CN110133254A - A kind of plug-in can realize the Raman chromatographic test paper of the secondary amplification of signal - Google Patents
A kind of plug-in can realize the Raman chromatographic test paper of the secondary amplification of signal Download PDFInfo
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- CN110133254A CN110133254A CN201810104302.1A CN201810104302A CN110133254A CN 110133254 A CN110133254 A CN 110133254A CN 201810104302 A CN201810104302 A CN 201810104302A CN 110133254 A CN110133254 A CN 110133254A
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- card
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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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- 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 invention discloses the Raman chromatographic test papers that a kind of plug-in can realize the secondary amplification of signal.The Test paper is divided into A card, B card and C card three parts, and connection may be implemented by bayonet between A, B card and disconnect, C card can be placed directly on B card；A card is made of bottom plate 1, sample pad, bonding pad 1, bonding pad 2, bonding pad 3.B card is made of reaction film, measurement line (immobilization versatility recognition component), nature controlling line, water suction paper washer, bottom plate 2.C card is made of reduction pad, enhancing pad；Plug-in design main advantage is that same B card can be applied in combination from different A cards, enhances signal comparativity.The secondary amplification of signal is realized in the reduction of Ag in the combination of two kinds of colloidal golds and reaction film.Test strips of the invention are easy to operate, detection time is short, storage is simple, long shelf-life.The present invention can be used for the numerous areas such as clinical diagnosis, inspection and quarantine, food safety detection, judicial expertise, illicit drugs inspection, have a vast market foreground.
Description
Technical field
The present invention relates to a kind of Test papers, and in particular to a kind of plug-in can realize the Raman chromatography of the secondary amplification of signal
Test paper belongs to field of biomedicine technology.
Background technique
Immune chromatography test paper based on ELISA testing principle is short, easy to operate with its minute, result interpretation is intuitive
Advantage becomes the most widely used chromatographic technique in practice.By taking common small molecule immune chromatographic test paper as an example, detection is former
It is a certain zone that determinand specific antibody is previously secured to nitrocellulose filter that reason is mostly, and sample to be tested is added drop-wise to test paper
One end, under capillarity, sample is moved forward along the film, to be measured in sample when the region by being fixed with antibody
Antigen can be specifically bound with the antibody.At this point, if with immune labeled nano particle such as colloidal gold or Immunoperoxidase Staining,
Or there is label Raman, upper conversion to shine, time-resolved fluorescence signal, chemiluminescent marker molecules, can realize and treat
Survey the qualitative and quantitative detection of object.
Due to the measurement of chromatography detection method rapidly, signal response it is intuitive, low in cost, medical treatment, food, drug, it is chemical with
And the multiple fields such as judicial expertise have been used widely, currently there are a series of colloidal gold tests, but this
A little test paper are without versatility feature, and detection sensitivity only reaches national standard.
Summary of the invention
The present invention is directed to the deficiency of above-mentioned existing immunochromatography product, and the secondary amplification of signal can be realized by establishing a kind of plug-in
Raman chromatographic test paper, it is therefore an objective to immunochromatography technique is combined with Raman detection, improves the sensitivity of chromatography detection and logical
The property used.In order to achieve the above object, the present invention adopts the following technical scheme:
Plug-in secondary singal amplifies the structure and purposes of Raman chromatographic test paper, which is divided into A card, B card and C card
Three parts may be implemented connection by bayonet between A, B card and disconnect, and C card can be placed directly on B card；A card is by bottom plate 1, sample
Product pad, bonding pad 1, bonding pad 2, bonding pad 3 form.B card is by reaction film, measurement line, nature controlling line, water suction paper washer, 2 groups of bottom plate
At.C card is made of reduction pad, enhancing pad；Plug-in design main advantage is A card and B card (cross-species transferability, not because to be measured
The change of object and change the card measurement line and nature controlling line composition) between can be achieved connection and disconnect, in the situation that B card is constant
Under, replace the A card of different target object.Since B card can be applied in combination from different A cards, enhance comparable between signal between Testing index
Property.The secondary amplification of the signal mainly reduction (second by the combination of two kinds of colloidal golds (a signal amplification) with Ag on reaction film
Secondary signal amplification) it realizes, it is intended to improve sensitivity.
