2%), under the protection of argon gas, it is heated to rare earth
Powder is completely dissolved, and rapidly plus 8~13mL 1- octadecylenes, continuation argon gas are protected, and are heated to 160 DEG C, 15~25min of constant temperature, by
50 DEG C are gradually cooled to, 4~8mL is slowly added into dropwise and contains 3mmol NH4F, 2.5mmol NaOH methanol solution.Drip
Finish, be stirred for 30min, be slowly warming up to 250 DEG C, react 60min, obtained upper conversion NaYF4Core is dissolved in 5mL hexamethylenes,
Save backup；
S2：2~4mL oleic acid is added in three neck round bottom flask, then adds 0.5mmol YCl3·6H2O solid powders,
Under the protection of argon gas, it is heated to rare earth powder and is completely dissolved, adds 8~13mL 1- octadecylenes rapidly, be heated to 160 DEG C, constant temperature
15~25min, is gradually cooled to 50 DEG C, and alternating is added dropwise the cyclohexane solution of the above-mentioned nano-particles of 5mL, and 4~8mL dissolved with
3mmolNH4F, 2.5mmol NaOH methanol solution.After dripping, stirring reaction 30min, temperature is slowly increased to 250
DEG C, 60min is reacted, obtained up-conversion nanoparticles, which are dissolved in 10mL chloroforms, to be preserved.
(2)NaYF4:Yb,Er/NaYF4High molecular cladding；
1~5mg amphipathic nature block polymers accurately are weighed in round-bottomed flask, and it is 1 to add volume ratio：3 chloroform and second
The mixed solution of alcohol, the up-conversion nanoparticles that 200uL steps (1) prepare then are added, be well mixed, mixture is placed in
On Rotary Evaporators, 35 DEG C of constant temperature water baths are kept, decompression makes it slowly be evaporated, it is molten to add the weakly alkaline phosphate-buffereds of 5mL
Liquid, suspension is white, centrifuge washing 5~10 times, centrifuges the particle 10mL pH 7.4 of bottom of the tube phosphate buffer solution
Dissolving, obtain the neutral Poly-NaYF of pH4:Yb,Er/NaYF4Suspension, 4 DEG C be kept in dark place it is standby.
(3) up-conversion nanoparticles labelled antibody；
The up-conversion nanoparticles suspension having been dissolved in alkaline solution that 200uL has been modified is taken in centrifuge tube, is centrifuged
After 20min, abandoning supernatant, nano-particle is dissolved in 1mL pH 6.5 MES cushioning liquid, ultrasound is mixed, and addition is now matched somebody with somebody
1mg/mL EDC and 1.5mg/mL NHS, EDC and NHS volume ratio be 3:4,20min is stored at room temperature, 15min is centrifuged, discards
Clear liquid, the nano-particle activated is dissolved in 1mL pH 7.4 PB, ultrasound mixes, and adds 15uL 5mg/mL Escherichia coli
O157:H7 monoclonal antibodies react 30min, centrifuge 30min again after completion of the reaction, abandoning supernatant, be coupled the nanometer of monoclonal antibody into PB
Particle is dissolved in 1mL pH 7.4 PB, and ultrasound mixes, and obtains 1mL suspensions, 4 DEG C of refrigerators are kept in dark place stand-by.
(4) making of the test strips of immunochromatographic method is changed on；
Treated NC films are pasted onto first the position of 2.1 × 30cm in adhesive base, carry out coating, drying；Knot
The position that pad is pasted onto 0.7 × 30cm in adhesive base along lower section line of cut is closed, upper side pressure analyzing film lower end is overlapping with analyzing film
About 1mm；Sample pad is pasted onto in adhesive base, lower end is concordant with PVC edge lines, upper side pressure pad lower end, it is overlapping about
1mm, blotting paper is pasted onto in adhesive base along above PVC bottom plates, lower side pressure analyzing film upper end, overlapping about 2mm；Paste
The band of 30cm length is cut into every a width of 3mm band with cutting machine, and each band is in plastic clip.
