CN112522368B - Immunochromatography test strip for detecting DNA and preparation method thereof - Google Patents
Immunochromatography test strip for detecting DNA and preparation method thereof Download PDFInfo
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 19
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 12
- 239000003638 chemical reducing agent Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000000020 Nitrocellulose Substances 0.000 claims description 10
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 10
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 10
- 239000012149 elution buffer Substances 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 10
- 229920001220 nitrocellulos Polymers 0.000 claims description 10
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- 101710134784 Agnoprotein Proteins 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- 230000008878 coupling Effects 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 8
- 229960002685 biotin Drugs 0.000 claims description 7
- 239000011616 biotin Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 6
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- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 abstract description 2
- 230000000007 visual effect Effects 0.000 abstract description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
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- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
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- 239000011734 sodium Substances 0.000 description 4
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
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- 238000004321 preservation Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 229910004042 HAuCl4 Inorganic materials 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention provides an immunochromatography test strip for detecting DNA, belonging to the technical field of immunochromatography detection; conjugate of detection probes and gold nanorods is sprayed on a binding pad of the immunochromatographic test strip; the detection probe is single-stranded DNA modified by 5' -end sulfhydryl; the detection probes are complementarily paired with the nucleotide sequence of the DNA to be detected. In the present invention, gold nanorods (AuNRs) have tunable and stronger visible/near infrared absorption, higher surface enhanced raman scattering cross section, possibility of unidirectional plasma propagation, and enhanced photoluminescence. The AuNRs-based immunochromatographic test strip can carry out sensitive and quantitative visual detection on DNA in a short time. Through a comparative test, auNRs-based lateral flow sensors were able to detect a minimum concentration of 10pM of DNA with the naked eye, 20 times lower than gold nanoparticles (AuNPs).
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to an immunochromatography test strip for detecting DNA and a preparation method thereof.
Background
The gene is a basic unit of inheritance, and a DNA or RNA sequence carrying genetic information is replicated to transfer the genetic information to the next generation, so that the synthesis of proteins is guided to express the genetic information carried by the gene, and the expression of the characteristics of an individual organism is controlled. Gene detection is a technique of detecting DNA by blood, other body fluids, or cells. DNA detection is important in gene therapy, clinical diagnosis, and various biomedical research. The most common technique for detecting DNA is the Polymerase Chain Reaction (PCR). PCR has high sensitivity and accuracy, but is not very effective in the case of too small a sample concentration to be detected due to the need for trained personnel, expensive instrumentation, and a relatively clean environment.
Disclosure of Invention
The invention aims to provide an immunochromatography test strip for detecting DNA and a preparation method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides an immunochromatography test strip for detecting DNA, which comprises a bottom plate, and a sample pad, a combination pad, a test pad and an absorption pad which are sequentially connected to the bottom plate;
The conjugate of the detection probe and the gold nanorods is sprayed on the binding pad; the detection probe is single-stranded DNA modified by 5' -end sulfhydryl; the detection probes are complementarily paired with the nucleotide sequence of the DNA to be detected;
the detection pad is provided with a detection line and a quality control line; the detection line is sprayed with a conjugate of a capture probe and streptavidin; the 3' end of the capture probe is marked by biotin;
The quality control line is sprayed with a conjugate of a quality control probe and streptavidin; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementarily paired with the nucleotide sequence of the DNA to be detected.
Preferably, the length of the gold nanorods is 20-200 nm.
Preferably, the preparation method of the conjugate of the detection probe and the gold nanorods comprises the following steps:
adding dATP, sodium dodecyl sulfate aqueous solution, sodium chloride aqueous solution and detection probe into the gold nanorods in sequence, performing coupling reaction, centrifuging, collecting precipitate, washing, and re-suspending to obtain a solution containing the conjugate of the detection probe and the gold nanorods.
Preferably, the volume ratio of the gold nanorods, dATP, sodium dodecyl sulfate aqueous solution, sodium chloride aqueous solution and detection probe is as follows: 500:5:7.5:25:85; the concentration of dATP is 1mM; the mass concentration of the sodium dodecyl sulfate aqueous solution is 1%; the concentration of the aqueous sodium chloride solution was 2M.
