CN103048449B - Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof - Google Patents
Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof Download PDFInfo
- Publication number
- CN103048449B CN103048449B CN201310012751.0A CN201310012751A CN103048449B CN 103048449 B CN103048449 B CN 103048449B CN 201310012751 A CN201310012751 A CN 201310012751A CN 103048449 B CN103048449 B CN 103048449B
- Authority
- CN
- China
- Prior art keywords
- aptamer
- streptavidin
- pad
- detection
- nature controlling
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a chromatographic detection kit based on an aptamer, as well as a preparation method and detection method of the kit. The test paper of the kit includes a bottom board, and a sample pad, a bonding pad, a cellulose nitrate film and a water absorption pad, which are adhered on the bottom board and overlap one another; a detection line is arranged on the side of the cellulose nitrate film close to the bonding pad, and a quality control line is arranged on the side of the cellulose nitrate film close to the water absorption pad; an A aptamer labeled by colloidal gold is applied on the bonding pad; a composite containing a B aptamer and streptavidin is applied on the detection line; streptavidin is applied on the quality control line; and both of the A aptamer and the B aptamer are aptamers labeled by biotin and capable of specifically identifying a same test object. The chromatographic detection kit can display the result in five minutes only by directly dropping a diluted sample to be tested into a sample hole, is simple, quick, sensitive, easy to read result, and convenient and simple to prepare, and so on, and has the advantages of simplicity in operation and high specificity without using antibodies and instruments.
Description
Technical field
The present invention relates to a kind of medical testing product and preparation method thereof, particularly relate to a kind of chromatography detection kit based on aptamer and preparation method thereof and detection method.
Background technology
Common immunodetection is that special labelled antibody is first fixed on solid support, after determinand specific bond, then be combined with another antibody, like this by naked eyes or specific detecting instrument, just can qualitative detection determinand, thus realize specific immunodiagnosis.
The biomacromolecule of the specific recognition detection thing that conventional immunochromatographic method is used is all antibody, reacted by antigen-antibody specific recognition, the immune colloidal solid be marked on antigen or antibody makes detection line and nature controlling line show certain color, thus realizes specific immunodiagnosis.But the method detects not fast with sensitive, and also needs to use corresponding instrument and equipment with antibody test, operates not easy.
Summary of the invention
The present invention is intended to overcome the deficiencies in the prior art, a kind of chromatography detection kit based on aptamer and preparation method thereof and detection method.Detecting thing by providing a kind of by aptamer specific recognition, on conventional colloidal gold immunochromatographykit kit basis, making colloidal gold immunochromatographiassay assay reagent box, by developing the color on detection line and nature controlling line, realizing detecting.
For achieving the above object, technical scheme provided by the invention is:
The invention provides a kind of chromatography detection kit based on aptamer, comprise Test paper; Described Test paper comprises base plate, to be bonded on base plate and the sample pad overlapped successively, pad, nitrocellulose filter and adsorptive pads; Side near pad on described nitrocellulose membrane is provided with detection line, and the side near adsorptive pads on nitrocellulose membrane is provided with nature controlling line; Described pad scribbles the A aptamer of nanometer particle to mark; Described detection line scribbles the compound of B aptamer and Streptavidin; Described nature controlling line scribbles Streptavidin; Described A aptamer and B aptamer be can specific recognition same detection thing by biotin labeled aptamer; Wherein, described nano particle is nm of gold, tri-iron tetroxide or latex.
Wherein, the DNA sequence dna 5 ' of described A aptamer is held by marking sulfhydryl, and 3 ' end is by biotin labeling; The DNA sequence dna 3 ' of described B aptamer is held by biotin labeling.
The sequence of described A aptamer and B aptamer can carry out designing and adjusting according to thing to be detected, the DNA sequence dna of described A aptamer is as shown in SEQ ID NO.1 in an embodiment of the present invention, and the DNA sequence dna of described B aptamer is as shown in SEQ ID NO.2; The DNA sequence dna of described A aptamer is as shown in SEQ ID NO.3 in another embodiment of the invention, and the DNA sequence dna of described B aptamer is as shown in SEQ ID NO.4; The DNA sequence dna of described A aptamer is as shown in SEQ ID NO.5 in another embodiment of the invention, and the DNA sequence dna of described B aptamer is as shown in SEQ IDNO.4.
Described base plate is PVC board; The material of sample pad is poly-Acetate Film; The material of described pad is glass fibre; Described adsorptive pads is absorbent filter; Described Test paper is packed by rigid plastic card.
Present invention also offers a kind of method preparing mentioned reagent box, comprise the steps:
(1) with nanometer particle to mark A aptamer;
(2) the sample pad treating fluid of PBS sample pad put into containing BSA and tween soaks more than 1h, dry for standby;
(3) step (1) is disperseed with the PBS buffer diluent containing sucrose, BSA and tween again by the A aptamer of nanometer particle to mark, and be sprayed on pad, dry for standby;
(4) compound of detection line position specking B aptamer and Streptavidin on nitrocellulose membrane, at nature controlling line position specking Streptavidin, dry for standby;
(5) sample pad, pad, nitrocellulose filter and adsorptive pads overlapped successively and be bonded on base plate, being cut into test strip, test strip rigid plastic card is packed.
