CN107796938A - A kind of aptamer fluorescent test paper strip and its preparation method and application - Google Patents
A kind of aptamer fluorescent test paper strip and its preparation method and application Download PDFInfo
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- CN107796938A CN107796938A CN201710969466.6A CN201710969466A CN107796938A CN 107796938 A CN107796938 A CN 107796938A CN 201710969466 A CN201710969466 A CN 201710969466A CN 107796938 A CN107796938 A CN 107796938A
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- G—PHYSICS
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- 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
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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Abstract
The invention provides a kind of aptamer fluorescent test paper strip and its preparation method and application, it is related to technical field of biological.A kind of aptamer fluorescent test paper strip, including bottom plate, sample pad, nano material pad, reaction film and adsorptive pads, reaction film are provided with detection line, and detection line is disposed in proximity to one end of nano material pad；Nano material pad is attached with the aptamer with catenation sequence of fluorescein-labeled aptamer and/or fluorescence labeling and free fluorescein, detection line are fixed with the complementary strand of aptamer or the complementary strand of aptamer catenation sequence.Test strips structure is simple, and detection is sensitive, and the degree of accuracy is high.A kind of preparation method of aptamer fluorescent test paper strip, including：Sample pad, nano material pad, reaction film and adsorptive pads are affixed to the same side of bottom plate successively.Simple to operate, flexibility is strong.
Description
Technical field
The present invention relates to technical field of biological, in particular to a kind of aptamer fluorescent test paper strip and its
Preparation method and application.
Background technology
Test strips have been widely used for clinical diagnosis, food security and environment as a kind of simple and quick analysis tool
In monitoring.Test strips mainly have two kinds of detection modes of collaurum and fluorescence at present, and wherein colloidal gold strip passes through chromogenic reaction
To show result, have naked eyes it is visible without other instruments the advantages of；But its shortcoming is that sensitivity is low, and can only be by comparing face
The shallow purpose for reaching sxemiquantitative of color depth.Relative to colloidal gold strip, fluorescent test paper strip high sensitivity, portable fluorescence of arranging in pairs or groups
Detector can carry out accurate quantification to the target in sample, therefore as the direction of the development of test strips in recent years.
But no matter colloidal gold strip or fluorescent test paper strip, be substantially the test paper based on antigen-antibody reaction at present
Bar, antigen-antibody needs low temperature accumulating to preserve as activated protein, therefore shelf life is shorter, limits the technology in remote condition
The application of backward areas.And because many small-molecule substances prepare haptens difficulty, it is difficult to stimulate and produce the anti-of high-affinity
Original antibody, even more limit the application of this technology.
Aptamer is a kind of identification molecule newfound in recent years, is primarily referred to as length between 10-100 bases
Single stranded DNA, combined after being folded by conformation with target molecules high-affinity high specific.Aptamer is by external
Screening is got, therefore compared with antibody, its target is unrestricted, small to metal ion to arrive protein molecule greatly and screen
Aptamers, the aptamers of screening can in batches synthesize after sequencing, be easy to modify, and stability is good, can be used for instead of antibody
In miscellaneous antibody analysis method, therefore also it is known as chemical antibody.
Same aptamer is also replaced antibody to be used in test strips exploitation, and its principle is similar to traditional test paper
Bar.But the collaurum or fluorescent test paper strip and few of aptamer are currently based on, its main cause is different aptamers sequences
Row length differs, and most of aptamers sequence is long, thus with complementary strand formed double-strand affinity be greater than aptamers and
Target forms the affinity of compound, therefore causes the aptamers for having been incorporated with target still to be dissociated at detection line,
The accurate detection to target can not be realized.Although detection line immobilized antigen is so that competitiveness is consistent, for many small molecule targets
Limited after being combined for mark with coupling protein and the combination of aptamers so that aptamers can not combine at detection line, can not
Realize sensitive and accurate detection.
The content of the invention
In view of the shortcomings of the prior art, the present invention proposes a kind of aptamer fluorescent test paper strip, simple in construction, detection
Sensitive, the degree of accuracy is high.