(5) configuration of conjugate solution and coating；
1%Tween-20,0.5%BSA, 0.5%PVP-K30,1.5% sucrose, 1% aqueous trehalose volume ratio are taken respectively
For (1~3)：(2~4)：(2~4)：(0.5~2)：(1~5), and take out the final nano-particle suspensions marked of 100uL
Into above-mentioned solution, water or PB is added to be configured to the conjugate suspension that cumulative volume is 1mL, this conjugate configured is suspended
Liquid, it is uniformly dropped on pad, be placed in 25 DEG C of baking ovens and thoroughly dry 0.5h.
(6) configuration of Quality Control solution and detection solution and coating；
The MAbF1 PBS for being 50mM with the concentration of pH 7.4 are diluted to 1mg/mL by the step (6), as Quality Control solution,
Escherichia coli O 157:H7 monoclonal antibodies pH 7.4, concentration are that 50mM PBS is diluted to 1mg/mL, and as detection solution, NC films are glued
It is attached on plastic bottom board, Quality Control solution is drawn film instrument with three-dimensional planar with detection solution respectively and uniformly draws the nature controlling line on NC films
With the position of detection line, pull to be placed in 30 DEG C of baking ovens entire plate after film and dry 30min.
(7) double-antibody sandwich pattern UCPs-ICA quantitatively detects Escherichia coli O 157 in mouse serum:H7；
Escherichia coli O 157 in mouse serum is prepared with 50mM, pH=7.4 PBS:H7 standard liquid, standard liquid contain
Escherichia coli O 157:H7 concentration is followed successively by 0.1mg/mL, 1mg/mL, 10mg/mL, 50mg/mL, 100mg/mL, with pH=7.4
PBS do blank control test, take 40uL measurement solution to be added drop-wise in test strips and determine every time, after reacting 30min, be total to laser
Focusing microscope scans to ELISA test strip band and quality control band, fluorescence intensity.
Relative to prior art, detection Escherichia coli O 157 of the present invention:H7 upper conversion-immuno-chromatographic test paper strip
And detection method has the advantage that：
(1) sensitivity of the Immunofluorescence test paper strip of the present invention based on up-conversion nano material is low, detection range
It is wider, the advantages of UCPs is as fluorescence probe, such as signal stabilization are given full play to, no photobleaching, biological sample is at NIR without certainly
Body fluorescence, therefore background is low, signal is sensitive, as a result accurately and reliably.
(2) Immunofluorescence test paper strip of the present invention based on up-conversion nano material has higher specificity, on
Quick determination method associated with conversion-immuno-chromatographic test paper strip has larger practicality.
Brief description of the drawings
The accompanying drawing for forming the part of the present invention is used for providing a further understanding of the present invention, schematic reality of the invention
Apply example and its illustrate to be used to explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the principle schematic of the present invention；
Fig. 2 is the structural representation of the test strips described in the embodiment of the present invention；
It is that test strips form structural representation wherein to scheme A, and figure B is the pictorial diagram that test strips are being loaded in plastic clip
Fig. 3 is that the TEM of the up-conversion nanoparticles described in the embodiment of the present invention is characterized；
Wherein：Scheme (A) NaYF4:Yb, Er/NaYF4 TEM are characterized, and a left side is the figure under 0.2um sizes, and the right side is saturating for high-resolution
Radio mirror characterizes,
Scheme (B) NaYF4:Yb, Er/NaYF4 TEM are characterized, and a left side is non-bag macromolecule, and the right side is bag macromolecule；
Escherichia coli O 157 in Fig. 4 UCPs-ICA detection cushioning liquid:H7 fluorescence intensity comparison diagrams.
Embodiment
The present invention is described in detail with reference to embodiment and accompanying drawing.