The bonding pad 1 contains the Raman labels gold core silver shell for being coupled bovine serum albumin(BSA) antibody；The bonding pad 2 contains
There is the Raman labels gold core silver shell for being coupled determinand antibody；Conjugate of the bonding pad 3 containing determinand and biotin；Instead
It answers and measures line and nature controlling line on film, be in the ribbon perpendicular with the length of the test paper, detection zone is coated with Avidin, Quality Control
Area is coated with recombinant G protein (SPG), and the reduction pad contains sodium citrate buffer solution；The enhancing pad contains silver nitrate solution.
The Raman labels gold core silver shell for being coupled bovine serum albumin(BSA) is prepared by active ester method；The Raman
Golden core silver shell is marked to prepare by Seed inducement method.
The Raman labels gold core silver shell for being coupled determinand antibody is prepared by active ester method；The Raman mark
Remember that golden core silver shell is prepared by Seed inducement method.
The conjugate of the determinand and biotin is prepared by active ester method.
The bottom plate is PVC or other hard nonabsorbent materials；The sample absorption pad is suction strainer paper；The water suction paper washer
For blotting paper.
It is a further object to provide a kind of methods for preparing above-mentioned colloidal gold strip comprising step:
1) preparation has the reaction film of the detection zone of coating Avidin and the quality control region of coating recombinant G protein；Prepare C card；
2) preparation is coated with the Raman labels gold core silver shell of bovine serum albumin(BSA) antibody respectively, has been coupled determinand antibody
The bonding pad of Raman labels gold core silver shell and determinand and biotin conjugates；
3) 1) reaction film after the coating of preparation and the assembling of bottom plate 2 are prepared into B card；
4) 2) bonding pad and bottom plate 1 assembling of preparation are prepared into A card；A card and B card are assembled into test strips；
Specifically, step includes:
1) it is reacted with gold chloride with sodium citrate, prepares colloidal gold；
2) colloidal gold is marked using Raman molecular；
3) it is that solution is resuspended with water, extra Raman molecular is removed by centrifugation；
4) sodium citrate and silver nitrate are sequentially added in the colloidal gold solution of Raman molecular label, prepares golden core silver shell nanometer
Material；
5) golden core silver shell nanometer material is marked using Raman molecular；
6) it is that solution is resuspended with water, extra Raman molecular is removed by centrifugation；
7) bovine serum albumin(BSA) antibody-solutions are added in the golden core silver shell nanometer material of preparation, with oralbumin or
Skim milk carries out the closing in unbonded site, has been coupled the Raman labels gold core silver shell nanometer of bovine serum albumin(BSA) antibody
Material is simultaneously sprayed at bonding pad, is successively prepared into bonding pad 1 after 37 DEG C of drying, freeze-drying；
8) determinand antibody-solutions are added in the golden core silver shell nanometer material of preparation, are carried out not with bovine serum albumin(BSA)
The closing of binding site has been coupled the Raman labels gold core silver shell nanometer material of determinand antibody and has been sprayed at bonding pad,
Successively bonding pad 2 is prepared into after 37 DEG C of drying, freeze-drying；
9) determinand and biotin conjugates are prepared by active ester method and is sprayed at bonding pad, 37 DEG C of drying 2h are prepared into
Bonding pad 3；
10) sodium citrate solution containing hydroquinone is sprayed at bonding pad, 37 DEG C of drying 2h are prepared into reduction pad；
11) silver nitrate solution is sprayed at bonding pad, 37 DEG C of drying 2h are prepared into enhancing pad；
12) bonding pad 3, bonding pad 2, bonding pad 1, sample pad are pasted in order on bottom plate 1 and is prepared into detection card A；
13) reaction film is pasted in order on a base plate 2, water absorption pad is prepared into detection card B；
14) reduction pad is stacked on enhancing pad, is prepared into C card；
A kind of plug-in of the present invention can realize the Raman chromatographic test paper of the secondary amplification of signal by plug-in design with
Believe the secondary amplification of bow and the cross-species transferability of B card, highly sensitive versatility immunochromatography Raman detection may be implemented.This
The plug-in of invention can realize that the Raman chromatographic test paper of the secondary amplification of signal is easy to operate, detection time is short, storage is simple, guarantee the quality
Phase is long.A, the signal enhancing of the versatility feature C card in addition of the plug-in design and B card of B card, significantly increases to chromatograph and detect
Sensitivity and versatility.