The present invention has also been proposed a kind of preparation method of aptamer fluorescent test paper strip, and simple to operate, flexibility is strong.
What the present invention was realized in：
The present invention proposes a kind of aptamer fluorescent test paper strip, including bottom plate, sample pad, nano material pad, reaction film
And adsorptive pads, sample pad, nano material pad, the end of reaction film and adsorptive pads are in turn connected to form detection layers, detection layers
It is connected with bottom plate, reaction film is provided with detection line and nature controlling line, and detection line is disposed in proximity to one end of nano material pad, and nature controlling line is set
It is disposed adjacent to one end of adsorptive pads；
Nano material pad is attached with the core with catenation sequence of fluorescein-labeled aptamer and/or fluorescence labeling
Sour aptamers and free fluorescein, detection line be fixed with aptamer complementary strand or aptamer catenation sequence it is mutual
Chain is mended, the anti-fluorescein antibody corresponding with fluorescein is provided with nature controlling line.
The present invention proposes a kind of preparation method of aptamer fluorescent test paper strip, including：By sample pad, nano material
Pad, reaction film and adsorptive pads affix to the same side of bottom plate successively.
The present invention proposes a kind of aptamer fluorescent test paper strip in food security, environmental monitoring and clinical diagnosis
Using.
The beneficial effect of such scheme：
A kind of aptamer fluorescent test paper strip provided in an embodiment of the present invention, conventional test paper is instead of with nano material pad
Gold standard pad in bar, nano material point can tightly adsorb fluorescein-labeled aptamer and its fluorescence are quenched, when having
During target, the aptamers only combined with target can just dissociate, and fluorescence recovers and is forwarded to detection line with sample, passes through adaptation
Body and complementary strand or the corresponding complementary strand of aptamers catenation sequence combine, and detection line fluorescence is occurred and with target concentration
Increase and strengthen.This method has versatility, can apply to the aptamers of various length, is also applied for various molecular sizes
Target；This method measurement result is sensitive and accurate reliable, avoid aptamers complementary strand and aptamers target affinity it is inconsistent with
And small molecule target coupled antigen makes to measure inaccuracy caused by affinity change.The test strips are by fluorescein-labeled aptamers
Nano material is adsorbed in, the quenching effect by nano material to fluorescein, improves the stability of fluorescein.
This test strips employs fluorescence labeling, has higher sensitivity, and fluorescence intensity is determined by fluorescence chart scanner, can
To realize that the accurate quantification to target molecules detects.
This test strips, as Quality Control, by nature controlling line fluorescence labeling, is carried using free fluorescein for the validity of test strips
For ensureing.
This test strips, as testing result foundation, is avoided using the ratio of detection line fluorescence signal and nature controlling line fluorescence signal
Difference caused by different test strips and different operating environmental condition, improves the comparativity between different operating, makes result more
Add accurately and reliably.
The test strips structure is simple, easy to detect, and detection is sensitive and accurate, can be with room temperature storage, before having a wide range of applications
Scape.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below by embodiment it is required use it is attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the structural representation of aptamer fluorescent test paper strip provided by the invention；
Fig. 2 is the standard curve of the detection sulfadimethoxine of the embodiment of the present invention 2；
Fig. 3 is the standard curve of the detection ochratoxin A of the embodiment of the present invention 3.
Icon：100- aptamer fluorescent test paper strips；110- sample pads；120- nano material pads；130- reaction films；
131- detection lines；133- nature controlling lines；140- adsorptive pads；150- detection layers；160- bottom plates.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
Part of the embodiment of the present invention, rather than whole embodiments.Unreceipted actual conditions person in embodiment, according to normal condition or
The condition that manufacturer suggests is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is that can be obtained by commercially available purchase
Conventional products.
Have below to a kind of aptamer fluorescent test paper strip of the embodiment of the present invention and its preparation method and application
Body explanation.