Wherein, described step (1) is the nanometer particle to mark A aptamer of 10-40nm with diameter; Specking described in step (4) is that B aptamer concentration is 0.5-1.5 μMs in the B aptamer of detection line position and the compound of Streptavidin, preferably the solution of 1 μM and concentration are 0.5-1.5mg/mL, Streptavidin 1:8-12 by volume of preferred 1mg/mL, preferred 1:10 is mixed with and forms; The Streptavidin of described specking in nature controlling line position is concentration is 1.5-2.5mg/mL, the Streptavidin dilution of preferred 2mg/mL.
Present invention also offers a kind of detection method based on aptamer:
Described method adopts two aptamers identifying same detection thing, on Conventional chromatography kit basis, make above-mentioned chromatography detection kit, detect by developing the color the chromatography realized without antibody after A aptamer and B aptamer and determinand specific bond on detection line and nature controlling line: if nature controlling line does not develop the color, then test result is all invalid; If detection line colour developing and nature controlling line do not develop the color also be considered as invalid; If nature controlling line and detection line all develop the color, then represent detection sample in have determinand.
Below in conjunction with the detailed manufacturing process of kit and principle, the invention will be further described:
The concrete manufacturing process of kit of the present invention is as follows:
(1) with diameter be the nanometer particle to mark aptamer 1 of 10-40nm, with sterilized water, nano particle is diluted, add aptamer again, centrifugal 15 minutes of 10000rpm, remove supernatant, colloid redissolves in 0.l M PBS damping fluid, such repeatable operation three times, finally the aptamer 1 marked is redissolved in the PBS damping fluid of the 0.0l M pH7.4 containing 20mM Na3PO4,1%PEG6000;
(2) select poly-Acetate Film as sample pad material, put it in the sample pad treating fluid containing the 0.0l M pH7.4PBS damping fluid of 3%BSA, 0.05% polysorbas20 and soak after 1 hour, dry for 37 DEG C and dry for 2 hours, 4 DEG C save backup;
(3) select glass fibre as pad material, colloid gold label A aptamer step (1) prepared is scattered in the 0.0l M PBS damping fluid containing 5% sucrose, 1%BSA, 0.05% polysorbas20 again, be sprayed on the pad of glass fibre material with some film machine again, 37 degree of oven dry 2 hours, 4 DEG C save backup;
(4) point sample on nitrocellulose filter: be dissolved in sterilized water with the B aptamer being marked with biotin of synthesis, concentration is 1 μM, the volume ratio being the streptavidin 1:10 of 1mg/ml with concentration mixes, room temperature reaction is after one hour, add the PBS damping fluid of the 0.01M pH7.4 that monoploid amasss again, then the centrifugal ultrafiltration pipe of molecular cut off 30000 is moved into, centrifugal 15 minutes of 6000rpm, add the PBS damping fluid with the 0.01M pH7.4 of above-mentioned same volume again, centrifugal 15 minutes of 6000rpm, such repeatable operation three times, obtain the B aptamer streptavidin compound being equivalent to original volume, be dissolved in by streptavidin in sterilized water, making concentration is that the bag of 2mg/ml is by nature controlling line streptavidin dilution.B aptamer streptavidin compound and streptavidin dilution are put detection line (detection line is positioned at the midline position of whole piece Test paper) on nitrocellulose filter and nature controlling line position by some film machine respectively, two lines are separated by three millimeters, then this nitrocellulose filter is put vacuum dryer, dry half an hour, dry 1 hour for 37 DEG C again, good seal to be placed in room temperature for subsequent use;
(5) sample pad handled well, pad, nitrocellulose filter and absorbent filter are pasted onto respectively in order after in PVC sheet, then the detector bar being cut into four mm in width loads in plastic clip, is assembled into chromatography detection kit.
During use, sample to be measured is joined loading wells, sample to be measured is under capillary action along sample pad, pad, nitrocellulose filter is to absorbent filter extreme direction chromatography, in chromatography process, detection thing in sample to be measured first makes it to redissolve with the nano particle-A aptamer compound specific bond in glass fiber conjugate pad, potpourri after redissolution continues chromatography on nitrocellulose filter, with the B aptamer generation immune response of detection line region bag quilt on nitrocellulose filter, under the effect of certain density detection thing, show the lines of corresponding color, unnecessary nano particle-A aptamer compound continues forward, and when the biotin that A aptamer marks arrives nature controlling line region, combine with the streptavidin of bag quilt, nano particle generation enrichment, shows the colored line of nano particle.If do not exist when analyzing thing in sample, nano particle-A aptamer compound and B aptamer can not act in detection line region, and continue chromatography forward and reacted by biological streptavidin.Therefore detection line is colourless, and nature controlling line shows the color of nano particle.Nature controlling line is for judging the quality condition of kit: when the detection substrate concentration in sample is too high, or there is high salt, nano particle-A aptamer can be caused all to be enriched in detection line, unnecessary collaurum is not had to continue forward, nature controlling line would not have the enrichment of nanoparticle interest, be shown as colourless, the scope of application of microenvironment beyond this detection box of detection is described, result is invalid.Departing from from nanoparticle surface when aptamer or be degraded, or other reason causes kit quality to change, and all can cause detection line and nature controlling line all there will not be nano particle, and detection line and nature controlling line are all colourless, show that testing result is invalid.Therefore, in the detection, detection line and nature controlling line show the color of nano particle simultaneously, just show the credible of testing result.