The present invention can be used for many necks such as clinical diagnosis, inspection and quarantine, food safety detection, judicial expertise, illicit drugs inspection
Domain has a vast market foreground.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, according to this
Other attached drawings that a little attached drawings obtain shall fall within the protection scope of the present invention.
Fig. 1 is the secondary amplification Raman chromatographic test paper assembling figure of pluggable signal of the present invention
In attached drawing, list of materials representated by each label is as follows: 1, bottom plate 1；2, sample pad；3, bonding pad 1；4, bonding pad
2；5, bonding pad 3；6, reaction film；7, line is measured；8, nature controlling line；9, reduction pad；10, enhancing pad；11, absorb water paper washer；12, bottom plate
2；
Fig. 2 is the secondary amplification Raman chromatographic test paper A card of pluggable signal of the present invention
In attached drawing, list of materials representated by each label is as follows: bottom plate 1；2, sample pad；3, bonding pad 1；4, bonding pad 2；
5, bonding pad 3；
Fig. 3 is the secondary amplification Raman chromatographic test paper B card of pluggable signal of the present invention
In attached drawing, list of materials representated by each label is as follows: 6, reaction film；7, line is measured；8, nature controlling line；11, it absorbs water
Paper washer；12, bottom plate 2；
Fig. 4 is the secondary amplification Raman chromatographic test paper C card of pluggable signal of the present invention
In attached drawing, list of materials representated by each label is as follows: 9, reduction pad；10, enhancing pad；
Fig. 5 is that the secondary amplification Raman chromatographic test paper back side of pluggable signal of the present invention is seen
In attached drawing, list of materials representated by each label is as follows: 1, bottom plate 1；12, bottom plate 2；
Fig. 6 is the secondary amplification Raman chromatographic test paper testing result interpretation of pluggable signal of the present invention
Fig. 6 a, 6b, 6c, 6d, 6e, 6f are test paper testing result process decision chart.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts all other
Embodiment shall fall within the protection scope of the present invention.
The composition of the secondary amplification Raman chromatograph test strip of 1 pluggable signal of embodiment:
The test paper is made of bottom plate, sample pad, reaction film, water absorption pad；
The bonding pad 3, bonding pad 2, bonding pad 1, sample pad are successively pasted on bottom plate 1, are prepared into detection card A, sample
The beginning of product pad is aligned with the beginning of bottom plate 1；(Fig. 2)
The reaction film, water absorption pad are successively pasted is prepared into detection card B on a base plate 2；The end of water absorption pad and bottom plate 2
End alignment；(Fig. 3)
Before use, detection card A passes through card by the protruding portion of 3 one end of bonding pad and the groove of detection card B reaction film one end
Mouth is assembled into complete reaction test paper；(Fig. 1, Fig. 5)
Detection zone and quality control region on the reaction film are in the ribbon perpendicular with the long axis of the test paper, detection zone
Close to bottom plate side with groove, quality control region is far from bottom plate side with groove.Detection zone is coated with Streptavidin, quality control region
It is coated with recombinant G protein.
The bottom plate is PVC bottom plate；The sample absorption pad is suction strainer paper；The reaction film is nitrocellulose membrane.
Reduction pad is stacked on enhancing pad, C card is prepared into；
By above-mentioned A card, B card, C card, saved in 2~8 DEG C of environment, validity period 12 months.
