CN108333348B - Test strips and preparation method thereof for detecting chloramphenicol - Google Patents
Test strips and preparation method thereof for detecting chloramphenicol Download PDFInfo
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- CN108333348B CN108333348B CN201810027812.3A CN201810027812A CN108333348B CN 108333348 B CN108333348 B CN 108333348B CN 201810027812 A CN201810027812 A CN 201810027812A CN 108333348 B CN108333348 B CN 108333348B
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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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
- G01N33/543—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
- G01N33/553—Metal or metal coated
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The present invention relates to a kind of test strips and preparation method thereof for detecting chloramphenicol, it include wherein support plate for detecting the test strips of chloramphenicol, it arranges in support plate nitrocellulose membrane, it arranges from bottom to top from nitrocellulose membrane bonding pad and sample pad one end of nitrocellulose membrane, water absorption pad is laid on the nitrocellulose membrane other end opposite with bonding pad, detection line and control line are drawn in nitrocellulose membrane, nanogold-chloramphenicol adaptor complex is added on bonding pad, detection line is made by the complementary probe solution A for combining the biotin modification of Streptavidin；Control line is crossed by the probe B solution for combining Streptavidin and is made；There is preferable consistency, stability and sensibility using test strips made from above method.
Description
Technical field
The present invention relates to use Lateral Flow Strip to identify chloramphenicol, and in particular to a kind of for detecting the test strips of chloramphenicol
And preparation method thereof.
Background technique
Chloramphenicol belongs to a kind of antibiotic of high-efficiency broad spectrum, but chloramphenicol has stronger toxic side effect, long-term to take in
The a variety of bacterial strains such as Escherichia coli, salmonella can be made to generate drug resistances, be easy to cause animal body flora imbalance, under resistance
Drop.China provides detect chloramphenicol in the edible tissues of all food animals, therefore, it is necessary to it is a kind of quickly, spirit
The method of chloramphenicol in quick detection biotic environment.
Antibody test chloramphenicol is generallyd use, using antibody as the bio-identification factor, is known using the specificity of antigen-antibody
It carry out not chloramphenicol detection.But antibody higher cost, it has not been convenient to need to generate in living body when saving, and preparing antibody and exempt from
Epidemic disease, lot stability are poor.Currently, more research steerings detect chloramphenicol using aptamer, that is, pass through one section of process
The oligonucleotide sequence or short polypeptide that in-vitro screening obtains can carry out high-affinity and strong specific with corresponding ligand
In conjunction with.If the applying date is on June 10th, 2015, application publication number is that one kind disclosed in the Chinese patent of CN104865216A is based on
The method of nanogold aptamer colorimetric detection chloramphenicol, by gold nano grain, can be to chloramphenicol specific recognition
Aptamer and cationic polymer are mixed to form stable compound；Solution and compound to be measured combine release cation poly-
Close object；Gold nano grain agglomerates into larger particles under the action of cationic polymer, causes gold particle cluster not
The color of the cylinder shown at co-wavelength；The concentration of chloramphenicol in sample to be tested is determined according to the variation of color.But it is used
Be solution colour developing method, that is, need situ configuration solution, carry out chromogenic reaction in the solution.In practical applications more not
Just.
Summary of the invention
The object of the present invention is to provide a kind of for detecting the test strips of chloramphenicol, with preferable consistency, stabilization
Property and sensibility.
Above-mentioned technical purpose of the invention has the technical scheme that a kind of for detecting chloramphenicol
Test strips, including support plate, nitrocellulose membrane of arranging in support plate, one end of nitrocellulose membrane is from nitrocellulose membrane
It arranges from bottom to top bonding pad and sample pad, is laid with water absorption pad on the nitrocellulose membrane other end opposite with bonding pad, nitrification
Detection line and control line are drawn on cellulose membrane,
Bonding pad is coated with nanogold-chloramphenicol adaptor complex, and the nucleotides sequence of chloramphenicol aptamers is classified as 3 '-AGC-
AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-TTT-
TTT-TTT-5 ', 5 ' ends use sulfydryl modification；
Detection line dressing probe A compound, probe A compound by Streptavidin and biotin modification probe solution A
It is mixed to prepare, the nucleotides sequence of probe A is classified as 5 '-CGC-CAT-CTC-TCC-ATG-CCC-TCC-TGT-CCG-TGC-CCT-
Biotin modification is used at CAG-TCA-TCT-GAC-CTC-TGT-GCT-GCT-3 ', 3 ' ends；
Control line dressing probe B compound, probe B compound by Streptavidin and biotin modification probe B solution
It is mixed to prepare, the sequence of probe B is 3 '-AAA-AAA-AAA-5 ', and 3 ' ends use biotin modification.