Preferably, the reaction procedure of the coupling reaction comprises: incubate in a 60℃water bath for 3h.
Preferably, the reagents used for the resuspension include an elution buffer; the elution buffer comprises the following components in parts by mass: 304mg of Na 3PO4·12H2 O, 2.0g of bovine serum albumin, 4.0g of sucrose, 200.1g of tween-and 40g of water.
Preferably, the preparation method of the gold nanorods comprises the following steps:
1) Adding HAuCl 4 aqueous solution into hexadecyl trimethyl ammonium bromide aqueous solution, adding NaBH 4 solution in the stirring process, and continuously stirring for 2min to obtain gold nano seed solution;
2) And mixing hexadecyl trimethyl ammonium bromide aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent and the gold nano seed solution, and carrying out reduction reaction to obtain the gold nano rod.
Preferably, the preparation method of the conjugate of the capture probe and streptavidin comprises the following steps: mixing a capture probe and a streptavidin aqueous solution, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cut-off of 30000, centrifuging at 600 rpm for 20min to remove unreacted capture probe, and collecting supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of capture probe to aqueous streptavidin solution was 50nmol: 80. Mu.L.
Preferably, mixing a quality control probe and a streptavidin aqueous solution with the concentration of 2.5mg/mL, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture into a sample concentration tube with the molecular weight cut-off of 30000, centrifuging at 6000rpm for 20min to remove unreacted capture probes, and collecting supernatant to obtain a quality control probe solution containing a conjugate of the quality control probe and the streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is 4:1.
The invention also provides a preparation method of the immunochromatographic test strip, which comprises the following steps:
s1, sticking a nitrocellulose membrane on a PVC bottom plate, and sequentially fixing a combination pad, a sample pad and an absorption pad;
S2, adsorbing the conjugate of the detection probe and the gold nanorod on the binding pad, spraying the conjugate of the capture probe and the streptavidin on a detection line of a nitrocellulose membrane, and spraying the conjugate of the quality control probe and the streptavidin on a quality control line to obtain an immunochromatographic test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorods is 8-12 cm 2.
The invention has the beneficial effects that: the invention provides an immunochromatographic test strip for detecting DNA, wherein a conjugate of a detection probe and a gold nanorod is sprayed on a binding pad of the immunochromatographic test strip; the detection probe is single-stranded DNA modified by 5' -end sulfhydryl; the detection probes are complementarily paired with the nucleotide sequence of the DNA to be detected. In the present invention, gold nanorods (AuNRs) have tunable and stronger visible/near infrared absorption, higher surface enhanced raman scattering cross section, possibility of unidirectional plasma propagation, and enhanced photoluminescence. The AuNRs-based immunochromatographic test strip can carry out sensitive and quantitative visual detection on DNA in a short time. Through a comparative test, auNRs-based lateral flow sensors were able to detect a minimum concentration of 10pM of DNA with the naked eye, 20 times lower than gold nanoparticles (AuNPs). The immunochromatographic test strip provides a new thought for DNA detection, and has wide prospects in clinical application and biomedical diagnosis.
Drawings
FIG. 1 is a gold nanorod having an average length of 25 nm;
FIG. 2 is a gold nanorod having an average length of 60 nm;
FIG. 3 is a gold nanorod having an average length of 80 nm;
FIG. 4 is a graph comparing the results of detecting target DNA (target concentration of 0.2 nM) with three gold nanorods of different lengths;
FIG. 5 is a graph showing the result of DNA detection using a AuNR (average length of 60 nm) based test strip;
FIG. 6 shows the detection of DNA (target concentration of 10 pM) using gold nanoparticles (A) and gold nanorods (B) in a test strip.