The preparation method of the secondary amplification Raman chromatographic test paper of pluggable signal described in 2 embodiment 1 of embodiment
One, the preparation of test strips (for detecting chloramphenicol)
The preparation method of test strips mainly comprises the steps that
1) preparation has the reaction film of the detection zone of coating Avidin and the quality control region of coating recombinant G protein；Prepare C card；
2) preparation respectively the Raman labels gold core silver shell containing bovine serum albumin(BSA) antibody, be coupled the drawing of chloramphenicol antibody
The graceful bonding pad for marking golden core silver shell and chloramphenicol and biotin conjugates；
3) 1) reaction film after the coating of preparation and the assembling of bottom plate 2 are prepared into B card；
4) 2) bonding pad and bottom plate 1 assembling of preparation are prepared into A card；A card and B card are assembled into test strips；
Substep narration in detail below:
(1) preparation of each component
1) preparation of Jin Heyin shell
The conical flask containing 100ml MilliQ water is added in 1% chlorauric acid solution of 1ml.It is heated with thermostatic electromagnetic blender
To boiling, it is persistently added with stirring a certain amount of 1% sodium citrate, continuation stops when being at the uniform velocity heated with stirring to solution in bright red
Only, 100ml is settled to MilliQ water after being cooled to room temperature.1ml 10mmol/L 5,5- bis- thiobis (2- nitrobenzoyl is added
Acid) (DTNB), 3h is stirred at room temperature.The a certain amount of sodium citrate solution refers to the corresponding sodium citrate of different-grain diameter nanogold
Additional amount specially prepares 15nm nanogold and 4ml sodium citrate is added；It prepares 35nm nanogold and 1.3ml sodium citrate is added；
9000rpm is used for 15nm nano material, is centrifuged 6min, supernatant is abandoned, is resuspended with MilliQ water by nano material；
7500rpm is used for 35nm nano material, is centrifuged 6min, supernatant is abandoned, is resuspended with MilliQ water by nano material, to remove not
The DTNB of reaction.It is heated to boiling under the conditions of nanomaterial solution magnetic agitation, 1ml sodium citrate is added, be then added certain
The 10mmol/L silver nitrate solution of amount, solution boiling 40min, then cools to room temperature, is settled to 100ml with MilliQ water.It is right
The material that Yu Jinhe is 15nm uses 7000rpm, is centrifuged 6min, abandons supernatant, is resuspended with MilliQ water by nano material；For
35nm nano material uses 5500rpm, is centrifuged 6min, abandons supernatant, is resuspended with MilliQ water by nano material, to remove unreacted
DTNB.The a certain amount of silver nitrate solution refers to the corresponding silver nitrate additional amount of different-grain diameter nanogold, and specially 15nm receives
4ml silver nitrate solution is added in rice gold；1ml silver nitrate solution is added in 35nm nanogold；The golden core silver housing chamber temperature prepared is protected from light guarantor
It deposits.The golden core silver shell prepared is pure, it is bright, without precipitating and floating material.
2) preparation of chloramphenicol monoclonal antibody-Jin Heyin shell marker
The golden core silver shell material that golden core is 15nm is used into 7000rpm, 6min is centrifuged, abandons supernatant, use 0.2mol/L
The PB buffer of pH8.4 is resuspended by nano material.Under magnetic agitation, the mark of 10 μ g antibody is added by every milliliter of golden core silver shell solution
Chloramphenicol antibody is added into golden core silver shell solution in standard, continues to stir and evenly mix 10min, be added 10% bovine serum albumin(BSA) (BSA)
Component V makes its final concentration of 1% (volumn concentration) in golden core silver shell solution, stands 10min.9000rpm, 4 DEG C of centrifugations
20min abandons supernatant, and precipitating is washed twice with redissolution buffer, will with the redissolution liquid that volume is initial golden core silver shell volume 1/10
Precipitating is resuspended, 4 DEG C of storages, spare.
Redissolve buffer: the 0.01mol/L pH's 7.2 containing 0.05% Tween-80 and 0.5% ovalbumin (OVA)
Phosphate buffer.
3) preparation of BSA monoclonal antibody-Jin Heyin shell marker
The golden core silver shell material that golden core is 35nm is used into 5500rpm, 6min is centrifuged, abandons supernatant, use 0.2mol/L
The PB buffer of pH8.4 is resuspended by nano material.Under magnetic agitation, the mark of 10 μ g antibody is added by every milliliter of golden core silver shell solution
Chloramphenicol antibody is added into golden core silver shell solution in standard, continues to stir and evenly mix 10min, 10% ovalbumin is added, makes it in gold
Final concentration of 1% (volumn concentration) in core silver shell solution stands 10min.9000rpm, 4 DEG C of centrifugation 20min abandon supernatant,
Precipitating is washed twice with redissolution buffer, precipitating is resuspended with the redissolution liquid that volume is initial golden core silver shell volume 1/10,4 DEG C of storages
It deposits, it is spare.
4) preparation of sample absorption pad
Sample absorption pad is placed in the phosphate buffer of the 0.01mol/L pH 7.2 containing ovalbumin 0.5% and is impregnated
2h, 37 DEG C of baking 2h are spare.Under room temperature, kept dry.
5) preparation of bonding pad 1
BSA monoclonal antibody-Jin Heyin shell marker is sprayed at bonding pad, dries 15min, 37 degrees celsius at room temperature
Lower dry 20min, is put into freeze drier, under the conditions of condenser temperature is -50 DEG C, pre-freeze 3h, then be dried in vacuo 15h, i.e.,
It can be taken off, obtained bonding pad 1 is put into plastic packaging bag, drying machine is added, is sealed.