The application detects chloramphenicol using chloramphenicol aptamers, and uses test strips method, from aptamers library
Suitable chloramphenicol aptamers are screened, the chloramphenicol aptamers a part and chloramphenicol complementary pairing, another part and probe B are mutual
It recruits pair；Its middle probe A and chloramphenicol and chloramphenicol aptamers competitive binding.The nucleotide sequence of the chloramphenicol aptamers of selection
Centainly, preferable using this method detection chloramphenicol consistency.
In addition, the application use test strips method, determinand drip in sample pad, determinand solution successively pass through bonding pad,
Detection line and control line develop the color.If containing chloramphenicol in determinand, when determinand flows through bonding pad, with the chlorine on bonding pad
Mycin aptamers combine；When continuing to flow to detection line and control line in conjunction with the determinands of chloramphenicol aptamers, probe B and chloramphenicol
Aptamers combine, and develop the color at control line.If being free of chloramphenicol in determinand, determinand flows through bonding pad, chloramphenicol aptamers after
At afterflow to detection line and control line, develop the color at detection line and control line.It is easy to operate using this method, it is only necessary to will be to be measured
Sample drop is in sample pad.
Further, nanogold-chloramphenicol adaptor complex is made as follows:
Chloramphenicol aptamers and nano-Au solution are mixed with volume ratio for the ratio of 1:5-6, are incubated at room temperature 6.5-8h, are added
Sodium chloride to concentration is 45-50mmol/L；Continue to activate 11-12h at room temperature, be centrifuged under the revolving speed of 6000-7000r/min
10-15min, obtained concentrate are nanogold-chloramphenicol adaptor complex.
Further, the partial size of nanogold is 15-35nm.
It is verified by test of many times, it, can be further in the nanogold in the particle size range in conjunction with chloramphenicol aptamers
Improve the detection sensitivity of test strips.
Further, nanogold is made using trisodium citrate reduction gold chloride, and trisodium citrate and gold chloride are with volume
Than the ratio mixing for 2-2.5:1, under the revolving speed of 300-350r/min, temperature is to stir 11-12min at 100-110 DEG C, is made
Obtain nano-Au solution.
It is preferable using the activity of nanogold particle made from above method, convenient for being combined with the chloramphenicol aptamers of sulfhydrylation.
Further, nanogold-chloramphenicol adaptor complex is coated on bonding pad, and in 35-37 DEG C of drying 7-
12min。
Using above technical scheme, preparation nanogold-chloramphenicol is combined to be adapted to nanogold and chloramphenicol aptamers in advance
Nanocrystal composition, nanogold-chloramphenicol adaptor complex are transferred on bonding pad according to above-mentioned condition, and the activity of nanogold is kept
Relatively persistently.
Further, probe A compound is made as follows: 18-20 μ L concentration is that the strepto- of 2-3mg/mL is affine
Element mixes 2-2.5h with the probe solution A for the biotin modification that 8-10 μ L concentration is 90-100 μm of ol/L at room temperature.
Using above technical scheme, probe A significantly improves detection sensitivity in conjunction with Streptavidin.
Further, Streptavidin uses non-sulfhydrylation Streptavidin.
The compatibility of the Streptavidin and biotin is more preferable, can be in conjunction with more biotins.
Further, the detection range of chloramphenicol is 0.5-160 μ g/mL.
Another object of the present invention is to provide a kind of for detecting the preparation method of the test strips of chloramphenicol, has inspection
Survey it is sensitive, can directly observe the characteristics of testing result.