Detailed Description
The invention provides an immunochromatography test strip for detecting DNA, which comprises a bottom plate, and a sample pad, a combination pad, a test pad and an absorption pad which are sequentially connected to the bottom plate;
The conjugate of the detection probe and the gold nanorods is sprayed on the binding pad; the detection probe is single-stranded DNA modified by 5' -end sulfhydryl; the detection probes are complementarily paired with the nucleotide sequence of the DNA to be detected;
the detection pad is provided with a detection line and a quality control line; the detection line is sprayed with a conjugate of a capture probe and streptavidin; the 3' end of the capture probe is marked by biotin;
The quality control line is sprayed with a conjugate of a quality control probe and streptavidin; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementarily paired with the nucleotide sequence of the DNA to be detected.
In the invention, the length of the DNA to be detected is preferably 20-30 bp; in the present invention, the 5' end of the quality control probe is preferably further modified with (CH 2)6 [ 6 methylene ].
In the present invention, the gold nanorods preferably have a length of 20 to 200nm, more preferably 25 to 80nm, still more preferably 50 to 60nm.
In the present invention, the preparation method of the conjugate of the detection probe and the gold nanorod preferably includes: adding dATP, sodium dodecyl sulfate aqueous solution, sodium chloride aqueous solution and detection probe into the gold nanorods in sequence, performing coupling reaction, centrifuging, collecting precipitate, washing, and re-suspending to obtain a solution containing the conjugate of the detection probe and the gold nanorods.
In the invention, the volume ratio of the gold nanorods, dATP, the sodium dodecyl sulfate aqueous solution, the sodium chloride aqueous solution and the detection probe is preferably as follows: 500:5:7.5:25:85; the concentration of dATP is preferably 1mM; the mass concentration of the sodium dodecyl sulfate aqueous solution is preferably 1%; the concentration of the sodium chloride aqueous solution is preferably 2M; the concentration of the detection probe is preferably 3. Mu.g/mL. In the invention, each substance is added into the gold nanorods, and vibration mixing is preferably carried out; the time of each mixing is preferably 10 to 30 minutes, more preferably 20 minutes, and the temperature is preferably 25 ℃. In the invention, the sodium chloride aqueous solution is preferably added for 5 times, and is added for the next time after shaking and mixing after each time of adding. The invention has no special requirement on the addition rate of dATP, sodium dodecyl sulfate aqueous solution, sodium chloride aqueous solution and detection probe. In the present invention, the procedure of the coupling reaction is preferably incubation in a water bath at 60℃for 3 hours. In the present invention, the rotational speed of the centrifugation is preferably 8000rpm, and the time is preferably 8min; the reagent used for washing the precipitate is preferably Phosphate Buffered Saline (PBS); the number of times of washing is preferably 3. In the present invention, the reagent used for the resuspension preferably comprises an elution buffer; the elution buffer comprises the following components in parts by mass: 304mg of Na 3PO4·12H2 O, 2.0g of Bovine Serum Albumin (BSA), 4.0g of sucrose, 200.1g of tween-and 40g of water; the preparation method of the elution buffer preferably comprises the following steps: na 3PO4·12H2 O, bovine serum albumin, sucrose 4.0g and tween-200.1 g are added into water in sequence and evenly mixed to obtain an elution buffer solution.
In the present invention, the storage temperature of the conjugate of the detection probe and the gold nanorods is preferably 4 ℃.
In the invention, the preparation method of the gold nanorods preferably comprises the following steps:
1) Adding HAuCl 4 aqueous solution into hexadecyl trimethyl ammonium bromide aqueous solution, adding NaBH 4 solution in the stirring process, and continuously stirring for 2min to obtain gold nano seed solution;
2) And mixing hexadecyl trimethyl ammonium bromide aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent and the gold nano seed solution, and carrying out reduction reaction to obtain the gold nano rod.
Firstly, adding HAuCl 4 aqueous solution into hexadecyl trimethyl ammonium bromide aqueous solution, adding NaBH 4 solution in the stirring process, and continuously stirring for 2min to obtain gold nano seed solution.
In the present invention, the ratio of the aqueous cetyl trimethylammonium bromide solution, the aqueous HAuCl 4 solution and the NaBH 4 solution is preferably (3 to 8) mL: (3-8) mL (500-600) mu L, more preferably 5mL:5mL: 600. Mu.L; the concentration of the aqueous solution of cetyltrimethylammonium bromide is preferably 180 to 220nM, more preferably 200nM; the concentration of the aqueous HAuCl 4 solution is preferably 0.3-0.8 nM, more preferably 0.5nM; the concentration of NaBH 4 solution is preferably 8 to 12nM, more preferably 10nM. In the present invention, the rotation speed of the stirring is preferably 1000rpm.