The nature controlling line of immunochromatographic method display of the present invention and p-wire are the color of nano particle.Aptamer also can specific recognition detect thing, two same detection things of energy specific recognition be coated on the glass fiber conjugate pad in colloidal gold immunochromatographimethod and the aptamer on nitrocellulose membrane respectively.Compared with prior art, the present invention uses conventional chromatography detector bar, realize without antibody chromatography: after the A aptamer of mark and detection thing combine, under capillarity, sample will move forward along nitrocellulose membrane, when move to be fixed with another also can specific recognition detect thing cellulose nitrate on detection line (being coated with B aptamer region) time, the detection thing combining A aptamer in sample again with B aptamer generation specific binding, with this, the A aptamer of nanoparticle interest mark, detect thing, another also can specific recognition detect thing B aptamer formed compound, for the color of visible nano particle, so just achieve specific immunochromatographyassay assay.When the present invention prepares test strips, without the need to using antibody, other reagent are not needed during detection yet, get 0.12mL after only needing directly will to hold test sample product thin up and instill well, result can be gone out after five minutes, there is the features such as simple, quick, sensitive, this detection kit without the need to using antibody, do not use instrument and equipment, simple to operate, specificity good, the easy interpretation of result, manufacture convenient and easy.
Accompanying drawing explanation
Fig. 1 is Test paper structural representation in kit of the present invention;
Fig. 2 is principle schematic of the present invention, wherein, has detection thing: represent and detect substrate concentration higher than minimal detectable concentration, and detection line is the color (for dark in figure) of nano particle; Without detecting thing: represent and detect substrate concentration higher than minimal detectable concentration, detection line position colourless (being blank in figure);
Fig. 3 is immunochromatographyassay assay result process decision chart of the present invention, and wherein, T represents detection line, and C represents nature controlling line.
In figure: 1, base plate; 2, sample pad; 3, pad; 4, nitrocellulose filter; 5, detection line; 6, nature controlling line; 7 adsorptive pads.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further:
Embodiment 1
See Fig. 1 to Fig. 3, a kind of colloidal gold immunity chromatography detection kit based on aptamer, comprise Test paper, described Test paper comprises base plate 1, to be bonded on base plate 1 and the sample pad 2 overlapped successively, pad 3, nitrocellulose filter 4 and adsorptive pads 7; Side near pad 3 on described nitrocellulose membrane 4 is provided with detection line 5, and the side near adsorptive pads 7 on nitrocellulose membrane 4 is provided with nature controlling line 6; Described pad 3 scribbles the A aptamer of nanometer particle to mark; Described detection line 5 scribbles the compound of B aptamer and Streptavidin; Described nature controlling line 6 scribbles Streptavidin; Described A aptamer and B aptamer be can specific recognition same detection thing by biotin labeled aptamer; Wherein, described nano particle is collaurum.
Wherein, the DNA sequence dna 5 ' of described A aptamer is held by marking sulfhydryl, and 3 ' end is by biotin labeling; The DNA sequence dna 3 ' of described B aptamer is held by biotin labeling; The DNA sequence dna of described A aptamer is as shown in SEQ ID NO.1, and the DNA sequence dna of described B aptamer is as shown in SEQ IDNO.2; Described base plate 1 is PVC board; The material of sample pad 2 is poly-Acetate Film; The material of described pad 3 is glass fibre; Described adsorptive pads 7 is absorbent filter; Described Test paper is packed by rigid plastic card.