Embodiment 1
One kind detection Escherichia coli O 157:H7 upper conversion-immuno-chromatographic test paper strip, the test strips include sample pad,
Pad, analyzing film, adsorptive pads, the sample pad and pad are glass fibre element film, and the analyzing film is nitrocellulose
Film, the adsorptive pads are cellulose membrane, and the analyzing film is provided with detection line and nature controlling line, the sample pad, pad, water suction
Pad is pasted onto in PVC board successively；
Up-conversion luminescence nano-particle-Escherichia coli O 157 is fixed with the pad:H7 monoclonal antibody IgG conjugates；
Detection line on the analyzing film is fixed with Escherichia coli O 157:H7 antigens, the nature controlling line are fixed with MAbF1.
Further, the sample pad, pad, analyzing film, adsorptive pads are successively with overlapping 1~2mm spacing alternation of bed
Folded to paste in PVC board, the width of the test strips is 4mm.
Described detection Escherichia coli O 157:The preparation method of H7 upper conversion-immuno-chromatographic test paper strip includes following step
Suddenly：
(3) NaYF of green light core shell structure4:Yb,Er/NaYF4The preparation of up-conversion nanoparticles；
S1：4mL oleic acid is added into round-bottomed flask, adds 3mmol rare earth ion chlorides, the mol ratio of rare earth element
Example is Y:Yb:Tm=60%:10%:1%, under the protection of argon gas, it is heated to rare earth powder and is completely dissolved, rapidly plus 8~
13mL 1- octadecylenes, continue argon gas protection, be heated to 160 DEG C, constant temperature 20min, be gradually cooled to 50 DEG C, be slowly added into dropwise
6mL contains 3mmol NH4F, 2.5mmol NaOH methanol solution.It is added dropwise, is stirred for 30min, is slowly warming up to 250
DEG C, react 60min, obtained upper conversion NaYF4Core is dissolved in 5mL hexamethylenes, is saved backup；
S2：3mL oleic acid is added in three neck round bottom flask, then adds 0.5mmol YCl3·6H2O solid powders,
Under the protection of argon gas, it is heated to rare earth powder and is completely dissolved, adds 10mL 1- octadecylenes rapidly, be heated to 160 DEG C, constant temperature
18min, 50 DEG C are gradually cooled to, the cyclohexane solution of the above-mentioned nano-particles of 5mL is added dropwise in alternating, and 6mL is dissolved with 3mmol
NH4F, 2.5mmol NaOH methanol solution.After dripping, stirring reaction 30min, temperature is slowly increased to 250 DEG C, reaction
60min, obtained up-conversion nanoparticles, which are dissolved in 10mL chloroforms, to be preserved.
(4)NaYF4:Yb,Er/NaYF4High molecular cladding；
2mg amphipathic nature block polymers accurately are weighed in round-bottomed flask, and it is 1 to add volume ratio：3 chloroform and ethanol
Mixed solution, the up-conversion nanoparticles that 200uL steps (1) prepare then are added, be well mixed, mixture is placed in rotation
On evaporimeter, 35 DEG C of constant temperature water baths are kept, decompression makes it slowly be evaporated, adds the weakly alkaline phosphate buffer solutions of 5mL, hangs
Turbid is white, centrifuge washing 10 times, and the particle for centrifuging bottom of the tube is dissolved with 10mL pH 7.4 phosphate buffer solution, is obtained
The Poly-NaYF neutral to pH4:Yb,Tm/NaYF4Suspension, 4 DEG C be kept in dark place it is standby.
(3) up-conversion nanoparticles labelled antibody；
The up-conversion nanoparticles suspension having been dissolved in alkaline solution that 200uL has been modified is taken in centrifuge tube, is centrifuged
After 20min, abandoning supernatant, nano-particle is dissolved in 1mL pH 6.5 MES cushioning liquid, ultrasound is mixed, and addition is now matched somebody with somebody
1mg/mL EDC and 1.5mg/mL NHS, EDC and NHS volume ratio be 3:4,20min is stored at room temperature, 15min is centrifuged, discards
Clear liquid, the nano-particle activated is dissolved in 1mL pH 7.4 PB, ultrasound mixes, and adds 15uL 5mg/mL Escherichia coli
O157:H7 monoclonal antibodies react 30min, centrifuge 30min again after completion of the reaction, abandoning supernatant, be coupled the nanometer of monoclonal antibody into PB
Particle is dissolved in 1mL pH 7.4 PB, and ultrasound mixes, and obtains 1mL suspensions, 4 DEG C of refrigerators are kept in dark place stand-by.