After the gold nano seed solution is obtained, the invention mixes hexadecyl trimethyl ammonium bromide (CTAB) water solution, HAuCl 4 water solution, agNO 3 water solution, reducing agent and the gold nano seed solution for reduction reaction to obtain the gold nano rod. In the present invention, the order of the mixing is preferably that cetyl trimethylammonium bromide aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent and the gold nano-seed solution are sequentially mixed. In the present invention, the reducing agent is preferably selected from vitamin C or hydroquinone; the reducing agent acts to reduce HAuCl 4. In the present invention, the ratio of the CTAB aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent, and gold nano-seed solution is preferably: 5mL:5mL: (30-80) mu L: (55-250) mu L: (8.2-80) mu L; the concentration of the CTAB aqueous solution is preferably 0.1-0.2M; the concentration of Au 3+ in the HAuCl 4 aqueous solution is preferably 10mM; the concentration of Ag + in the AgNO 3 aqueous solution is preferably 10mM; when the reducing agent is vitamin C, the concentration of vitamin C is preferably 78.8mM; when the reducing agent is hydroquinone, the concentration of hydroquinone is preferably 0.1M. In the present invention, the procedure of the reduction reaction preferably includes: stirring and mixing for 0-2 min; the rotation speed of the stirring is preferably 200 to 500rpm, more preferably 300rpm; the mixture is stirred and mixed and then kept stand for preservation; the temperature of the preservation is preferably 28 ℃.
In the present invention, the preparation method of the conjugate of the capture probe and streptavidin preferably comprises: the preparation method of the conjugate of the capture probe and the streptavidin comprises the following steps: mixing a capture probe and a streptavidin aqueous solution, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cut-off of 30000, centrifuging at 600 rpm for 20min to remove unreacted capture probe, and collecting supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of capture probe to aqueous streptavidin solution is preferably 50nmol:80 μl, i.e. the molar ratio of capture probe to streptavidin is preferably 4:1. in the present invention, the temperature of the coupling is preferably 1h, and the temperature is preferably 25 ℃. In the invention, the temperature of the centrifugation is preferably 4 ℃, the rotating speed is preferably 6000rpm, and the time is preferably 20min; the effect of the centrifugation is to remove unbound capture probes. In the practice of the present invention, the step of mixing the coupling reaction solution with PBS at a concentration of 500mg/L to collect the supernatant is repeated three times, i.e., the supernatant is collected and then added with PBS to repeat centrifugation, thereby removing the probe that has not been coupled.
In the present invention, the preparation method of the conjugate of the quality control probe and streptavidin preferably comprises the following steps: mixing a quality control probe and a streptavidin aqueous solution with the concentration of 2.5mg/mL, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cut-off of 30000, centrifuging at 6000rpm for 20min to remove unreacted capture probes, and collecting supernatant to obtain a quality control probe solution containing a conjugate of the quality control probe and the streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is preferably 4:1. in the present invention, the temperature of the coupling is preferably 1h, and the temperature is preferably 25 ℃. In the invention, the temperature of the centrifugation is preferably 4 ℃, the rotating speed is preferably 6000rpm, and the time is preferably 20min; the effect of the centrifugation is to remove unbound capture probes. In the practice of the present invention, the step of mixing the coupling reaction solution with PBS at a concentration of 500mg/L to collect the supernatant is repeated three times, i.e., the supernatant is collected and then added with PBS to repeat centrifugation, thereby removing the probe that has not been coupled.
The invention also provides a preparation method of the immunochromatographic test strip, which comprises the following steps:
s1, sticking a nitrocellulose membrane on a PVC bottom plate, and sequentially fixing a combination pad, a sample pad and an absorption pad;
S2, adsorbing the conjugate of the detection probe and the gold nanorod on the binding pad, spraying the conjugate of the capture probe and the streptavidin on a detection line of a nitrocellulose membrane, and spraying the conjugate of the quality control probe and the streptavidin on a quality control line to obtain an immunochromatographic test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorods is 8-12 cm 2, preferably 10cm 2.