Above-mentioned purpose of the invention is achieved by the following technical programs: a kind of for detecting the test strips of chloramphenicol
Preparation method includes the following steps:
Nitrocellulose membrane, bonding pad, sample pad and water absorption pad are separately fixed in support plate, sample pad, bonding pad it
Between be overlapped 1.5-2.5mm, support board width is 4-4.5mm；Detection line and control line are drawn in support plate；
It is spare in 35-37 DEG C of drying 7-12min that nanogold-chloramphenicol adaptor complex is infiltrated on bonding pad；
2-2.5h is incubated at room temperature with the probe solution A and Streptavidin of chloramphenicol aptamers partial complementarity, and spy is made
Needle A compound, and be coated at detection line；
The probe B solution complementary with chloramphenicol aptamers another part and Streptavidin are incubated at room temperature 2-2.5h system
Probe B compound is obtained, and is coated at control line；
Nitrocellulose membrane is placed on 37 DEG C of dry 8-12h, is put in kept dry at 4 DEG C.
Test strips are made according to method as above, it is easy to operate, and largely improve the detection spirit of test strips
Sensitivity.
Further, sample pad uses bovine serum albumin Seal treatment in vain.
Using above technical scheme, nanogold is closed, the non-specific binding of nanogold and sample pad is reduced, mentions
The detection sensitivity of high test strips.
In conclusion the invention has the following advantages:
1, test strips produced by the present invention are more sensitive to color change, restore gold chloride system by using trisodium citrate
Standby nanogold, while cooperating different drying temperatures during preparing test strips, and use bovine serum albumin envelope in vain
Processing is closed, the non-specific binding of nanogold is reduced, so that the visual sensitivity of final test strips can reach 0.5ppm, readout instrument
Detection limit can reach 63.4ppb；
2, wider range of test strips detection chloramphenicol produced by the present invention, passes through the concentration to chloramphenicol aptamers, chain
The particle size of the type of mould Avidin, the type of sealer and concentration and nanogold is adjusted, final test paper obtained
Item is able to detect the range of chloramphenicol between 0.5-160 μ g/mL；
3, test strips consistency produced by the present invention is preferable, selects the chloramphenicol adaptation that the nucleic acid sequence by screening is certain
Body, using competitive binding method, final testing result is observable by naked eyes, easy to operate.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of test strips；
Fig. 2 is the test schematic of test strips；
Fig. 3 is chloramphenicol standard curve；
In figure, 1, support plate；2, nitrocellulose membrane；3, bonding pad；4, sample pad；5, water absorption pad；6, detection line；7, it controls
Line processed.
Specific embodiment
Invention is further described in detail with reference to embodiments.
Embodiment one: it is a kind of for detecting the test strips of chloramphenicol, as shown in Figure 1, including the support plate 1 of strip, branch
1 upper surface of fagging fixes and supports plate 1 with wide nitrocellulose membrane 2, and nitrocellulose membrane 2 is located at the middle part of support plate 1.Nitre
The end of the top of cellulose film 2 along its length is arranged bonding pad 3 and sample upwards in turn from nitrocellulose membrane 2
Product pad 4, sample pad 4 are located at the top of bonding pad 3 and are staggered arrangement with bonding pad 3.Bonding pad 3 and nitrocellulose membrane 2, knot
The width that laminates for closing pad 3 and sample pad 4 is 2mm, laminates width also and can choose 1.5mm or 2.5mm；Nitrocellulose membrane
The width that laminates of fixed water absorption pad 5 above 2 the other end along its length, water absorption pad 5 and nitrocellulose membrane 2 is 2mm.
The detection line 6 and control line 7 vertical with the length direction of nitrocellulose membrane 2, inspection are printed in nitrocellulose membrane 2
Survey line 6 is close to sample pad 4, detection line 6 and control line 7 at a distance of 5mm.
The width of support plate 1 can select between 4-4.5mm in the test strips, the coating detection target in sample pad 4,
When detecting length direction flowing of the target along test strips, the liquid velocity of edge and intermediate flow keeps almost the same.
In the present embodiment support plate 1 be PVC material, sample pad 4 use the materials such as glass fibre, polyester film, be convenient for and to
Sample combines；Absorption pad is oriented to sample to be tested with imbibition effect using filter paper.
Be coated on bonding pad 3 combines the nanogold formed-chloramphenicol to be adapted to bluk recombination by nanogold and chloramphenicol aptamers
Object, chloramphenicol aptamers can be in conjunction with chloramphenicol molecular pairs；
It is compound that the probe A formed after being combined by Streptavidin and probe A is coated in nitrocellulose membrane 2 at detection line 6
Object, the number of base sequence complementary pairing of middle probe A and chloramphenicol aptamers；
It is compound that the probe B formed after being combined by Streptavidin and probe B is coated at 2 upper control line 7 of nitrocellulose membrane
Another part base sequence complementary of object, middle probe B and chloramphenicol aptamers matches；
Detection target is added in sample pad 4, and flowing buffer solution is added, so that detection target is along nitrocellulose membrane 2
Length direction it is mobile (as shown in figure 1 shown in arrow direction) to detection line 6 and 7 direction of control line, observation colour developing situation, and exempting from
It is read on epidemic disease chromatography readout instrument.