A kind of aptamer fluorescent test paper strip provided by the invention, including bottom plate, sample pad, nano material pad, reaction
Film and adsorptive pads, sample pad, nano material pad, the end of reaction film and adsorptive pads are in turn connected to form detection layers, detected
Layer is connected with bottom plate, and reaction film is provided with detection line and nature controlling line, and detection line is disposed in proximity to one end of nano material pad, nature controlling line
It is disposed in proximity to one end of adsorptive pads；
Nano material pad is attached with the core with catenation sequence of fluorescein-labeled aptamer and/or fluorescence labeling
Sour aptamers and free fluorescein, detection line be fixed with aptamer complementary strand or aptamer catenation sequence it is mutual
Chain is mended, the anti-fluorescein antibody corresponding with fluorescein is provided with nature controlling line.
Sample pad adsorbs testing sample, and sample is from sample pad by chromatography action flow to nano material pad, preferably, sample
Product pad is made by cellulose membrane.Adsorptive pads are used to absorb sample, can be absorbent filter.
Preferably, nano material pad can be for porous nanometer material film or by nanometer-material-modified glass fibre element film.
Nano material includes but is not limited in graphene oxide, reduced graphene, nanogold, molybdenum disulfide and CNT at least
It is a kind of.Cellulose membrane includes but is not limited at least one of glass fibre, cellulosic filter paper and polyester film.
The gold standard pad for focusing on instead of conventional test strips with nano material pad of the present invention, can be applicable various length
Aptamer and various targets.And collaurum or fluorescein-labeled aptamers are fixed to gold by conventional test strips
On mark pad, then carry out reaction solution with the antigen in detection line or complementary strand or send fluorescence.This method is frequently subjected to limit
System：First if fixing complementary strand in detection line, this is for the long aptamers of sequence, due to aptamers and complementary strand
Affinity be more than the affinity of aptamers and target, so as to which competitive reaction can not be realized；If secondly fix target in detection line
Mark, it is necessary to be fixed again with after carrier protein couplet for small molecule target, because this coupling limits aptamers and target
The abundant combination of molecule is marked, reduces affinity, or even can not finally be combined, ultimately resulting in detect.
ELISA test strip principle of the present invention is：
When detecting sample with the aptamer fluorescent test paper strip of the present invention, testing sample is added drop-wise in sample pad,
Sample can chromatograph under capillary action it is up, when sample enters nano material pad, target meeting and fluorescence labeling in sample
Aptamer combine, formed compound simultaneously make fluorescein-labeled aptamer from nano-material surface dissociate, simultaneously
Free fluorescein in nano material pad chromatographs nitrocellulose under sample drive together with target-adaptor complex
On film.
, can be with the nucleic acid adaptation of biotin modification fixed in detection line when target-adaptor complex reaches detection line
The complementary strand of body or the complementary strand of aptamer catenation sequence combine, and fluorescence occurs in detection line.And target amount is more, dissociation
Target-adaptor complex is more, and the aptamers for the fluorescence labeling being attached in detection line are more, and detection line fluorescence signal is got over
By force；Otherwise the target in sample is fewer, and fluorescence signal is lower at detection line, so as to by fluorescence to signal strength to sample
In target quantified.
If being free of target to be detected in sample, fluorescein-labeled aptamer is closely tied with nano material
Close, without being dissociated, so as to there is no fluorescence at detection line.Whether no matter to be detected target is had in sample, on nano material pad
Free fluorescein can be combined by the anti-fluorescein antibody at nature controlling line when being up to nature controlling line, fluorescence occur in nature controlling line.If matter
There is no fluorescence appearance at control line, then show that this test strips fails, change test strips and redeterminate.Nature controlling line can know to try in time
Whether paper slip fails, and improves the accuracy of detection.
A kind of preparation method of aptamer fluorescent test paper strip includes：
The preparation method of nanometer-material-modified nano material pad：Cellulose membrane is chosen as the original for preparing nano material pad
Material, cellulose membrane is sprayed to by nano material aaerosol solution, and 7~9h is dried under conditions of 110~130 DEG C, removes glass fibers
The moisture on plain film is tieed up, is obtained through nano-material modified cellulose membrane.Cut as needed and produce nano material pad.