6) preparation of bonding pad 2
Chloramphenicol monoclonal antibody-Jin Heyin shell marker is sprayed at bonding pad, at room temperature dry 15min, and 37 degrees Celsius
Under the conditions of dry 20min, be put into freeze drier, under the conditions of condenser temperature is -50 DEG C, pre-freeze 3h, then be dried in vacuo
15h can be taken off, obtained bonding pad 1 is put into plastic packaging bag, drying machine is added, is sealed.
7) preparation of bonding pad 3
Chloramphenicol biotin conjugates are sprayed at bonding pad, 37 DEG C of baking 2h are spare.Under room temperature, kept dry.
8) preparation of reaction film
Coating process: being diluted to 1mg/ml for Streptavidin with the phosphate buffer of 0.01mol/L pH7.4, uses
Isoflow point film instrument is coated in the detection zone (T) on nitrocellulose membrane, and package amount is 1.0 μ l/cm；Use 0.01mol/L
Recombinant G protein is diluted to 1mg/ml by the phosphate buffer of pH7.4, is coated in cellulose nitrate with Isoflow point film instrument
Quality control region (C) on film, package amount are 1.0 μ l/cm.Dry 16h, spare under the conditions of the reaction film being coated with is placed in 37 DEG C
9) preparation of reduction pad
By 0.5mol/L pH4.0 and the sodium citrate solution containing 3% hydroquinone is sprayed at fiberglass packing, 37 DEG C of bakings
It is spare that reduction pad is made in 2h.Under room temperature, it dries, be protected from light, be sealed.
10) preparation of enhancing pad
20mmol/L silver nitrate solution is sprayed at fiberglass packing, 37 DEG C of baking 2h are spare, and that enhancing pad is made is spare.Room temperature
Under the conditions of, it dries, be protected from light, be sealed.
(2) assembling of each component
1, the assembling of test paper
It pastes bonding pad 3, bonding pad 2, bonding pad 1, sample pad in order on bottom plate 1 and is prepared into detection card A.In bottom plate
Reaction film is pasted in order on 2, water absorption pad is prepared into detection card B.Reduction pad is stacked on enhancing pad, C card is prepared into；
2, the assembling of test strips
A, B card prepared in above-mentioned steps 1 and C card are individually stored.Using preceding A, B card by bottom plate 1 and bottom plate 2 it
Between card slot connection.It is stored in 2~8 DEG C of environment, validity period 12 months.
Embodiment 1, the detection of middle chloramphenicol (CAP) in dairy products
Material requested: 1. for the Raman chromatographic test paper of the secondary amplification of plug-in signal of chloramphenicol, (this laboratory is certainly
It is standby)；2. chloramphenicol mark-on plain chocolate is a (providing for oneself in this laboratory)
1) test strips test sample is used
Operating procedure: 1. immunity chromatography detection test paper is balanced to room temperature, connects A card and B card；2. the pure ox of chloramphenicol mark-on
Milk is spare after lead acetate method pre-treatment；3. sample to be tested is added drop-wise to sample pad；4. waiting 10min under constant temperature；At this point, sample
Product solution is chromatographed under capillarity, substance on bonding pad 1,2,3 on dissolution A card, and carries out competitive binding reaction；Instead
Solution is answered to be acted on when reaching B card detection zone in conjunction with immobilization versatility recognition component through chromatography, unbonded part continues to chromatograph
It is combined when to quality control region with corresponding recognition component.5. C is placed in above detection zone and quality control region, 100 μ l aqueous solutions are added,
10min is waited under constant temperature, silver nitrate is restored by the sodium citrate solution of hydroquinone on C card at this time, and Argent grain is attached to
On detection zone and quality control region, enhance Ramam effect；6. visualizing interpretation result combination Raman detector measures detection zone, analysis note
Record result.The lead acetate pre-treating method specifically, 100 μ l acetic acid lead solutions are added in 1ml milk sample, after oscillating reactions 3min plus
Enter Potassium Oxalate Solution 50 μ l, 3000rpm, 3min, draws supernatant as sample solution.The acetic acid lead solution preparation method: 20g
Lead acetate water dissolves and is settled to 100ml；The potassium oxalate-disodium phosphate soln preparation method: potassium oxalate 3g and phosphorus
Sour disodium hydrogen 7g is dissolved in water and is settled to 100ml.