In the present invention, the method for using the immunochromatographic test strip preferably includes: the sample pad was immersed in a buffer containing the DNA to be detected, the liquid migrated toward the absorbent pad, and the test and control areas were visually evaluated within 10 minutes. In the present invention, SSC (sodium citrate buffer) at a concentration of 1/4 is preferable in the buffer; the SSCs are commercially available. The present invention is not particularly limited in terms of immersion time. In the invention, when the quality control area and the detection area are both provided with strips, the detection area is positive; when the quality control area has stripes, and the detection area has no stripes and is negative; and if the quality control area is not provided with a strip, the test strip detection is invalid.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 gold nanorod preparation
1. Preparation of gold nano seeds
Freshly prepared NaBH 4 solution (10 ml,0.5 mM) was added to HAuCl 4 solution (10 ml,0.5 mM) containing 0.1M cetyltrimethylammonium bromide (CTAB) with vigorous stirring (1000 rpm). (CTAB, naBH4 and HAuCl4 in a molar ratio of 200:1:1) for 2min, and preserving at 28 ℃ for 2h to obtain a gold nano seed solution.
2. Growth of gold nanorods
A proper amount of CTAB aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent, gold nano seed solution were added sequentially into a round bottom flask, and the amounts are shown in table 1. The specific method comprises the following steps: the aqueous CTAB, aqueous HAuCl 4 and aqueous AgNO 3 were added sequentially to the round bottom flask. The reducer is added into the solution, and the solution is gently stirred under the condition of 200-500 rpm, and the solution is changed from bright yellow to colorless. And finally, adding the gold nano seed solution, slightly stirring for 2min at 200-500 rpm, and standing at 28 ℃ for 12h to obtain a solution containing gold nano rods.
TABLE 1 reaction System for reduction reactions
See fig. 1-3 for transmission electron microscope images of three gold nanorods of different lengths, respectively. FIG. 1 is a gold nanorod having an average length of 25 nm; FIG. 2 is a gold nanorod having an average length of 60 nm; FIG. 3 is a gold nanorod having an average length of 80 nm.
Example 2 detection of cervical cancer marker HPV16
HPV16 sequence of interest: 5-ggc att tgt tgg ggt aac caa ctattt gtt-3, the nucleotide sequence is shown as SEQ ID No. 1;
HPV16 detection probe sequence: SH-C6-5-aac aaa tag ttg-3, the nucleotide sequence is shown as SEQ ID No. 2;
HPV16 capture probe sequence: 5-cca aca aat gcc-3-Biotin, the nucleotide sequence is shown as SEQ ID No. 3;
HPV16 quality control probe sequence: biotin-5-caa cta ttt gtt-3 has a nucleotide sequence shown as SEQ ID No. 4.
1. Preparation of conjugate of HPV 16 detection Probe and gold nanorod
500. Mu.L of gold nanorods with lengths of 25nm, 60nm and 80nm prepared by the method are respectively taken, 15. Mu.L of 1mM dATP is added, and then the mixture is oscillated for 30min at 25 ℃, 7.5. Mu.L of 1% Sodium Dodecyl Sulfate (SDS) aqueous solution is added, and then the mixture is oscillated for 10min at 25 ℃, then 25. Mu.L of 2M sodium chloride aqueous solution is added in batches, and then 85. Mu.L of HPV 16 detection probe is added. Finally, the mixture was incubated in a water bath at 60℃for 3h. Centrifugation at 8000rpm for 8min, washing with Phosphate Buffered Saline (PBS) for 3 times, and re-suspending in elution buffer (specific configuration method comprises sequentially adding Na 3PO4·12H2 O304 mg, bovine Serum Albumin (BSA) 2.0g, sucrose 4.0g, tween-200.1 g in 40mL of water, and mixing to obtain conjugates of 3 HPV 16 detection probes and gold nanorods, wherein the conjugates are respectively a first conjugate (prepared from gold nanorods with a size of 25 (+/-10) x 8 (+/-3), a second conjugate (prepared from gold nanorods with a size of 60 (+/-15) x 28 (+/-5) and a third conjugate (prepared from gold nanorods with a size of 80 (+/-20) x 10 (+/-1)). The conjugate solution of HPV 16 detection probe and gold nanorods was stored at 4 ℃ for later use.