It is adsorbed with the preparation method of the nano material pad of fluorescence labeling aptamers：By the aptamer of fluorescence labeling and/
Or the aptamer with catenation sequence of fluorescence labeling and free fluorescein are added dropwise to nano material, it is incubated 15~
After 25min, dried under conditions of 35~40 DEG C, remove the moisture of nano material.Preferably, fluorescein can use different sulphur cyanogen
Sour fluorescein (FITC), phycoerythrin (PE), anthocyanidin 5 (Cy5), Fluoresceincarboxylic acid (FAM), quantum dot, upper transfer and fluorescence
Microballoon etc., the present invention are not limited it.Aptamer from specific aptamer and designs according to target difference
Corresponding catenation sequence.Preferably, aptamer catenation sequence includes one in polyTn, polyAn, polyCn, polyGn
Kind, wherein n is 6~25 integer.
The part that reaction film reacts for generation fluorescence developing.In embodiments of the present invention, using nitrocellulose filter conduct
Reaction film, in other embodiments of the invention, reaction film can use other materials.Reaction film is provided with detection line and is used to detect
Target.
Reaction film detection line preparation method：The complementary strand of the aptamer of biotin modification or aptamer are connected
The complementary strand of sequence is incubated 25~35min with Avidin, is then directly sprayed onto at detection line.Preferably, connect with aptamer
Connecing the complementary strand of the corresponding aptamer catenation sequence of sequence includes one in polyAn, polyTn, polyGn, polyCn
Kind, wherein n is 6~25 integer.
Reaction film is additionally provided with nature controlling line close to one end of adsorptive pads, for verifying whether test strips are effective.React film quality
Control the preparation method of line：The anti-fluorescein antibody corresponding with the fluorescein for marking aptamers is sprayed onto at nature controlling line, it is to be dried
.
After the completion of nano material pad and reaction film preparation, by sample pad, nano material pad, reaction film and adsorptive pads successively
Affix to the same side of bottom plate.The fluorescent test paper strip is simple in construction, and detection is sensitive, and the degree of accuracy is high.
Present invention also offers above-mentioned aptamer fluorescent test paper strip in food security, environmental monitoring and clinical diagnosis
In detection application, including：
S1. ELISA test strip target standard items are used, obtain standard items in the detection line of test strips and the fluorescence of nature controlling line
Signal intensity rate, the concentration for making target standard items correspond to the linear regression curves of signal intensity rate, calculate regression equation；
S2. ELISA test strip testing sample is used, obtains testing sample in the detection line of test strips and the fluorescence letter of nature controlling line
Number intensity rate, the regression equation obtained according to S1, obtains the content of target in testing sample.
The method of the fluorescence signal intensity ratio of detection detection line and nature controlling line includes：Take target standard items or testing sample
It is added drop-wise on the sample absorption pad of test paper, after 8~12min, test strips is put into ELISA test strip instrument ESEQuant-LR3
Detected, determine the fluorescence signal intensity of detection line and nature controlling line respectively；By the fluorescence signal intensity (T) of detection line than upper matter
The fluorescence signal intensity (C) of line is controlled, obtains the fluorescence signal ratio of T/C values, as detection line and nature controlling line.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
Fig. 1 is refer to, the invention provides a kind of aptamer fluorescent test paper strip 100, including bottom plate 160, sample pad
110th, nano material pad 120, reaction film 130 and adsorptive pads 140, sample pad 110, nano material pad 120, reaction film 130 with
And the end of adsorptive pads 140 is in turn connected to form detection layers 150, detection layers 150 are connected with bottom plate 160.
In an embodiment of the present invention, aptamer fluorescent test paper strip 100 can be conventional strip, sample pad
110th, nano material pad 120, reaction film 130 and adsorptive pads 140 are corresponding strip, in other embodiments, core
Sour aptamers fluorescent test paper strip 100 can be circular or other shapes, sample pad 110, nano material pad 120, reaction film 130 with
And the shape of adsorptive pads 140 is also changed accordingly, the present invention is not limited its profile.
Reaction film 130 is provided with detection line 131 and nature controlling line 133, and detection line 131 is disposed in proximity to nano material pad 120
One end, nature controlling line 133 are disposed in proximity to one end of adsorptive pads 140.Certain distance is kept between detection line 131 and nature controlling line 133,
To distinguish the colour developing reason of fluorescence, target and free fluorescein are distinguished.