Embodiment 2
Prepare the method for kit described in embodiment 1:
(1) aptamer 1 marks colloid gold particle: by centrifugal 15 minutes of the 13 nano-colloid gold solution 10000rpm of synthesis, sucking-off supernatant, add the sterilized water of original volume, repetitive operation twice, the ratio of 0.5 O D aptamer is added according to every milliliter of collaurum, A aptamer DNA sequence dna is: 5 '-SHC6-ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG TTT TTT TTT T-3 ' Biotin(SEQ ID NO.1), this adaptive physical efficiency specific recognition lysozyme, slowly join under magnetic stirring in colloidal gold solution, after room temperature places 24 hours, add 0.l M PB solution, regulate PB final concentration to 10mM, add PBS damping fluid again, regulate NaCl concentration in solution to be 0.1M, aged at room temperature 24 hours, finally add PBS damping fluid, regulate NaCl final concentration in solution to be 0.3M, aged at room temperature after 24 hours 10000rpm centrifugal, remove supernatant, colloid redissolves in 0.l M PBS damping fluid, such repeatable operation three times, redissolves in the PBS damping fluid of the 0.0l M pH7.4 containing 20mM Na3PO4,1%PEG 6000 by the aptamer 1 marked,
(2) select poly-Acetate Film as sample pad material, the sample pad treating fluid put into by poly-Acetate Film containing the 0.0l M pH7.4PBS damping fluid of 3%BSA, 0.05% polysorbas20 soaked after 1 hour, and dry for 37 DEG C and dry for 2 hours, 4 DEG C save backup;
(3) preparation of pad: during use, collaurum prepared by step (1) is scattered in again in the 0.0l M pH7.4PBS damping fluid containing 5% sucrose, 5%BSA, 1%PEG20000,0.05% polysorbas20, be sprayed on the pad of glass fibre material with some film machine again, 37 degree of oven dry 2 hours, 4 DEG C save backup;
(4) point sample on nitrocellulose filter: with the B aptamer being marked with biotin of synthesis, its DNA sequence dna is: 5 '-GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGCATG CGG CTT TTT TTT TT-3 ' Biotin(SEQ ID NO.2), be dissolved in sterilized water, concentration is 1 μM, the volume ratio being the streptavidin 1:10 of 1mg/ml with concentration mixes, room temperature reaction is after one hour, add the PBS damping fluid of the 0.01M pH7.4 that monoploid amasss again, then the daltonian centrifugal ultrafiltration pipe of molecular cut off 30000 is moved into, centrifugal 15 minutes of 6000rpm, add the PBS damping fluid with the 0.01MpH7.4 of above-mentioned same volume again, centrifugal 15 minutes of 6000rpm, such repeatable operation three times, obtain the B aptamer streptavidin compound being equivalent to original volume, be dissolved in by streptavidin in sterilized water, making concentration is that the bag of 2mg/ml is by nature controlling line streptavidin dilution.Aptamer 2 streptavidin compound and streptavidin dilution are put detection line (detection line is positioned at the midline position of whole piece Test paper) on nitrocellulose filter and nature controlling line position by some film machine respectively; two lines are separated by three millimeters; then this nitrocellulose filter is put vacuum dryer; dry half an hour; dry 1 hour for 37 DEG C again, good seal to be placed in room temperature for subsequent use;
(5) sample pad handled well, pad, nitrocellulose filter and absorbent filter are pasted onto respectively in order after in PVC sheet, then the detector bar being cut into four mm in width loads in plastic clip, is assembled into chromatography detection kit;
(6) detect: get 0.12mL after drawing sample thin up to be measured and instill well.If nature controlling line does not develop the color, test result is all invalid, if the aobvious red and nature controlling line of detection line do not develop the color also be considered as invalid; If nature controlling line and detection line all occur redness, represent and detecting in sample the lysozyme had higher than minimal detectable concentration (1 μM).
Embodiment 3
See Fig. 1 to Fig. 3, a kind of colloidal gold immunity chromatography detection kit based on aptamer, comprise Test paper, described Test paper comprises base plate 1, to be bonded on base plate 1 and the sample pad 2 overlapped successively, pad 3, nitrocellulose filter 4 and adsorptive pads 7; Side near pad 3 on described nitrocellulose membrane 4 is provided with detection line 5, and the side near adsorptive pads 7 on nitrocellulose membrane 4 is provided with nature controlling line 6; Described pad 3 scribbles the A aptamer of nanometer particle to mark; Described detection line 5 scribbles the compound of B aptamer and Streptavidin; Described nature controlling line 6 scribbles Streptavidin; Described A aptamer and B aptamer be can specific recognition same detection thing by biotin labeled aptamer; Wherein, described nano particle is collaurum.
Wherein, the DNA sequence dna 5 ' of described A aptamer is held by marking sulfhydryl, and 3 ' end is by biotin labeling; The DNA sequence dna 3 ' of described B aptamer is held by biotin labeling; The DNA sequence dna of described A aptamer is as shown in SEQ ID NO.3, and the DNA sequence dna of described B aptamer is as shown in SEQ IDNO.4; Described base plate 1 is PVC board; The material of sample pad 2 is poly-Acetate Film; The material of described pad 3 is glass fibre; Described adsorptive pads 7 is absorbent filter; Described Test paper is packed by rigid plastic card.