2. Preparation of Capture Probe solution
50Nmol of HPV16 capture probe was mixed with 80uL 2.5mg/mL streptavidin at a molar ratio of about 4: incubating for 1h at 1, 25 ℃; adding 500mg/L PBS500, μl, 4deg.C, centrifuging at 6000rpm with sample concentration tube with molecular weight cut-off of 30000 for 20min, removing unbound capture probe, and repeating the above steps for 3 times; the upper layer solution of the concentrated tube is the rest solution of the capture probe solution, namely the capture probe solution.
3. Preparation of quality control Probe solution
50Nmol of HPV16 capture probe was mixed with 80uL 2.5mg/mL streptavidin at a molar ratio of approximately 100:1 and incubated for 1h at 25 ℃; adding 500mg/LPBS into the mixed solution, centrifuging at 4deg.C with sample concentration tube with molecular weight cut-off of 30000 at 6000rpm for 20min, removing unbound capture probe, and repeating the above steps for 3 times; the upper layer solution of the concentration tube is a quality control probe solution.
4. Test strip preparation method
1) Sticking a nitrocellulose membrane on a PVC bottom plate, and sequentially fixing a bonding pad, a sample pad and an absorption pad;
2) Adsorbing conjugate (10 mu L/cm 2) of HPV 16 detection probe and gold nanorod on the binding pad, and spraying 1 mu L of capture probe solution and 1 mu L of quality control probe solution on the detection line and the quality control line of the nitrocellulose membrane respectively to obtain gold nanorod lateral flow chromatography test strips for detecting three nucleic acids.
Example 3
3. Mu.L of conjugate of HPV16 detection probe and gold nanorods was added dropwise to the sample pad of the three gold nanorod lateral flow chromatography test strips prepared in example 2, and the sample pad was immersed in 1 (1/4 concentration SSC, commercially available from 1/4 diluted with deionized water to the original concentration) of 100. Mu.L of buffer containing target HPV16 DNA (0.2 nM) and migrated toward the absorbent pad. The test and control zones may be visually assessed within 10 minutes. As a result of detection, referring to FIG. 4, three kinds of gold nanorods (25 nM, 60nM, 80 nM) having different lengths were used as labels to detect DNA, respectively, and the 60nM gold nanorod test wire was the most concentrated when the DNA concentration was 0.2 nM. Therefore, the 60nm gold nanorods can be used as the optimal labels.
Example 4
3 Mu L of conjugate of HPV16 detection probe and gold nanorods is dripped into the binding pad of the prepared test strip, the sample pad is immersed in 1100 mu L buffer (1/4 SSC) containing target HPV16 DNA with different concentrations, and the liquid migrates to the absorption pad. The test and control zones may be visually assessed within 10 minutes. The quality control area and the test area are positive in strip occurrence; the quality control area has stripes, and the test area has no stripes and is negative; and if the quality control area is not provided with a strip, the test strip detection is invalid. The results are shown in FIG. 5. In FIG. 5, 1 to 8 represent target DNA concentrations of 0, 10pM,20pM,50pM,100pM,200pM,500pM and 1nM, respectively. When the concentration of the target object is zero, no obvious strip exists in the test area, the higher the concentration is, the darker the color of the strip is, and all the quality control areas of the test strips are provided with strips, so that the prepared test strip can work normally and well, and the macroscopic detection limit is 10pM. Therefore, the prepared test strip can rapidly, accurately and sensitively detect DNA.
Comparative example 1
1. Preparation of gold nanoparticles
100ML of a 0.01% chloroauric acid (HAuCl 4) solution was heated to boiling, and 2mL of a 1% by mass sodium citrate solution was added while stirring at high speed (1000 rpm), followed by stirring and boiling for 15min, and cooling to 25 ℃.