(4) making of the test strips of immunochromatographic method is changed on；
Treated NC films are pasted onto first the position of 2.1 × 30cm in adhesive base, carry out coating, drying；Knot
Close the position that pad is pasted onto 0.7 × 30cm in adhesive base along lower section line of cut, upper side pressure analyzing film lower end, with analyzing film weight
Folded about 1mm；Sample pad is pasted onto in adhesive base, lower end is concordant with PVC edge lines, upper side pressure pad lower end, it is overlapping about
1mm, blotting paper is pasted onto in adhesive base along above PVC bottom plates, lower side pressure analyzing film upper end, overlapping about 2mm；Paste
The band of 30cm length is cut into every a width of 3mm band with cutting machine, and each band is in plastic clip.
(5) configuration of conjugate solution and coating；
1%Tween-20,0.5%BSA, 0.5%PVP-K30,1.5% sucrose, 1% aqueous trehalose volume ratio are taken respectively
For 2：3：3：1：3, and the final nano-particle suspensions marked of 100uL are taken out into above-mentioned solution, add water or PB to be configured to
Cumulative volume is 1mL conjugate suspension, this conjugate suspension configured, it is uniformly dropped on pad, put
0.5h is thoroughly dried in 25 DEG C of baking ovens.
(6) configuration of Quality Control solution and detection solution and coating；
The MAbF1 PBS for being 50mM with the concentration of pH 7.4 are diluted to 1mg/mL by the step (6), as Quality Control solution,
Escherichia coli O 157:H7 monoclonal antibodies pH 7.4, concentration are that 50mM PBS is diluted to 1mg/mL, and as detection solution, NC films are glued
It is attached on plastic bottom board, Quality Control solution is drawn film instrument with three-dimensional planar with detection solution respectively and uniformly draws the nature controlling line on NC films
With the position of detection line, pull to be placed in 30 DEG C of baking ovens entire plate after film and dry 30min.
(7) double-antibody sandwich pattern UCPs-ICA quantitatively detects Escherichia coli O 157 in mouse serum:H7；
Escherichia coli O 157 in mouse serum is prepared with 50mM, pH=7.4 PBS:H7 standard liquid, standard liquid is containing big
Enterobacteria O157:H7 concentration is followed successively by 0.1mg/mL, 1mg/mL, 10mg/mL, 50mg/mL, 100mg/mL, with pH=7.4's
PBS does blank control test, takes 40uL measurement solution to be added drop-wise in test strips every time and determines, after reacting 30min, is copolymerized with laser
Focusing microscope scans to ELISA test strip band and quality control band, fluorescence intensity.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (10)
1. detect Escherichia coli O 157:H7 upper conversion-immuno-chromatographic test paper strip, it is characterised in that：The test strips include sample
Product pad, pad, analyzing film, adsorptive pads, the sample pad are glass fibre element film with pad, and the analyzing film is that nitric acid is fine
Tie up plain film, the adsorptive pads are cellulose membrane, and the analyzing film is provided with detection line and nature controlling line, the sample pad, pad,
Adsorptive pads are pasted onto in PVC board successively；
Up-conversion luminescence nano-particle-Escherichia coli O 157 is fixed with the pad:H7 monoclonal antibody IgG conjugates；
Detection line on the analyzing film is fixed with Escherichia coli O 157:H7 antigens, the nature controlling line are fixed with MAbF1.