Embodiment 2
Present embodiments provide a kind of sulfadimethoxine aptamer test strips, preparation method and its detection and answer
With.
First, the preparation method of sulfadimethoxine aptamer test strips includes：
Glass fibre element film is chosen as the raw material for preparing nano material pad, graphene oxide aaerosol solution is sprayed to glass
Glass cellulose membrane, 8h is dried under conditions of 120 DEG C, remove the moisture on glass fibre element film, obtain oxidized graphene and change
The glass fibre of property.Cut and produce nano material.
By the fluorescein-labeled sulfadimethoxine aptamers (Cy5-GAGGG- containing catenation sequence polyT15 of Cy5
CAACGAGTGTTTATAGATTTTTTTTTTTTTTT) it is added drop-wise on nano material pad, is incubated 20 minutes, under conditions of 37 DEG C
Dry, remove the moisture of nano material.
Nitrocellulose filter is chosen as reaction film.By the complementary series of the aptamers connects chain of biotin modification
PolyA15 (Btiotin-AAAAAAAAAAAAAAA) is incubated 30min with Avidin, is then sprayed onto at detection line；By anti-Cy5's
Antibody is sprayed onto at nature controlling line.
Sample pad, nano material pad, reaction film and adsorptive pads are affixed to the same side of bottom plate successively.
2nd, the Cleaning Principle of test strips：
As shown in figure 1, when the sample containing sulfadimethoxine is added in sample pad, sample flows under capillary action
Move nano material pad, the sulfadimethoxine in sample is combined with the aptamer on nano material pad, makes it from receiving
Dissociated in rice material cushion, and continue flow forward together with free fluorescein.When flowing to detection line, target is combined with
Aptamer on catenation sequence can be combined with the complementary series of the catenation sequence in detection line, so as to going out at detection line
Existing fluorescence, and the sulfadimethoxine in sample is more, and the fluorescence intensity in detection line is stronger；Otherwise the sulfanilamide (SN) in sample
SDM is fewer, and the fluorescence intensity in detection line is lower, so as to according to the height of fluorescence intensity to the sulphur in sample
Amine SDM carries out quantitative detection.The fluorescein that dissociates simultaneously in liquid continues to go upward at nature controlling line and by antibody herein
, there is fluorescence signal in capture.Fluorescence in detection line and nature controlling line can use ELISA test strip instrument ESEQuant-LR3 to detect
Go out fluorescence signal intensity.
As shown in figure 1, when not containing sulfadimethoxine in sample, aptamer can not be from nano material pad
Dissociation, therefore combined at detection line without aptamers, just there is no fluorescence signal yet.And the free fluorescein on nano material pad is still
It can be up at the nature controlling line of reaction film under the drive of sample liquids and be captured by anti-fluorescein antibody, nature controlling line occurs glimmering
Optical signal.
3rd, the detection method of sulfadimethoxine aptamer test strips.
Take series concentration sulfadimethoxine standard items to be added drop-wise on the sample absorption pad of test paper, after 10 minutes, will try
Paper slip is put into ELISA test strip instrument ESEQuant-LR3, is detected, and determines the fluorescence letter of detection line and nature controlling line respectively
Number intensity；By fluorescence signal intensity (C) of the fluorescence signal intensity (T) of detection line than upper nature controlling line, T/C values are obtained, make sulphur
The log concentration of amine SDM standard items corresponds to the linear regression curves of fluorescence signal T/C ratios, calculates regression equation；
Amine SDM standard items are replaced with into testing sample, testing sample is detected with above-mentioned test paper, is recorded in test paper
Detection line and nature controlling line fluorescence signal ratio, according to the regression equation of step (1), obtain aflatoxin in testing sample
Concentration.
4th, sulfadimethoxine test strips performance study.