2. Preparation of conjugate of detection probe and gold nanoparticle
500 Mu L of gold nano-particles are taken, 15 mu L of 1mM dATP is added, and then the mixture is oscillated for 30min at 25 ℃, 7.5 mu L of 1% Sodium Dodecyl Sulfate (SDS) aqueous solution is added, and then the mixture is oscillated for 10min at 25 ℃, and then 25 mu L of 2M sodium chloride solution and 85 mu L of HPV 16 detection probe are slowly and sequentially added. Finally, the mixture was incubated in a water bath at 60℃for 3h. Centrifuging at 8000rpm for 8min, washing with Phosphate Buffer Solution (PBS) for 3 times, and re-suspending in elution buffer (specifically, the preparation method comprises sequentially adding Na 3PO4·12H2 O304 mg, bovine Serum Albumin (BSA) 2.0g, sucrose 4.0g, tween-200.1 g into 40mL water, and mixing. The conjugate solution of the detection probe and gold nanoparticles was stored at 4 ℃ for later use.
3. Preparation of Capture Probe solution
50NM HPV 16 capture probe was mixed with 2.5mg/mL streptavidin and incubated at 25℃for 1h; adding 500mg/LPBS into the mixed solution, centrifuging at 6000rpm for 20min at 4 ℃, removing unbound capture probes, and repeating the above steps for 3 times; the upper layer solution of the concentrated tube is the quality control probe solution.
4. Preparation of quality control probe solution
50NM HPV16 quality control probe was mixed with 2.5mg/mL streptavidin and incubated at 25℃for 1h; adding 500mg/LPBS into the mixed solution, centrifuging at 6000rpm for 20min at 4 ℃, removing unbound capture probes, and repeating the above steps for 3 times; the upper layer solution of the concentrated tube is the quality control probe solution.
5. Test strip preparation method
1) Sticking a nitrocellulose membrane on a PVC bottom plate, and sequentially fixing a bonding pad, a sample pad and an absorption pad;
2) And (3) adsorbing conjugate solution (10 mu L/cm 2) of the detection probes and gold nanoparticles on the binding pad, and respectively spraying 1 mu L of capture probe solution and 1 mu L of quality control probe solution on a detection line and a quality control line of the nitrocellulose membrane to obtain the gold nanoparticle lateral flow chromatography test strip for detecting nucleic acid.
Example 5
The gold nanorod lateral flow chromatography test strip prepared in example 2 and the sample pad of the gold nanoparticle lateral flow chromatography test strip prepared in comparative example 1 were immersed in 1 buffer (1/4 SSC) containing 100. Mu.L of the target HPV16 DNA (10 pM), respectively, (purchased SSC buffer, diluted to 1/4 of the original concentration with deionized water) and the liquid migrated toward the absorbent pad. The test and control zones may be visually assessed within 10 minutes. The detection results (the quality control area and the test area are positive, the quality control area is provided with a strip, the test area is not provided with a strip, the quality control area is not provided with a strip, and the test strip detection is not effective) are shown in fig. 6. As can be seen from FIG. 6, the same conditions compare gold nanoparticles with gold nanorod test strips to detect DNA, and the gold nanorods can be detected at least 10pM by naked eyes.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Anhui academy of science and technology
<120> An immunochromatography test strip for detecting DNA and a preparation method thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
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<210> 2
<211> 12
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
aacaaatagt tg 12
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<211> 12
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
ccaacaaatg cc 12
<210> 4
<211> 12
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
caactatttg tt 12
Claims (5)
1. An immunochromatography test strip for detecting cervical cancer marker HPV16 is characterized by comprising a bottom plate, and a sample pad, a binding pad, a test pad and an absorption pad which are sequentially connected to the bottom plate;
the conjugate of the detection probe and the gold nanorods is sprayed on the binding pad; the detection probe is single-stranded DNA modified by 5' -end sulfhydryl; the detection probes are complementarily paired with the nucleotide sequence of the DNA to be detected; the nucleotide sequence of the detection probe is shown as SEQ ID No. 2;