2. detection Escherichia coli O 157 according to claim 1:H7 upper conversion-immuno-chromatographic test paper strip, its feature exist
In：The sample pad, pad, analyzing film, adsorptive pads paste PVC board with overlapping 1~2mm spacing interaction cascading successively
On, the width of the test strips is 4mm.
3. the detection Escherichia coli O 157 described in any one of claim 1~2:The system of H7 upper conversion-immuno-chromatographic test paper strip
Preparation Method, it is characterised in that：Comprise the following steps：
(1) NaYF of green light core shell structure4:Yb,Er/NaYF4The preparation of up-conversion nanoparticles；
(2)NaYF4:Yb,Er/NaYF4High molecular cladding；
(3) up-conversion nanoparticles labelled antibody；
(4) making of the test strips of immunochromatographic method is changed on；
(5) configuration of conjugate solution and coating；
(6) configuration of Quality Control solution and detection solution and coating；
(7) double-antibody sandwich pattern UCPs-ICA quantitatively detects Escherichia coli O 157 in mouse serum:H7.
4. detection Escherichia coli O 157 according to claim 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The preparation method of step (1) up-conversion nanoparticles is as follows：
S1：2~4mL oleic acid is added into round-bottomed flask, adds 2~5mmol rare earth ion chlorides, mole of rare earth element
Ratio is Y:Yb:Tm=(50~70%):(5~15%):(0.5~2%), under the protection of argon gas, it is heated to rare earth powder
It is completely dissolved, rapidly plus 8~13mL 1- octadecylenes, continuation argon gas are protected, and are heated to 160 DEG C, 15~25min of constant temperature, gradually cold
But to 50 DEG C, 4~8mL is slowly added into dropwise and contains 3mmol NH4F, 2.5mmol NaOH methanol solution.It is added dropwise, then
30min is stirred, is slowly warming up to 250 DEG C, reacts 60min, obtained upper conversion NaYF4Core is dissolved in 5mL hexamethylenes, is preserved standby
With；
S2：2~4mL oleic acid is added in three neck round bottom flask, then adds 0.5mmol YCl3·6H2O solid powders, in argon
Under the protection of gas, it is heated to rare earth powder and is completely dissolved, rapidly plus 8~13mL 1- octadecylenes, is heated to 160 DEG C, constant temperature 15~
25min, 50 DEG C are gradually cooled to, replace the cyclohexane solution that the above-mentioned nano-particles of 5mL are added dropwise and 4~8mL dissolved with 3mmol
NH4F, 2.5mmol NaOH methanol solution, after dripping, stirring reaction 30min, temperature is slowly increased to 250 DEG C, reaction
60min, obtained up-conversion nanoparticles, which are dissolved in 10mL chloroforms, to be preserved.
5. detection Escherichia coli O 157 according to claim 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The preparation method of step (2) is as follows：
1~5mg amphipathic nature block polymers accurately are weighed in round-bottomed flask, and it is 1 to add volume ratio：3 chloroform and ethanol
Mixed solution, the up-conversion nanoparticles that 200uL steps (1) prepare then are added, be well mixed, mixture is placed in rotation
On evaporimeter, 35 DEG C of constant temperature water baths are kept, decompression makes it slowly be evaporated, adds the weakly alkaline phosphate buffer solutions of 5mL, hangs
Turbid is white, centrifuge washing 5~10 times, and the particle for centrifuging bottom of the tube is dissolved with 10mL pH 7.4 phosphate buffer solution,
Obtain the neutral Poly-NaYF of pH4:Yb,Er/NaYF4Suspension, 4 DEG C be kept in dark place it is standby.