One, chloramphenicol aptamers, the probe B at probe A and control line 7 at detection line 6 are selected
1, the nucleotide sequence (Sequence ID No.1) of chloramphenicol aptamers are as follows: 3 '-AGC-AGC-ACA-GAG-
GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG- GCG-TTT-TTT-TTT-5 ', 5 '
End uses sulfydryl modification, and in order to combine with nanogold, which is synthesized by Shanghai Sangon Biotech Company.
2, the probe A(Sequence ID No.2 at detection line 6), the nucleotide sequence of probe A are as follows: 5 '-CGC-CAT-
CTC-TCC-ATG-CCC-TCC-TGT-CCG-TGC-CCT-CAG-TCA-TCT-GAC-CTC- TGT-GCT-GCT-3 ', 3 ' ends are adopted
It with biotin modification, is combined with facilitating with Streptavidin, which is synthesized by Shanghai Sangon Biotech Company.
3, the probe B at control line 7, the nucleotide sequence of probe B are as follows: 3 '-AAA-AAA-AAA-5 ', 3 ' using biology
Element modification.
Two, application method
Chloramphenicol aptamers and nanogold self assembly, which combine, forms nanogold-chloramphenicol adaptor complex, and is coated on
At bonding pad 3；
Probe A forms Streptavidin-probe A compound after mixing with Streptavidin, is coated at detection line 6.
Probe B forms Streptavidin-probe B compound after mixing with Streptavidin, is coated at control line 7.
Detection target is added at sample pad 4, and flowing buffer solution is added, so that detection target is to detection line 6 and controls
7 direction of line is mobile.
Three, test strips principle
The testing principle of test strips is as shown in Figure 2.If detecting and not including chloramphenicol in target, detection target and nanogold-
Chloramphenicol adaptor complex reaches at detection line 6 and control line 7, in conjunction with the probe A in detection line 6, and with control line 7
In probe B combine；Final detection line 6 and control line 7 develop the color jointly, and result is feminine gender.
If in target solution including chloramphenicol, target solution and nanogold-chloramphenicol adaptor complex reach detection line 6
Before, the chloramphenicol in target solution is in conjunction with adaptor complex, when it is reached at detection line 6, can not in conjunction with probe A or
Person and probe A weak binding；Finally detection line 6 does not develop the color or develops the color and is unobvious, and control line 7 develops the color, and result is the positive.
Embodiment two: it is a kind of for detecting the preparation method of the test strips of chloramphenicol, include the following steps:
One, aptamers and nanogold are self-assembly of adaptor complex
1, the preparation of nanogold
1mL1% gold chloride is diluted to 100mL, the trisodium citrate that 2mL mass percent is 1% is added, 350r/min's
Under revolving speed, temperature is to stir 11min at 100 DEG C, and the nano-Au solution that average grain diameter is 15nm is made；
2, nanogold-chloramphenicol adaptor complex preparation
5 ' ends of chloramphenicol aptamers (Sequence ID No.1) use sulfydryl modification (Shanghai Sangon Biotech Company's synthesis)
Nano-Au solution in step 1 modulates pH=7.4 using the NaOH of 0.01mol/L, under conditions of 10000r/min
It is centrifuged 15min, discards supernatant liquid, it is spare that concentration nanogold is made；
10 μ L concentration are that the chloramphenicol aptamers of 10 μm of ol/L and the concentration nanogold of 500 μ L are stirred, and are incubated in room temperature
Educating 8h 2mol/LNaCl is added later to concentration is 50mmol/L, and after activating 12h at room temperature, mixture is in 6500r/min
Revolving speed under be centrifuged 15min, discard supernatant liquid be made nanogold-chloramphenicol adaptor complex it is spare.Nanogold-chloramphenicol is suitable
Ligand complex is coated on bonding pad 3, and in 37 DEG C of dry 7min.