Embodiment 4
Prepare the method for kit described in embodiment 3:
(1) aptamer 1 marks colloid gold particle: by centrifugal 15 minutes of the 10 nano-colloid gold solution 10000rpm of synthesis, sucking-off supernatant, add the sterilized water of original volume, repetitive operation twice, the ratio of 0.5OD aptamer 1 is added according to every milliliter of collaurum, the A aptamer DNA sequence dna of specific recognition human thrombin is: 5 ' SH C6-TTT TTT TTT TGG TTG GTG TGG TTG GTT TTTTTT TT3 ' Biotin(SEQ ID NO.3), slowly join under magnetic stirring in colloidal gold solution, after room temperature places 24 hours, add 100mM PB solution, regulate final concentration to 10mM, add PBS damping fluid again, regulate NaCl concentration in solution to be 0.1M, aged at room temperature 24 hours, finally add PBS damping fluid, NaCl final concentration in solution is regulated to be 0.3M, aged at room temperature after 24 hours 10000rpm centrifugal, remove supernatant, colloid redissolves in 0.l M PBS damping fluid, such repeatable operation three times, finally redissolves in the PBS damping fluid of the 0.0l M pH7.4 containing 20mM Na3PO4,1%PEG6000 by the aptamer 1 marked,
(2) select poly-Acetate Film as sample pad material, the sample pad treating fluid put into by poly-Acetate Film containing the 0.0l M pH7.4PBS damping fluid of 3%BSA, 0.05% polysorbas20 soaked after 1 hour, and dry for 37 DEG C and dry for 2 hours, 4 DEG C save backup;
(3) preparation of pad: during use, collaurum prepared by step (1) is scattered in again in the 0.0l M pH7.2PBS damping fluid containing 5% sucrose, 2%BSA, 2%Dextran, 0.05% polysorbas20, be sprayed in glass fiber conjugate pad with some film machine again, dry 2 hours for 37 DEG C, 4 DEG C save backup;
(4) point sample on nitrocellulose filter: the specific recognition human thrombin B aptamer being marked with biotin with one end of synthesis, its DNA sequence dna is: 5 '-AGT CCG TGG TAG GGC AGG TTGGGG TGA CTT TTT TTT TTT-3 ' Biotin(SEQ ID NO.4), be dissolved in sterilized water, concentration is 1 μM, the streptavidin being 1mg/ml with concentration mixes with the volume ratio of 1:10, room temperature reaction one hour, adding the PBS damping fluid of the 0.02M pH7.2 that monoploid amasss, then the daltonian centrifugal ultrafiltration pipe of molecular cut off 30000 is moved into, centrifugal 15 minutes of 6000rpm, add the PBS damping fluid with the 0.02M pH7.2 of above-mentioned same volume again, centrifugal 15 minutes of 6000rpm, such repeatable operation three times, obtain the aptamer 2 streptavidin compound being equivalent to original volume, be dissolved in by streptavidin in sterilized water, wrapping by nature controlling line streptavidin diluent concentration is 2mg/ml.Aptamer 2 and streptavidin compound and streptavidin dilution are put detection line (detection line is positioned at whole test strip midline position) on nitrocellulose filter and nature controlling line position by some film machine respectively; two lines are separated by three millimeters; then nitrocellulose filter is placed in 37 DEG C to dry 2 hours, good seal to be placed in room temperature for subsequent use;
(5) sample pad handled well, pad, nitrocellulose filter and absorbent filter are pasted onto respectively in order after in PVC sheet, then the detector bar being cut into four mm in width loads in plastic clip, is assembled into chromatography detection kit;
(6) detect: get 0.12mL after drawing sample thin up to be measured and instill well.If nature controlling line does not develop the color, test result is all invalid, if the aobvious red and nature controlling line of detection line do not develop the color also be considered as invalid; If nature controlling line and detection line all occur redness, represent and detecting in sample the lysozyme had higher than minimal detectable concentration (1 μM).
Embodiment 5
See Fig. 1 to Fig. 3, a kind of colloidal gold immunity chromatography detection kit based on aptamer, comprise Test paper, described Test paper comprises base plate 1, to be bonded on base plate 1 and the sample pad 2 overlapped successively, pad 3, nitrocellulose filter 4 and adsorptive pads 7; Side near pad 3 on described nitrocellulose membrane 4 is provided with detection line 5, and the side near adsorptive pads 7 on nitrocellulose membrane 4 is provided with nature controlling line 6; Described pad 3 scribbles the A aptamer of nanometer particle to mark; Described detection line 5 scribbles the compound of B aptamer and Streptavidin; Described nature controlling line 6 scribbles Streptavidin; Described A aptamer and B aptamer be can specific recognition same detection thing by biotin labeled aptamer; Wherein, described nano particle is ferroferric oxide nano granules.
Wherein, the DNA sequence dna 5 ' of described A aptamer is held by marking sulfhydryl; The DNA sequence dna 3 ' of described B aptamer is held by biotin labeling; The DNA sequence dna of described A aptamer is as shown in SEQ IDNO.5, and the DNA sequence dna of described B aptamer is as shown in SEQ ID NO.4; Described base plate 1 is PVC board; The material of sample pad 2 is poly-Acetate Film; The material of described pad 3 is glass fibre; Described adsorptive pads 7 is absorbent filter; Described Test paper is packed by rigid plastic card.