1st, sulfadimethoxine sensitivity and the range of linearity are detected
The various concentrations sulfadimethoxine aqueous solution is prepared, the test strips prepared respectively with embodiment 2 are measured.With
ELISA test strip instrument ESEQuant-LR3 determines the fluorescence intensity at detection line and nature controlling line respectively.It is strong using detection line fluorescence
Degree and nature controlling line fluorescence intensity ratio (table 1), the standard curve of detection sulfadimethoxine is drawn as shown in Fig. 2 result table
It is bright, the sensitivity 20ng/mL of test strips；The range of linearity is 20ng/mL~500ng/mL.Regression equation is y=1.4064x-
1.8343 R2=0.9693.
The testing result of the sulfadimethoxine standard items of table 1
2nd, special Journal of Sex Research
Respectively compound concentration be the sulfadimethoxine solution for 100ng/mL, chloromycetin solution, tetracycline,
Enrofloxacin solution and amantadine solution, are measured with the test strips, and as a result (table 2) shows, only sulfanilamide (SN) dimethoxy
There is significant fluorescence signal at T lines in pyrimidine, does not almost have fluorescence signal at other drugs detection line, therefore show this examination
Paper slip has specificity well.
The sulfadimethoxine test strips specific detection result of table 2
5th, application of the sulfadimethoxine test strips in sulfadimethoxine in detecting milk
1st, Specification Curve of Increasing
It is bent with the standard detected in the present embodiment Part I in sulfadimethoxine sensitivity and range of linearity research
Line method, obtained regression equation are：Y=1.4064x-1.8343, R2=0.9693, it is sulfadimethoxine standard curve
Regression equation with one unknown.
2nd, sample extraction
5 parts of milk samples that sulfadimidine is not contained through national standard method (LC-MSMS) measure of selection, are separately added into not
With the sulfadimethoxine of concentration, sample extraction is then carried out, extracting method is：2mL milk is taken in pvc pipe, Ran Houjia
Enter 2mL deionized waters, (27 DEG C in complete warm shaken cultivation case；After 180r) vibration shakes up 10min, 7mL ethyl acetate is added,
Under the same conditions, vibration shakes up 15min, centrifuges 15min under the conditions of 5000r/4 DEG C afterwards, then takes supernatant to add
7mL ethyl acetate, vibration being carried out again under the same terms and shakes up and centrifuges, second of centrifugation takes its supernatant after terminating, and nitrogen is blown to
Precipitation, finally with 2mL deionized water dissolvings.
3rd, sample determines
The above-mentioned μ L of testing sample 100 prepared are taken to be added drop-wise on the sample absorption pad of test strips, observation sample chromatography edge
Nitrocellulose membrane flow, until by adsorptive pads absorption above；After 10 minutes, test strips are put into ELISA test strip instrument
In ESEQuant-LR3, detected, determine the fluorescence signal intensity of detection line and nature controlling line respectively；The fluorescence of detection line is believed
Number intensity (T) substitutes into obtained calculated value the regression equation of above-mentioned steps 1, meter than the fluorescence signal intensity (C) of upper nature controlling line
Calculation obtains the concentration of sulfadimethoxine in testing sample.
4th, testing result：
5 parts of milk sample testing results are as shown in table 3.From the results, it was seen that the rate of recovery of 5 parts of samples 89%~
Between 105%, the rate of recovery is preferable, illustrates that test strips of the present invention have higher accuracy.Other this method standard deviation is less than
10%, illustrate that this method has good stability.Therefore it can be used for the detection of sulfadimethoxine in actual sample.
Sulfadimethoxine result in the ELISA test strip milk of the present invention of table 3
Embodiment 3
A kind of ochratoxin A test strips are present embodiments provided to prepare and apply.
First, the preparation method of ochratoxin A test strips：
Glass fibre element film is chosen as the raw material for preparing nano material pad, graphene oxide aaerosol solution is sprayed to glass
Glass cellulose membrane, 7h is dried under conditions of 110 DEG C, remove the moisture on glass fibre element film, obtain oxidized graphene and change
The glass fibre of property.Cut and produce nano material.