Two, at detection line 6 probe A compound preparation
The Streptavidin that the probe A for the biotin modification that 10 μ L concentration are 100 μm of ol/L and 20 μ L concentration are 2mg/mL
It is stirred, reacts 2 hours obtained probe A compounds at normal temperature.It is test strips scribing line using colloidal gold spotting system, and
In 37 DEG C of drying and processings.
Three, at control line 7 probe B compound preparation
The Streptavidin that the probe B that 10 μ L concentration are 100 μm of ol/L is 2mg/mL with 20 μ L concentration is stirred, normal
Temperature is lower to react 2 hours obtained probe B compounds.It is test strips scribing line using colloidal gold spotting system, and at 37 DEG C of drying
Reason.
Four, the Seal treatment of test strips
In order to reduce test strips to the non-specific adsorption of nanogold, sample pad 4 is impregnated using sample pad sealer in advance,
Flowing buffer solution also uses sealer to do Seal treatment simultaneously.Sample pad sealer uses concentration molten for 1% bovine serum albumin(BSA)
Liquid, flowing buffer solution select containing mass percent to be 1% bovine serum albumin(BSA), the phosphoric acid buffer of 2% sucrose and 0.5% Tween-20
Liquid.
Five, it tests
5 μ L nanogold-chloramphenicol adaptor complex is coated on bonding pad 3, and in 37 DEG C of drying 10min；Detection
2 μ L probe A compounds are coated at line 6, control line 7 is coated with 2 μ L probe B compounds.
It takes detection target of the 5 μ L without chloramphenicol to drip in sample pad 4, and 55 μ L flowing is added dropwise in sample pad 4 and delays
Fliud flushing, so that detection target is mobile to detection line 6 and 7 direction of control line.After 20min, detection line 6 and control line are visually observed
7 common colour developings, and use immune chromatograph readout instrument test absorbance value for 357.3.
Comparative example
For the color developing effect for improving test strips, following comparative example is set respectively to the partial size of nanogold, chloramphenicol aptamers
Concentration, the type of sealer, the type of Streptavidin, the type of nitrocellulose membrane 2 be adjusted.Institute specific as follows
Show:
Comparative example 1: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is chlorine
It is different that mycin is adapted to bulk concentration.
(1) chloramphenicol adaptation bulk concentration is 50 μm of ol/L；Its final absorbance value is 482.0.
(2) chloramphenicol adaptation bulk concentration is 100 μm of ol/L, and final absorbance value is 522.7.
The chloramphenicol of various concentration can be detected using the test strips.
Comparative example 2: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is to seal
Close the type and concentration difference of agent.
(1) sealer selects mass percent for 0.25% bovine serum albumin(BSA), the phosphoric acid of 2% sucrose and 0.5% Tween-20
Buffer, final absorbance value are 506.8；
(2) sealer selects mass percent slow for the phosphoric acid of 0.5% bovine serum albumin(BSA), 2% sucrose and 0.5% Tween-20
Fliud flushing, final absorbance value are 481.3；
(3) sealer selects mass percent for 1% bovine serum albumin(BSA), the phosphoric acid buffer of 2% sucrose and 0.5% Tween-20
Liquid, final absorbance value are 456.1；
(4) sealer selects mass percent for 0.25% casein, the phosphate buffer of 2% sucrose and 0.5% Tween-20,
Its final absorbance value is 121.5；
(5) sealer selects mass percent for 0.5% casein, the phosphate buffer of 2% sucrose and 0.5% Tween-20,
Its final absorbance value is 51.1；
(6) sealer selects mass percent for 1% casein, the phosphate buffer of 2% sucrose and 0.5% Tween-20,
Final absorbance value is 28.7；
(7) sealer selects mass percent slow for the phosphoric acid of 0.25% chicken egg white, 2% sucrose and 0.5% Tween-20
Fliud flushing, final absorbance value are 51.5；
(8) sealer selects mass percent for 0.5% chicken egg white, the phosphoric acid buffer of 2% sucrose and 0.5% Tween-20
Liquid, final absorbance value are 12.3；
(9) sealer selects mass percent for 1% chicken egg white, the phosphoric acid buffer of 2% sucrose and 0.5% Tween-20
Liquid, final absorbance value are 7.06.
From the above results, select bovine serum albumin(BSA) as sealer, the absorbance value finally measured is relatively high, examination
The color developing effect of paper slip is preferable.