6. the detection Escherichia coli O 157 according to claim asks 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The labeling method of the step (3) is as follows：
The up-conversion nanoparticles suspension having been dissolved in alkaline solution that 200uL has been modified is taken in centrifuge tube, centrifuges 20min
Afterwards, abandoning supernatant, nano-particle is dissolved in 1mL pH 6.5 MES cushioning liquid, ultrasound mixes, and adds the 1mg/ now matched somebody with somebody
ML EDC and 1.5mg/mL NHS, EDC and NHS volume ratio are 3:4, it is stored at room temperature 20min, centrifuges 15min, abandoning supernatant,
The nano-particle activated is dissolved in 1mL pH 7.4 PB, ultrasound mixes, and adds 15uL 5mg/mL Escherichia coli O 157s:
H7 monoclonal antibodies react 30min, centrifuge 30min again after completion of the reaction, abandoning supernatant, be coupled the nano-particle of monoclonal antibody into PB
It is dissolved in 1mL pH 7.4 PB, ultrasound mixes, and obtains 1mL suspensions, 4 DEG C of refrigerators are kept in dark place stand-by.
7. the detection Escherichia coli O 157 according to claim asks 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The preparation method of step (4) test strips is as follows：
Treated NC films are pasted onto first the position of 2.1 × 30cm in adhesive base, carry out coating, drying；Pad
Be pasted onto the position of 0.7 × 30cm in adhesive base along lower section line of cut, upper side pressure analyzing film lower end, it is overlapping with analyzing film about
1mm；Sample pad is pasted onto in adhesive base, lower end is concordant with PVC edge lines, upper side pressure pad lower end, overlapping about 1mm,
Blotting paper is pasted onto in adhesive base along above PVC bottom plates, lower side pressure analyzing film upper end, overlapping about 2mm；The 30cm pasted
Long band is cut into every a width of 3mm band with cutting machine, and each band is in plastic clip.
8. the detection Escherichia coli O 157 according to claim asks 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The configuration of step (5) the conjugate solution and method for coating are as follows：
1%Tween-20,0.5%BSA, 0.5%PVP-K30,1.5% sucrose are taken respectively, and 1% aqueous trehalose volume ratio is (1
~3)：(2~4)：(2~4)：(0.5~2)：(1~5), and the final nano-particle suspensions marked of 100uL are taken out to upper
State in solution, add water or PB is configured to the conjugate suspension that cumulative volume is 1mL, this conjugate suspension configured,
It is uniformly dropped on pad, is placed in 25 DEG C of baking ovens and thoroughly dries 0.5h.
9. the detection Escherichia coli O 157 according to claim asks 3:The preparation side of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：The MAbF1 PBS for being 50mM with the concentration of pH 7.4 are diluted to 1mg/mL by the step (6), as Quality Control
Solution, Escherichia coli O 157:H7 monoclonal antibodies pH 7.4, concentration be 50mM PBS be diluted to 1mg/mL, as detection solution,
NC films are pasted onto on plastic bottom board, Quality Control solution is drawn film instrument with three-dimensional planar with detection solution respectively and uniformly drawn on NC films
Nature controlling line and detection line position, pull to be placed in 30 DEG C of baking ovens entire plate after film and dry 30min.