Embodiment 6
Prepare the method for kit described in embodiment 5:
(1) aptamer 1 marks ferroferric oxide nano granules: by the brown ferroferric oxide nano granules solution 14000rpm centrifugal 10 minute of surface with particle diameter 10 nanometer of carboxyl, sucking-off supernatant, adding concentration is in the MES solution of 10mM EDC, stirring at normal temperature 30 minutes, centrifugally remove supernatant, add the ratio of 0.5OD aptamer 1 according to every milliliter of nanoparticles solution, the A aptamer DNA sequence dna adding the specific recognition human thrombin of the PBS buffer solution of 10mM pH7.4 is: 5 '-TGG TTGGTG TGG TTG GTT TTT TTT TT-3 ' NH
2(SEQ ID NO.5), slowly join under magnetic stirring in ferroferric oxide nano granules, after room temperature places 2 hours, centrifugally remove supernatant, use the PBS buffer solution twice of 10mM pH7.4 again, finally the aptamer 1 marked is redissolved in the PBS damping fluid of the 0.0l M pH7.4 containing 20mM Na3PO4,1%PEG6000;
(2) select poly-Acetate Film as sample pad material, the sample pad treating fluid put into by poly-Acetate Film containing the 0.0l M pH7.4PBS damping fluid of 3%BSA, 0.05% polysorbas20 soaked after 1 hour, and dry for 37 DEG C and dry for 2 hours, 4 DEG C save backup;
(3) preparation of pad: during use, ferroferric oxide nano granules prepared by step (1) is scattered in again in the 0.0l M pH7.2PBS damping fluid containing 5% sucrose, 2%BSA, 2%Dextran, 0.05% polysorbas20, be sprayed in glass fiber conjugate pad with some film machine again, dry 2 hours for 37 DEG C, 4 DEG C save backup;
(4) point sample on nitrocellulose filter: the specific recognition human thrombin B aptamer being marked with biotin with one end of synthesis, its DNA sequence dna is: 5 '-AGT CCG TGG TAG GGC AGG TTGGGG TGA CTT TTT TTT TTT-3 ' Biotin(SEQ ID NO.4), be dissolved in sterilized water, concentration is 1 μM, the streptavidin being 1mg/ml with concentration mixes with the volume ratio of 1:10, room temperature reaction one hour, adding the PBS damping fluid of the 0.02M pH7.2 that monoploid amasss, then the daltonian centrifugal ultrafiltration pipe of molecular cut off 30000 is moved into, centrifugal 15 minutes of 6000rpm, add the PBS damping fluid with the 0.02M pH7.2 of above-mentioned same volume again, centrifugal 15 minutes of 6000rpm, such repeatable operation three times, obtain the aptamer 2 streptavidin compound being equivalent to original volume, be dissolved in by streptavidin in sterilized water, wrapping by nature controlling line streptavidin diluent concentration is 2mg/ml.Aptamer 2 and streptavidin compound and streptavidin dilution are put detection line (detection line is positioned at whole test strip midline position) on nitrocellulose filter and nature controlling line position by some film machine respectively; two lines are separated by three millimeters; then nitrocellulose filter is placed in 37 DEG C to dry 2 hours, good seal to be placed in room temperature for subsequent use;
(5) sample pad handled well, pad, nitrocellulose filter and absorbent filter are pasted onto respectively in order after in PVC sheet, then the detector bar being cut into four mm in width loads in plastic clip, is assembled into chromatography detection kit;
(6) detect: draw human blood to be measured reset and add sterilized water dilution after, get 0.12mL and instill well, if nature controlling line does not develop the color for invalid, if detection line show brown and nature controlling line do not develop the color also be considered as invalid, if nature controlling line and detection line all occur brown, represent and detecting in sample the human thrombin had higher than minimal detectable concentration (100nM).
Claims (7)
1., based on a colloidal gold immunity chromatography detection kit for aptamer, comprise Test paper; Described Test paper comprises base plate (1), is bonded in base plate (1) and goes up and the sample pad (2) overlapped successively, pad (3), nitrocellulose filter (4) and adsorptive pads (7); The upper side near pad (3) of described nitrocellulose membrane (4) is provided with detection line (5), and the upper side near adsorptive pads (7) of nitrocellulose membrane (4) is provided with nature controlling line (6); It is characterized in that, described pad (3) scribbles the A aptamer of colloid gold label; Described detection line (5) scribbles the compound of B aptamer and Streptavidin; (6) scribble Streptavidin to described nature controlling line; Described A aptamer and B aptamer be can specific recognition same detection thing by biotin labeled aptamer; The DNA sequence dna 5 ' of described A aptamer is held by marking sulfhydryl, and 3 ' end is by biotin labeling; The DNA sequence dna 3 ' of described B aptamer is held by biotin labeling; The DNA sequence dna of described A aptamer is as shown in SEQ ID NO.1, and the DNA sequence dna of described B aptamer is as shown in SEQ ID NO.2; Or the DNA sequence dna of described A aptamer is as shown in SEQ ID NO.3, the DNA sequence dna of described B aptamer is as shown in SEQ ID NO.4.
2. kit as claimed in claim 1, it is characterized in that, described base plate (1) is PVC board; The material of sample pad (2) is poly-Acetate Film; The material of described pad (3) is glass fibre; Described adsorptive pads (7) is absorbent filter.
3. kit as claimed in claim 1, it is characterized in that, described Test paper is packed by rigid plastic card.
4. prepare the method for kit described in any one of claims 1 to 3, comprise the steps:
(1) with colloid gold label A aptamer;
(2) the sample pad treating fluid of PBS sample pad put into containing BSA and tween soaks more than 1h, dry for standby;
(3) step (1) is scattered in the PBS buffer diluent containing sucrose, BSA and tween again by the A aptamer of colloid gold label, and is sprayed on pad, dry for standby;
(4) compound of detection line position specking B aptamer and Streptavidin on nitrocellulose membrane, at nature controlling line position specking Streptavidin, dry for standby;
(5) sample pad, pad, nitrocellulose filter and adsorptive pads overlapped successively and be bonded on base plate, being cut into test strip, test strip rigid plastic card is packed.