Comparative example 3: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is to seal
The detent position for closing agent is different, select concentration for 1% bovine serum albumin(BSA) as sealer.
(1) sealer infiltration is only added in sample pad 4, final absorbance value is 429.43；
(2) concentration that sealer is only added to bovine serum albumin(BSA) in buffer is 1%, and final absorbance value is
444.77；
(3) sealer is added in sample pad 4 and buffer, final absorbance value is 522.9.
It can be seen from the above result that sealer is all added in sample pad 4 and buffer, color developing effect is preferable, when actually detected,
Sealer is added in sample pad 4 and buffer.
Comparative example 4: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is chain
The type of mould Avidin is different.
(1) select the GSA of Pan Gu's gene biological Engineering stock Co., Ltd as Streptavidin, pH=7 are final to inhale
Shading value is 509.4；
(2) select the GSA of Pan Gu's gene biological Engineering stock Co., Ltd as Streptavidin, pH=8 are final to inhale
Shading value is 454.3；
(3) select the GSA of Pan Gu's gene biological Engineering stock Co., Ltd as Streptavidin, pH=9 are final to inhale
Shading value is 216.3；
(4) select the SA of Nanjing Pan Gu biotech firm as Streptavidin, pH=7, final absorbance value is 385.9；
(5) select the SA of Nanjing Pan Gu biotech firm as Streptavidin, pH=7, final absorbance value is 385.1.
When detecting using the test strips, select the GSA of Pan Gu's gene biological Engineering stock Co., Ltd affine as strepto-
The color developing effect of element, test strips is preferable.
Comparative example 5: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is nitre
The type of cellulose film 2 is different.
(1) select the Millopore 135 of the outstanding Bioisystech Co., Ltd in Shanghai as nitrocellulose membrane 2, finally
Absorbance value is 464.3；
(2) select the Sartorius 140 of the outstanding Bioisystech Co., Ltd in Shanghai as nitrocellulose membrane 2, finally
Absorbance value is 448.6.
When actually detected, select Millopore 135 as nitrocellulose membrane 2.
Comparative example 6: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is to receive
The partial size of meter Jin is different.It is specific as follows:
(1) gold chloride that 1mL mass percent is 1% is diluted to 100mL, and the lemon that 2.5mL mass percent is 1% is added
Sour trisodium stirs 12min at 100 DEG C under 320r/min revolving speed, and the nano-Au solution that average grain diameter is 25nm is made；
10 μ L concentration are that the chloramphenicol aptamers of 10 μm of ol/L and the concentration nanogold of 600 μ L are stirred, and are incubated in room temperature
Educating 6.5h 2mol/LNaCl is added later to concentration is 45mmol/L, and after activating 12h at room temperature, mixture is in 7000r/
It is centrifuged 10min under the revolving speed of min, it is spare to discard supernatant the obtained nanogold-chloramphenicol adaptor complex of liquid；The suction finally measured
Shading value is 506.7.
(2) gold chloride that 1mL mass percent is 1% is diluted to 100mL, and the citric acid that 2mL mass percent is 1% is added
Trisodium stirs 11min at 110 DEG C under 300r/min revolving speed, and the nano-Au solution that average grain diameter is 35nm is made；
10 μ L concentration are that the chloramphenicol aptamers of 10 μm of ol/L and the concentration nanogold of 600 μ L are stirred, and are incubated in room temperature
Educating 6.5h 2mol/LNaCl is added later to concentration is 45mmol/L, and after activating 12h at room temperature, mixture is in 7000r/
It is centrifuged 10min under the revolving speed of min, it is spare to discard supernatant the obtained nanogold-chloramphenicol adaptor complex of liquid；Final absorbance value
It is 660.
Average grain diameter is that the nanogold of 15-35nm all has preferable color developing effect.
Comparative example 7: a kind of for detecting the preparation method of the test strips of chloramphenicol, the difference with embodiment two is to read
The number time is different.It is specific as follows:
(1) after sample pad 4,5min is read detection target drop；Final absorbance value is 513.2；
(2) after sample pad 4,10min is read detection target drop；Final absorbance value is 684.9；
(3) after sample pad 4,15min is read detection target drop；Final absorbance value is 522.6；
(4) after sample pad 4,25min is read detection target drop；Final absorbance value is 519.6；
(5) after sample pad 4,35min is read detection target drop；Final absorbance value is 517.6.