By the fluorescein-labeled ochratoxin A aptamers (FAM- containing catenation sequence polyT15 of FAM
GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACATTTTTTTTTTTTTTT) it is added drop-wise on nano material pad, is incubated 15
Minute, dried under conditions of 35 DEG C, remove the moisture of nano material.
Nitrocellulose filter is chosen as reaction film.By the complementary series of the aptamers connects chain of biotin modification
PolyA15 (Btiotin-AAAAAAAAAAAAAAA) is sprayed onto at detection line, and anti-Cy5 antibody is sprayed onto at nature controlling line.
Sample pad, nano material pad, reaction film and adsorptive pads are affixed to the same side of bottom plate successively.
2nd, the Cleaning Principle of ochratoxin A test strips
Cleaning Principle of the Cleaning Principle with sulfadimethoxine test strips in embodiment 2.
3rd, the detection method of ochratoxin A test strips
Detection method of the detection method with sulfadimethoxine test strips in embodiment 2.
4th, ochratoxin A test strips performance study
1st, detect ochratoxin A sensitivity determination and be prepared by standard curve
Various concentrations ochratoxin A solution is prepared, the test strips prepared respectively with embodiment 3 are measured.Use test paper
Bar detecting instrument ESEQuant-LR3 determines the fluorescence intensity at detection line and nature controlling line respectively.Using detection line fluorescence intensity with
Nature controlling line fluorescence intensity ratio (table 4), the standard curve for drawing detection ochratoxin A are as shown in Figure 3, the results showed that, test paper
The sensitivity 2ng/mL of bar；The range of linearity is 2ng/mL~50ng/mL.Regression equation y=1.2593x-0.1874, R2=
0.941。
The testing result of the ochratoxin A standard items of table 4
2nd, special Journal of Sex Research
Compound concentration is the ochratoxin A for 10ng/mL, aflatoxin B1, Aflatoxins M1, vomiting respectively
Toxin, fumonisin and Gibberella zeae alcoholic solution, are measured with the test strips, and as a result (table 5) shows, only Aspergillus ochraceus poison
There is significant fluorescence signal at T lines in plain A, does not almost have fluorescence signal at other Mycotoxin identification lines, therefore show this examination
Paper slip has specificity well.
The ochratoxin A test strips specific detection result of table 5
5th, the application of ochratoxin A test strips ochratoxin A in feed is detected
1st, Specification Curve of Increasing
With the standard curve side detected in the present embodiment Part I in ochratoxin A sensitivity and range of linearity research
Method, obtained regression equation are y=1.2593x-0.1874, R2=0.941, it is ochratoxin A standard curve simple regression
Equation.
2nd, sample extraction
5 parts of selection contains ochratoxin A Feed Sample through national standard method (LC-MSMS) measure, carries out sample extraction, carries
The method is taken to be：Feed or grain sample are crushed and by 20 mesh sieves first.Take 0.5g handle after sample, be added to 15ml
In centrifuge tube.Pure water and each 2mL of ethyl acetate are accurately added into centrifuge tube, bottle stopper tightening seal is divided with forced oscillation 5
Clock, 4000rpm are centrifuged 1 minute；0.6mL supernatants are taken into small glass with suction pipe, are dried up filtrate, are then diluted with 0.3ml
Liquid redissolves bottom of a cup solid.This lysate is to detect liquid.
3rd, sample determines
The above-mentioned μ L of testing sample 100 prepared are taken to be added drop-wise on the sample absorption pad of test strips, observation sample chromatography edge
Nitrocellulose membrane flow, until by adsorptive pads absorption above；After 10 minutes, test strips are put into ELISA test strip instrument
In ESEQuant-LR3, detected, determine the fluorescence signal intensity of detection line and nature controlling line respectively；The fluorescence of detection line is believed
Number intensity (T) substitutes into obtained calculated value the regression equation of above-mentioned steps 1, meter than the fluorescence signal intensity (C) of upper nature controlling line
Calculation obtains the concentration of ochratoxin A in testing sample.