5. method as claimed in claim 4, is characterized in that, described step (1) marks A aptamer with the colloid gold particle that diameter is 13-40nm.
6. method as claimed in claim 5, it is characterized in that, specking described in step (4) by volume in the Streptavidin of the B aptamer of detection line position and the compound of the Streptavidin 0.5-1.5mg/mL that is that to be the solution of 0.5-1.5 μMs and concentration by B aptamer concentration be is mixed with and forms in 1:8-12; The Streptavidin dilution of the Streptavidin of described specking in nature controlling line position to be concentration be 1.5-2.5mg/mL.
7. method as claimed in claim 6, it is characterized in that, specking described in step (4) in the B aptamer of detection line position and the compound of the Streptavidin 1mg/mL that is that to be the solution of 1 μM and concentration by B aptamer concentration be Streptavidin by volume 1:10 be mixed with and form; The Streptavidin dilution of the Streptavidin of described specking in nature controlling line position to be concentration be 2mg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310012751.0A CN103048449B (en) | 2013-01-14 | 2013-01-14 | Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310012751.0A CN103048449B (en) | 2013-01-14 | 2013-01-14 | Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103048449A CN103048449A (en) | 2013-04-17 |
| CN103048449B true CN103048449B (en) | 2015-06-24 |
Family
ID=48061163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310012751.0A Active CN103048449B (en) | 2013-01-14 | 2013-01-14 | Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103048449B (en) |
Families Citing this family (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745586B (en) * | 2013-12-27 | 2020-02-04 | 上海市刑事科学技术研究院 | Cocaine aptamer, detection kit and application thereof |
| CN103983774A (en) * | 2014-06-03 | 2014-08-13 | 合肥工业大学 | Probe capable of specifically identifying bisphenol A nucleic acid aptamer and test strip detection application of probe |
| CN104606919B (en) * | 2014-12-31 | 2017-01-11 | 华南师范大学 | Preparation method of nucleic acid aptamer/nano-gold modified solid phase microextraction quartz capillary tube |
| CN104593372B (en) * | 2015-02-09 | 2017-07-28 | 湖南大学 | A kind of aptamer, kit and method for detecting Pancreatic neoplasms |
| JP2018519796A (en) * | 2015-04-29 | 2018-07-26 | ドッツ テクノロジー コーポレイションＤＯＴＳ Ｔｅｃｈｎｏｌｏｇｙ Ｃｏｒｐ． | Compositions and methods for allergen detection |
| CN105572393B (en) * | 2016-01-21 | 2017-11-17 | 武汉慧禹信息科技有限公司 | The aptamer and detection gold label test strip of a kind of estradiol based in saliva |
| CN106290831A (en) * | 2016-08-02 | 2017-01-04 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of competition law test strip based on antigen aptamers and application thereof |
| CN105784990B (en) * | 2016-05-17 | 2018-06-01 | 中国农业科学院农业质量标准与检测技术研究所 | It is a kind of to detect aflatoxin B1 or the test strips of M1 using aptamers |
| CN107164383B (en) * | 2016-09-29 | 2020-08-14 | 北京理工大学 | Creatine kinase isoenzyme nucleic acid aptamer and application thereof |
| CN106568951B (en) * | 2016-10-26 | 2019-01-01 | 北京农业质量标准与检测技术研究中心 | Escherichia coli O 157 based on aptamer: H7 colloidal gold strip and detection method |
| CN106970076A (en) * | 2017-04-17 | 2017-07-21 | 西安交通大学 | It is a kind of to be used for sxemiquantitative aptamer test strips that TNF α are detected and preparation method thereof |
| CN107478830A (en) * | 2017-06-14 | 2017-12-15 | 江苏大学 | A kind of ochratoxin A Rapid detection test strip based on aptamers identification |
| CN107462714B (en) * | 2017-07-17 | 2019-06-11 | 昆明理工大学 | With the aptamer Sf-A09 of sodium formaldehyde sulfoxylate specific bond and its application |
| CN108303415B (en) * | 2018-01-18 | 2020-10-27 | 北京勤邦生物技术有限公司 | Aptamer test strip for detecting aflatoxin B1, and preparation method and application thereof |
| CN109321577B (en) * | 2018-10-09 | 2020-12-08 | 安徽科技学院 | Aptamer group for detecting exosome, lateral flow aptamer biosensor and preparation method thereof |
| CN109187970B (en) * | 2018-12-05 | 2019-03-08 | 鲁东大学 | It is a kind of quickly to detect aquatic products disease gold mark nucleic acid test strip and preparation method thereof |
| CN109781994B (en) * | 2018-12-29 | 2021-12-03 | 长沙科雅生物科技有限公司 | Aptamer signal amplification-based platelet-derived growth factor PDGF-BB test strip and detection method |
| CN109765381B (en) * | 2018-12-29 | 2021-12-03 | 长沙科雅生物科技有限公司 | Aptamer signal amplification-based platelet-derived growth factor PDGF-BB test strip and detection method |
| CN109738624A (en) * | 2018-12-29 | 2019-05-10 | 中国科学院广州生物医药与健康研究院 | A kind of colloidal gold aptamer test strips of platelet derived growth factor PDGF-BB and preparation method thereof |
| CN109596827A (en) * | 2019-01-17 | 2019-04-09 | 长江师范学院 | Fluorescence detection test strip and its preparation method and application that is a kind of while detecting 4 kinds of pathogenic bacteria |