From the above data, it reads, develops the color more stable, detection effect is more preferably after 20min.
It verifies by test of many times, finally obtains:
(1) bovine serum albumin(BSA) of sealer selection 0.9-1.1%；
(2) closed optimum way be by be all added in buffer and sample pad 4 1% bovine serum albumin(BSA)；
(3) GSA, pH 7 of Pan Gu's gene biological Engineering stock Co., Ltd are selected；
(4) nitrocellulose membrane 2 selects the Millopore 135 of the outstanding Bioisystech Co., Ltd in Shanghai；
(5) select the partial size of nanogold for 15-35nm；
(6) test strips are preferably read after 20min.
Test strips for detecting chloramphenicol are tested under this condition, and final testing result has preferable consistent
Property, stability and sensibility.
Embodiment three: chloramphenicol Specification Curve of Increasing
Taking concentration is the chloramphenicol standard reserving solution of 1mg/mL, and being configured to concentration with acetonitrile is respectively 0.5 μ g/mL, 1 μ
g/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL、120μg/mL、160μg/
The standard curve of the standard solution of mL, 180 μ g/mL and 200 μ g/mL, specific concentration and absorbance value is as shown in Figure 3.
It can be seen from the above result that the detection range of chloramphenicol is between 0.5-160 μ g/mL.And detection is quick-eyed
Degree is 0.5ppm, and readout instrument detection is limited to 63.4ppb(, and 3 methods calculate according to international law).
Example IV: the chloramphenicol in detection water
Tap water is crossed, 10,80 and 160 μ g/mL standard chloramphenicol are added after film, the 5 μ L samples is taken to be added to sample pad 4
On, and add 55 μ L buffers that sample is made to flow through 6 position of detection line, test strips are put into chart scanner (C11787 MOD after 20min
L1 reading obtains absorbance value in), and all results are repeated 3 times, and concrete outcome is as shown in table 1.
1 recovery of standard addition test result of table
| Detection project | Chloramphenicol concentration (μ g/mL) | Absorbance | Absorbance corresponding chloramphenicol concentration (μ g/mL) on standard curve | Recovery of standard addition (%) |
| 1 | 10 | 449.9 | 8.575 | 85.75 |
| 2 | 80 | 315.7 | 88.566 | 110.71 |
| 3 | 160 | 175.7 | 172.013 | 107.51 |
Recovery of standard addition is finally obtained between 85.7%-110.68%, and relative standard deviation is 0.2707%-
0.7437%, it meets the requirements, illustrates that the method can be used for detecting the chloramphenicol in actual sample.
The calculation formula of recovery of standard addition are as follows: recovery of standard addition=actual measurement chloramphenicol concentration/standard chloramphenicol concentration * 100%.
Embodiment five: specific test
Taking Florfenicol, concentration that blank sample (tap water), concentration are 100nmol/L respectively is 100nmol/L methyl sulfone
Mycin, concentration are 100nmol/L aflatoxin B1 and concentration is that 100nmol/L chloramphenicol is added drop-wise in test strips, observation examination
Test phenomenon.