(2) analysis detection result.
Positive: when quality control region (C) display Raman signal, and detection zone (T) is weaker than C line without Raman signal or Raman signal and draws
Graceful signal, is judged to the positive, is indicated with "+", such as Fig. 6 a, 6b.
It is negative: when quality control region (C) and detection zone (T) shows that Raman signal is close, to be judged to feminine gender, indicated with "-", such as figure
6c、6d。
It is invalid: when quality control region (C) does not show Raman signal, test paper failure, such as Fig. 6 e.
It is quantitative: it is accurate to measure determinand content according to quality control region (C) and detection zone (T) Raman signal relative intensity, such as scheme
6f。
The determination of 2 Lateral Flow Strip parameter of embodiment
1, detection limit experiment
It is 0,0.01,0.1,1 μ g/L that mark-on chloramphenicol to total concentration is distinguished into blank milk sample, is carried out with test strips
Milk sample detection, as a result are as follows: when chloramphenicol concentration is 0,0.01 μ g/L, test paper is shown except macroscopic black, grey bar
Band, and Raman signal can be determined, measurement region signal is better than quality control region, is negative.When chloramphenicol concentration is 0.1,1 μ g/L,
Test paper is shown except macroscopic black, gray bars, and can determine Raman signal, and measurement region signal is weaker than quality control region, in sun
Property, by calculating the two Raman signal relative intensity, show that this test strips is limited to 0.03 μ g/L to chloramphenicol detection in milk.
2, false positive rate, false negative rate test
Take milk positive sample 20 part of the known chloromycetin content greater than 0.03 μ g/L and chloromycetin content less than 0.03 μ g/
20 parts of the milk positive sample of L is detected respectively with the test strips that 3 batches produce, the results are shown in Table 1, table 2.
Table 1 detects positive sample result
Table 2 detects negative sample result
The result shows that: when detecting positive milk sample with the test strips that 3 each batches produce, as a result it is all positive, it is known that sun
Property sample coincidence rate be 100%, false positive rate 0.When detecting 20 parts of negative milk samples, as a result it is all negative, it is known that negative
Coincidence rate is 100%, false negative rate 0.Detection chloramphenicol test strips of the invention can be to residual chloromycetin in milk sample
It is used for quickly detecting.
3, specificity experiments
The common cross reacting rate of specificity indicates, refer to antibody structure similar in energy in conjunction with antigenic determinant
Power.The test strips detect sulfamido (Huang peace dimethyl pyrimidine, sulphadiazine), fluoroquinolones (Ofloxacin, promise of 30 μ g/L
Flucloxacillin), erythromycin, the drugs such as aminoglycoside (gentamicin, spectinomycin), test strips detection zone and quality control region are aobvious
Color, and detection zone is not weaker than quality control region, illustrates this test strips to these drug no cross reactions.
The amplification Raman chromatographic test paper preparation of plug-in secondary singal is provided for the embodiments of the invention above to have carried out in detail
Thin to introduce, instrument can analyze sample with Raman detector etc..The secondary amplification Raman layer of pluggable signal of the present invention
Analysis detection is mainly for small molecules such as antibiotic, pesticide, heavy metal, hormones, it can also be used to the big grain such as albumen, cell, microorganism
Diameter material is detected, and can be used for clinical diagnosis, inspection and quarantine, food safety detection, judicial expertise, drug, illicit drugs inspection etc.
Field.
Used herein a specific example illustrates the principle and implementation of the invention, and above embodiments are said
It is bright to be merely used to help understand method and its core concept of the invention: at the same time, for those skilled in the art, foundation
Thought of the invention, there will be changes in the specific implementation manner and application range, in conclusion the content of the present specification is not
It is interpreted as limitation of the present invention.