4th, testing result：
6 parts of Feed Sample testing results are as shown in table 6.From the results, it was seen that the rate of recovery of 6 parts of samples 87%~
Between 105%, there is extraordinary uniformity with national standard method LC-MS/MS methods, it is higher to illustrate that test strips of the present invention have
Accuracy.Other this method standard deviation is less than 10%, illustrates that this method has good stability.Therefore it can be used for feed sample
The detection of ochratoxin A in product.
Ochratoxin A result in the ELISA test strip feed of the present invention of table 6
In summary, a kind of aptamer fluorescent test paper strip provided in an embodiment of the present invention, substituted with nano material pad
Gold standard pad in conventional test strips, solve the defects of current most of aptamer is not used to test strips exploitation, be
Numerous analytes provide a kind of new analysis method.And this method and LC-MS/ can be seen that by the embodiment of the present invention
The national standard methods such as MS have extraordinary uniformity, but this method is simple to operate, and cost is cheap, be more convenient for basic unit and Site Detection
Using.
Embodiments described above is part of the embodiment of the present invention, rather than whole embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
- A kind of 1. aptamer fluorescent test paper strip, it is characterised in that including bottom plate, sample pad, nano material pad, reaction film with And adsorptive pads, the sample pad, the nano material pad, the end of the reaction film and the adsorptive pads are in turn connected to form Detection layers, the detection layers are connected with the bottom plate, and the reaction film is provided with detection line and nature controlling line, and the detection line is arranged at Close to one end of the nano material pad, the nature controlling line is disposed in proximity to one end of the adsorptive pads；The nano material pad is attached with the core with catenation sequence of fluorescein-labeled aptamer and/or fluorescence labeling Sour aptamers and free fluorescein, the detection line are fixed with the complementary strand of the aptamer or the aptamer The complementary strand of catenation sequence, the anti-fluorescein antibody corresponding with the fluorescein is provided with the nature controlling line.
- 2. aptamer fluorescent test paper strip according to claim 1, it is characterised in that the aptamer connects sequence Row include one kind in polyTn, polyAn, polyCn and polyGn, the corresponding aptamer catenation sequence it is mutual Mending chain includes one kind in polyAn, polyTn, polyGn and polyCn, and wherein n is 6~25 integer.
- 3. aptamer fluorescent test paper strip according to claim 1, it is characterised in that the nano material pad is porous Nano-material film or by nanometer-material-modified cellulose membrane.
- 4. aptamer fluorescent test paper strip according to claim 3, it is characterised in that the nano material is included but not It is limited at least one of graphene oxide, reduced graphene, nanogold, molybdenum disulfide and CNT.
- 5. aptamer fluorescent test paper strip according to claim 3, it is characterised in that the cellulose membrane is included but not It is limited at least one of glass fibre, cellulosic filter paper and polyester film.
- 6. aptamer fluorescent test paper strip according to claim 1, it is characterised in that the fluorescein includes but unlimited In at least one of anthocyanidin 5 (Cy5), Fluoresceincarboxylic acid (FAM), quantum dot, upper transfer and fluorescent microsphere.
- 7. a kind of preparation method of aptamer fluorescent test paper strip as described in any one of claim 1 to 6, its feature exist In, including：The sample pad, the nano material pad, the reaction film and the adsorptive pads are affixed into the bottom successively The same side of plate.
- 8. the preparation method of aptamer fluorescent test paper strip according to claim 7, it is characterised in that the nanometer material The preparation method of backing strap includes：Graphene oxide aaerosol solution is sprayed to glass fibre element film, in 110~130 DEG C of condition 7~9h of lower drying produces nano material, and the fluorescein-labeled aptamer and free fluorescein are added dropwise into described receive Rice material, reacts 15~25min, is dried under conditions of 35~40 DEG C.
- 9. the preparation method of aptamer fluorescent test paper strip according to claim 8, it is characterised in that the reaction film Preparation method include：By the complementary strand of the aptamer of biotin modification or the aptamer catenation sequence Complementary strand is incubated 25~35min with Avidin, is sprayed onto at the detection line.
- 10. aptamer fluorescent test paper strip as described in any one of claim 1~6 food security, environmental monitoring and Detection application in clinical diagnosis, it is preferable that sulfadimethoxine in detection food, environment and clinical diagnosis should With.
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