10. the detection Escherichia coli O 157 according to claim asks 3:The preparation of H7 upper conversion-immuno-chromatographic test paper strip
Method, it is characterised in that：Step (7) the double-antibody sandwich pattern UCPs-ICA quantitatively detects Escherichia coli in mouse serum
O157:H7 specific detection method is as follows：
Escherichia coli O 157 in mouse serum is prepared with 50mM, pH=7.4 PBS:H7 standard liquid, standard liquid bar containing large intestine
Bacterium O157:H7 concentration is followed successively by 0.1mg/mL, 1mg/mL, 10mg/mL, 100mg/mL, and blank pair is done with pH=7.4 PBS
According to experiment, take 40uL measurement solution to be added drop-wise in test strips every time and determine, after reacting 30min, with laser confocal microscope pair
ELISA test strip band and quality control band scanning, fluorescence intensity.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441219A (en) * | 2018-03-16 | 2018-08-24 | 中肽生化有限公司 | A kind of upconverting fluorescent material preparation method of size tunable |
| CN109444422A (en) * | 2018-12-17 | 2019-03-08 | 福建省立医院 | A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II |
| CN109596827A (en) * | 2019-01-17 | 2019-04-09 | 长江师范学院 | Fluorescence detection test strip and its preparation method and application that is a kind of while detecting 4 kinds of pathogenic bacteria |
| CN111190012A (en) * | 2020-02-22 | 2020-05-22 | 南京申基医药科技有限公司 | Rare earth up-conversion fluorescent nano test strip for novel coronavirus detection and preparation method thereof |
| CN111308068A (en) * | 2020-03-06 | 2020-06-19 | 苏州缔因安生物科技有限公司 | Detection test paper based on up-conversion luminescent nano material and preparation method and application thereof |
| CN111735957A (en) * | 2020-06-24 | 2020-10-02 | 深圳市光与生物科技有限公司 | Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology |
| CN113056674A (en) * | 2018-09-25 | 2021-06-29 | 悉尼科技大学 | Quantification of analytes |
| CN113238047A (en) * | 2021-05-10 | 2021-08-10 | 深圳市光明区疾病预防控制中心 | Graphene quantum dot immunochromatography test strip for rapidly detecting Escherichia coli O157H 7 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375060A (en) * | 2010-08-19 | 2012-03-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Food borne pathogenic bacteria detection test paper disc based on up-converting phosphor ten-channel immunochromatography |
| CN104449662A (en) * | 2014-11-11 | 2015-03-25 | 合肥工业大学 | Surface modification method of oil-soluble upconversion luminescent nanometer material |
| CN105486854A (en) * | 2015-09-29 | 2016-04-13 | 南方医科大学 | Signal amplification method and related apparatus |
-
2017
- 2017-09-14 CN CN201710825322.3A patent/CN107632156A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375060A (en) * | 2010-08-19 | 2012-03-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Food borne pathogenic bacteria detection test paper disc based on up-converting phosphor ten-channel immunochromatography |
| CN104449662A (en) * | 2014-11-11 | 2015-03-25 | 合肥工业大学 | Surface modification method of oil-soluble upconversion luminescent nanometer material |
| CN105486854A (en) * | 2015-09-29 | 2016-04-13 | 南方医科大学 | Signal amplification method and related apparatus |
Non-Patent Citations (2)
| Title |
|---|
| GUICHENG，JIANG，ET AL.: "An Effective Polymer Cross-Linking Strategy To Obtain Stable Dispersions of Upconverting NaYF4 Nanoparticles in Buffers and Biological Growth Media for Biolabeling Applications", 《LANGMUIR》 * |
| 谭贵良等: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441219A (en) * | 2018-03-16 | 2018-08-24 | 中肽生化有限公司 | A kind of upconverting fluorescent material preparation method of size tunable |
| CN113056674A (en) * | 2018-09-25 | 2021-06-29 | 悉尼科技大学 | Quantification of analytes |
| CN109444422A (en) * | 2018-12-17 | 2019-03-08 | 福建省立医院 | A kind of detection card and its construction method and application based on UPT-LF quantitative determination blood-serum P IVKA-II |
| CN109596827A (en) * | 2019-01-17 | 2019-04-09 | 长江师范学院 | Fluorescence detection test strip and its preparation method and application that is a kind of while detecting 4 kinds of pathogenic bacteria |
| CN111190012A (en) * | 2020-02-22 | 2020-05-22 | 南京申基医药科技有限公司 | Rare earth up-conversion fluorescent nano test strip for novel coronavirus detection and preparation method thereof |
| CN111308068A (en) * | 2020-03-06 | 2020-06-19 | 苏州缔因安生物科技有限公司 | Detection test paper based on up-conversion luminescent nano material and preparation method and application thereof |
| CN111735957A (en) * | 2020-06-24 | 2020-10-02 | 深圳市光与生物科技有限公司 | Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology |
| CN113238047A (en) * | 2021-05-10 | 2021-08-10 | 深圳市光明区疾病预防控制中心 | Graphene quantum dot immunochromatography test strip for rapidly detecting Escherichia coli O157H 7 |
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