Only chloramphenicol display color absorption value is 292.3, identical as notional result；Blank sample, Florfenicol, methyl sulfone are mould
The color absorption value of element and aflatoxin B1 is respectively 514.2,500.567,503.0,506.1.It follows that the test strips
Only there is specificity to chloramphenicol.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
SEQUENCE LISTING
<110>Beijing University of Chemical Technology
<120>for detecting test strips of chloramphenicol and preparation method thereof
<130> 2018.1.11
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 66
<212> DNA
<213>artificial sequence
<400> 1
agcagcacag aggtcagatg actgagggca cggacaggag ggcatggaga gatggcgttt 60
tttttt 66
<210> 2
<211> 57
<212> DNA
<213>artificial sequence
<400> 2
cgccatctct ccatgccctc ctgtccgtgc cctcagtcat ctgacctctg tgctgct 57
Claims (6)
1. a kind of for detecting the test strips of chloramphenicol, including support plate (1), nitrocellulose membrane of arranging in support plate (1)
(2), one end of nitrocellulose membrane (2) is arranged bonding pad (3) and sample pad (4) from bottom to top from nitrocellulose membrane (2),
Water absorption pad (5) are laid in nitrocellulose membrane (2) and bonding pad (3) opposite other end, detection is drawn in nitrocellulose membrane (2)
Line (6) and control line (7), it is characterised in that:
Bonding pad (3) is coated with nanogold-chloramphenicol adaptor complex, the nucleotide sequence of chloramphenicol aptamers are as follows:
3'-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-
The end ATG-GCG-TTT-TTT-TTT-5', 5' is modified using base is dredged；
Detection line (6) dressing probe A compound, probe A compound are mixed by the probe solution A of Streptavidin and biotin modification
It closes and is made, the nucleotide sequence of probe A are as follows:
5'-CGC-CAT-CTC-TCC-ATG-CCC-TCC-TGT-CCG-TGC-CCT-CAG-TCA-TCT-GAC-CTC-TGT-
The end GCT-GCT-3', 3' biotin modification；
Control line (7) dressing probe B compound, probe B compound are mixed by the probe B solution of Streptavidin and biotin modification
It closes and is made, the sequence of probe B are as follows:
The end 3'-AAA-AAA-AAA-5', 3' uses biotin modification；
Nanogold-chloramphenicol adaptor complex is made as follows:
Chloramphenicol aptamers and nano-Au solution are mixed with volume ratio for the ratio of 1:5-6, are incubated at room temperature 6.5-8h, chlorination is added
Sodium to concentration is 45-50mmol/L；Continue to activate 11-12h at room temperature, is centrifuged 10- under the revolving speed of 6000-7000r/min
15min, obtained concentrate are nanogold-chloramphenicol adaptor complex；
The partial size of nanogold is 15-35nm, and nanogold is made using trisodium citrate reduction gold chloride, trisodium citrate and chlorine gold
Acid is mixed with volume ratio for the ratio of 2-2.5:1, and under the revolving speed of 300-350r/min, temperature is to stir 11- at 100-110 DEG C
Nano-Au solution is made in 12min；
Sample pad (4) uses bovine serum albumin Seal treatment in vain, and the concentration of bovine serum albumin solution is 1%.
2. according to claim 1 for detecting the test strips of chloramphenicol, it is characterised in that: nanogold-chloramphenicol adaptation
Nanocrystal composition is coated on bonding pad (3), and in 35-37 DEG C of drying 7-12min.
3. according to claim 1 for detecting the test strips of chloramphenicol, it is characterised in that: probe A compound is according to such as
Lower section method is made: the biology that the Streptavidin and 8-10 μ L concentration that 18-20 μ L concentration is 2-3mg/mL are 90-100 μm of o1/L
The probe solution A of element modification mixes 2-2.5h at room temperature.
4. according to claim 1-3 for detecting the test strips of chloramphenicol, it is characterised in that: Streptavidin
Using non-thin base Streptavidin.
5. according to claim 1-3 for detecting the test strips of chloramphenicol, it is characterised in that: the inspection of chloramphenicol
Survey range is 0.5-160 μ g/mL.
6. a kind of claim 1-5 is described in any item for detecting the preparation method of the test strips of chloramphenicol, it is characterised in that:
Include the following steps:
Nitrocellulose membrane (2), bonding pad (3), sample pad (4) and water absorption pad (5) are separately fixed on support plate (1), sample
1.5-2.5mm is overlapped between pad (4), bonding pad (3), support plate (1) width is 4-4.5mm；Detection line is drawn in support plate (1)
(6) and control line (7)；
It is spare in 35-37 DEG C of drying 7-12min that nanogold-chloramphenicol adaptor complex is infiltrated on bonding pad (3)；
2-2.5h is incubated at room temperature with the probe solution A and Streptavidin of chloramphenicol aptamers partial complementarity, and probe A is made
Compound, and be coated at detection line (6)；
The probe B solution complementary with chloramphenicol aptamers another part and Streptavidin are incubated at room temperature 2-2.5h and spy are made
Needle B compound, and be coated at control line (7)；
Nitrocellulose membrane (2) is placed on 37 DEG C of dry 8-12h, is put in kept dry at 4 DEG C.
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| CN102128832A (en) * | 2010-12-27 | 2011-07-20 | 江南大学 | Colloidal gold nucleic acid test strip for watermelon bacterial fruit blotches as well as preparation and application thereof |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
| CN106645679A (en) * | 2017-01-17 | 2017-05-10 | 吉林大学 | Method for detecting tetracycline (TET) based on colloidal gold (AuNPs) chromatography test strip of aptamer |
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