The detection pad is provided with a detection line and a quality control line; the detection line is sprayed with a conjugate of a capture probe and streptavidin; the 3' end of the capture probe is marked by biotin; the nucleotide sequence of the capture probe is shown as SEQ ID No. 3;
The quality control line is sprayed with a conjugate of a quality control probe and streptavidin; the 3' end of the quality control probe is modified by biotin; the quality control probe is complementary and paired with the nucleotide sequence of the detection probe; the nucleotide sequence of the quality control probe is shown as SEQ ID No. 4;
the length of the gold nanorods is 60+/-15 nm, and the width of the gold nanorods is 28+/-5 nm;
The preparation method of the gold nanorods comprises the following steps:
1) Adding HAuCl 4 aqueous solution into hexadecyl trimethyl ammonium bromide aqueous solution, adding NaBH 4 solution in the stirring process, and continuously stirring for 2 min to obtain gold nano seed solution;
2) Mixing hexadecyl trimethyl ammonium bromide aqueous solution, HAuCl 4 aqueous solution, agNO 3 aqueous solution, reducing agent and the gold nano seed solution, and carrying out reduction reaction to obtain gold nano rods;
The preparation method of the conjugate of the detection probe and the gold nanorods comprises the following steps:
sequentially adding dATP, sodium dodecyl sulfate aqueous solution, sodium chloride aqueous solution and detection probe into the gold nanorods, performing coupling reaction, centrifuging, collecting precipitate, washing, and re-suspending to obtain a solution containing a conjugate of the detection probe and the gold nanorods;
the reagent used for the resuspension is an elution buffer, and the elution buffer comprises the following components in parts by mass: na 3PO4·12H2 O304 mg, bovine serum albumin 2g, sucrose 4g, tween-20.1 g and water 40g.
2. The immunochromatographic test strip according to claim 1, wherein the reaction procedure of the coupling reaction comprises: incubate in a 60℃water bath for 3h.
3. The immunochromatographic test strip according to claim 1, wherein the preparation method of the conjugate of the capture probe and streptavidin comprises the following steps: mixing a capture probe and a streptavidin aqueous solution with the concentration of 2.5 mg/mL, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture in a sample concentration tube with the molecular weight cut-off of 30000, centrifuging at 6000 rpm for 20 min to remove unreacted capture probe, and collecting supernatant to obtain a capture probe solution containing a conjugate of the capture probe and the streptavidin; the ratio of capture probe to aqueous streptavidin solution at a concentration of 2.5 mg/mL was 50 nmol: 80. Mu.L.
4. The immunochromatographic test strip according to claim 1, wherein the preparation method of the conjugate of the quality control probe and streptavidin comprises the following steps: mixing a quality control probe and a streptavidin aqueous solution with the concentration of 2.5 mg/mL, performing coupling to obtain a coupling reaction solution, mixing the coupling reaction solution with PBS with the concentration of 500mg/L, placing the mixture into a sample concentration tube with the molecular weight cut-off of 30000, centrifuging 6000 rpm and 20 min to remove unreacted quality control probe, and collecting supernatant to obtain a quality control probe solution containing a conjugate of the quality control probe and streptavidin; the molar ratio of the quality control probe to the streptavidin in the streptavidin aqueous solution is 4:1.
5. The immunochromatographic test strip according to any one of claims 1 to 4, characterized in that the preparation method of the immunochromatographic test strip comprises the following steps:
s1, sticking a nitrocellulose membrane on a PVC bottom plate, and sequentially fixing a combination pad, a sample pad and an absorption pad;
S2, adsorbing the conjugate of the detection probe and the gold nanorod on the binding pad, spraying the conjugate of the capture probe and the streptavidin on a detection line of a nitrocellulose membrane, and spraying the conjugate of the quality control probe and the streptavidin on a quality control line to obtain an immunochromatographic test strip; the spraying amount of the conjugate of the capture probe and the streptavidin is 0.5-1 mu L; the spraying amount of the conjugate of the quality control probe and the streptavidin is 0.5-1 mu L; the adsorption capacity of the conjugate of the detection probe and the gold nanorods is 8-12 cm 2.
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