Claims (7)
1. the invention discloses the preparation methods that a kind of plug-in can realize the Raman chromatographic test paper of the secondary amplification of signal.The detection
Test paper is divided into A card, B card and C card three parts, and connection may be implemented by bayonet between A, B card and disconnect, C card can be placed directly within B
On card；A card is made of bottom plate 1, sample pad, bonding pad 1, bonding pad 2, bonding pad 3.B card is by reaction film, measurement line, Quality Control
Line, water suction paper washer, bottom plate 2 form.C card is made of reduction pad, enhancing pad；Plug-in design main advantage is A card and B card
The company's of can be achieved make and break between (cross-species transferability does not change the composition of card measurement line and nature controlling line because of the change of determinand)
It opens, in the case where B card is constant, replaces the A card of different target object.Due to B card cross-species transferability and can be combined with different A cards
It uses, enhances between different Testing index comparativity between signal.The secondary amplification of signal mainly passes through the combination (one of two kinds of colloidal golds
The amplification of secondary signal) it is realized with the reduction (second of signal amplifies) of Ag on reaction film, it is intended to enhance signal sensitivity.Institute of the present invention
Stating a kind of plug-in can realize that the Raman chromatographic test paper of the secondary amplification of signal passes through the secondary amplification of plug-in design and signal, real
The high sensitivity and versatility of existing immunochromatography.Test strips of the invention are easy to operate, detection time is short, storage is simple, protect
The matter phase is long.The present invention can be used for many necks such as clinical diagnosis, inspection and quarantine, food safety detection, judicial expertise, illicit drugs inspection
Domain has a vast market foreground.
2. the Raman chromatographic test paper that plug-in according to claim 1 can realize the secondary amplification of signal, it is characterised in that institute
The Test paper is divided into A card, B card and C card three parts, and connection may be implemented by bayonet between A, B card and disconnect, C card can be straight
It connects and is placed on B card；A card is made of bottom plate 1, sample pad, bonding pad 1, bonding pad 2, bonding pad 3.B card is by reaction film, measurement
Line, nature controlling line, water suction paper washer, bottom plate 2 form.C card is made of reduction pad, enhancing pad.
3. the secondary amplification Raman chromatographic test paper of pluggable signal according to claim 1, it is characterised in that the detection zone
Positioned at one end with groove that bottom plate 2 can be attached with bottom plate 1, the quality control region is located remotely from the one end with groove of bottom plate 2.
4. the secondary amplification Raman chromatographic test paper of pluggable signal according to claim 1, it is characterised in that the determinand
Antibody marks golden core silver shell to be coupled to obtain by active ester method with golden core silver shell nanometer material by determinand monoclonal antibody；It is described
Bovine serum albumin(BSA) antibody marks golden core silver shell to pass through work by bovine serum albumin(BSA) monoclonal antibody and golden core silver shell nanometer material
Property ester process is coupled to obtain.
5. a kind of method for preparing the secondary amplification Raman chromatographic test paper of the described in any item pluggable signals of claim 1-4,
Including step；
1) preparation has the reaction film of the detection zone of coating Avidin and the quality control region of coating recombinant G protein；Prepare C card；
2) preparation respectively the Raman labels gold core silver shell containing bovine serum albumin(BSA) antibody, be coupled the Raman mark of determinand antibody
Remember the bonding pad of golden core silver shell and determinand and biotin conjugates；
3) 1) reaction film after the coating of preparation and water suction paper washer, the assembling of bottom plate 2 are prepared into B card；
4) 2) bonding pad of preparation and sample pad, the assembling of bottom plate 1 are prepared into A card；A card and B card are assembled into test strips；
5) C card, enhancing response signal are placed in the region between after the colour developing of detection card above the quality control region and detection zone.
6. a kind of remaining method of determinand in detection milk comprising step:
1) it is detected with the described in any item colloidal gold strips of claim 1-4；
2) analysis detection result.
7. Test paper according to claim 1, it is characterised in that: plug-in design main advantage is A card and B card
The company's of can be achieved make and break between (cross-species transferability does not change the composition of card measurement line and nature controlling line because of the change of determinand)
It opens, in the case where B card is constant, replaces the A card of different target object.Due to B card cross-species transferability and can be combined with different A cards
It uses, enhances between different Testing index comparativity between signal.The secondary amplification of signal mainly passes through the combination (one of two kinds of colloidal golds
The amplification of secondary signal) it is realized with the reduction (second of signal amplifies) of Ag on reaction film.A kind of plug-in of the present invention can be realized
The Raman chromatographic test paper of the secondary amplification of signal realizes the highly sensitive of chromatography detection by the secondary amplification of plug-in design and signal
Property and versatility.Test strips of the invention are easy to operate, detection time is short, storage is simple, long shelf-life.The present invention can be used for facing
The numerous areas such as bed diagnosis, inspection and quarantine, food safety detection, judicial expertise, illicit drugs inspection, have a vast market foreground.
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