| CN109900905A (en) * | 2019-02-28 | 2019-06-18 | 中国科学院广州生物医药与健康研究院 | A kind of colloidal gold strip and preparation method detecting carcinomebryonic antigen |
| CN110441508B (en) * | 2019-05-06 | 2020-11-24 | 华南理工大学 | Test strip for rapidly detecting acrylamide in food based on nanogold composite coating |
| JP7241619B2 (en) * | 2019-06-19 | 2023-03-17 | アークレイ株式会社 | Target substance detection method, target substance detection kit, target substance detection system |
| CN111044729B (en) * | 2019-12-13 | 2023-05-02 | 西安医学院 | Nucleic acid aptamer-based high-sensitivity C-reactive protein nano immunochromatography detection method |
| CN113174447A (en) * | 2021-03-26 | 2021-07-27 | 江苏省农业科学院 | Gold-labeled rapid detection card for detecting CGMMV and preparation method thereof |
| CN114441521A (en) * | 2022-01-27 | 2022-05-06 | 宜宾五粮液股份有限公司 | Preparation method of white spirit ethyl carbamate quick-detection test paper |
| CN116008542A (en) * | 2022-10-21 | 2023-04-25 | 青岛农业大学 | Aptamer test strip, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851667A (en) * | 2009-04-03 | 2010-10-06 | 上海市公共卫生临床中心 | Fluorescent quantitative PCR detection kit of vibrio parahemolyticus pathogenicity marker gene tdh as well as detection method and application thereof |
| CN101942386A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nanogold biosensor and preparation method thereof |
| CN101942387A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses |
-
2013
- 2013-01-14 CN CN201310012751.0A patent/CN103048449B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851667A (en) * | 2009-04-03 | 2010-10-06 | 上海市公共卫生临床中心 | Fluorescent quantitative PCR detection kit of vibrio parahemolyticus pathogenicity marker gene tdh as well as detection method and application thereof |
| CN101942386A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nanogold biosensor and preparation method thereof |
| CN101942387A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses |
Non-Patent Citations (3)
| Title |
|---|
| Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for Protein Analysis;Hui Xu et al.;《Analytical Chemistry》;20081212;第81卷(第2期);669-675 * |
| Disposable nucleic acid biosensors based on gold nanoparticle probes and lateral strip;Xun Mao et al;《Analytical Chemistry》;20090121;第81卷(第4期);1160-1168 * |
| 以分子信标为报告分子的凝血酶检测新方法;仲志鸿 等;《生命科学研究》;20060930;第10卷(第3期);206-209 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103048449A (en) | 2013-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103048449B (en) | Chromatographic detection kit based on aptamer, as well as preparation method and detection method thereof | |
| CN104502586B (en) | A kind of immunochromatography detection method and test paper | |
| CN101326441B (en) | Test device for rapid diagnostics | |
| EP2574926B1 (en) | Chromatographic kit and chromatography method | |
| CN104820094A (en) | Immune-chromatographic detection test paper and detection method | |
| CN103604926B (en) | Method for detecting microcystin-LR by immunochromatographic test strip | |
| WO2010126011A1 (en) | Simple membrane assay device | |
| EP2042868A1 (en) | Immunochromatographic test device | |
| CN102507929B (en) | Preparation method of signal-enhancement type immunochromatographic gold-labeled test strip | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| CN101553729A (en) | Saturation assay | |
| CN107271669A (en) | Propepsin, helicobacter pylori antibody and G17 detection method and its kit | |
| CN107132359A (en) | Pepsinogen Cgene and PGⅡ detection method and its kit | |
| CN104897906A (en) | Dual quantitative protein toxin quantum dot fluorescence immunochromatographic strip and preparation method thereof | |
| CN104991058A (en) | Preparation method for gold labeled immucochromatographic test strip jointly marked through colloidal gold and latex microsphere | |
| JP2007064766A (en) | Detection method of substance to be detected, sensitizer and kit for immunochromatography | |
| CN101957369A (en) | The enhancing mark conjugate that is used for immunoassays | |
| CN106353503A (en) | Novel quality control method applied to immunochromatography test paper strip | |
| CN103940990A (en) | Sensitivity-enhanced gold-labeled immunochromatographic strip and preparation method thereof | |
| EP3290918A1 (en) | Immunochromatography device having reduced background noises, and method for reducing background noises in immunochromatographty device | |
| CN110231489A (en) | A method of improving the test strip biosensor detection sensitivity of carbon nanotube label | |
| Zeng et al. | Quantitative immunochromatographic strip biosensor for the detection of carcinoembryonic antigen tumor biomarker in human plasma | |
| US8110403B2 (en) | Immunoassay method | |
| JP2013205335A (en) | Immunochromatographic test tool using avidin-biotin linkage labeling reagent, and use thereof | |
| JP2013205336A (en) | Immunochromatographic test tool |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |