WO2022262135A1 - 一种检测小分子物质的通用型适配体胶体金侧向层析试纸 - Google Patents

一种检测小分子物质的通用型适配体胶体金侧向层析试纸 Download PDF

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WO2022262135A1
WO2022262135A1 PCT/CN2021/118498 CN2021118498W WO2022262135A1 WO 2022262135 A1 WO2022262135 A1 WO 2022262135A1 CN 2021118498 W CN2021118498 W CN 2021118498W WO 2022262135 A1 WO2022262135 A1 WO 2022262135A1
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polya
dna
pad
colloidal gold
test strip
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PCT/CN2021/118498
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French (fr)
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彭池方
常芮
李秀萍
钱志娟
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江南大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips

Definitions

  • the invention relates to a general-purpose aptamer colloidal gold lateral flow chromatography test paper for detecting small molecular substances, and belongs to the fields of analytical chemistry, medicine, environment, food safety detection, nanobiological sensing and the like.
  • Lateral flow chromatography a paper-based assay platform that can be used to detect a variety of target substances, is of great interest to researchers due to its potential to provide results within minutes. Because of its low cost and ease of development and production, lateral flow chromatography has been widely used in on-site detection and widely used in various fields, including biomedicine, food safety, quality control, and environmental hygiene. Lateral flow chromatography can be applied to a range of biological samples, including urine, saliva, sweat, serum, plasma, and blood. Therefore, lateral flow chromatography analysis technology has high application value in various fields.
  • the signal intensity of the T line is inversely proportional to the concentration of the detection target in the sample to be tested. Regardless of whether the sample to be tested contains the detection target or not, the C line has a visible independent signal. In recent years, more and more researchers have paid attention to the detection of small molecules by lateral flow chromatography.
  • Nucleic acid aptamer is a short structured oligonucleotide sequence (RNA or DNA) obtained through in vitro screening technology-exponential enrichment ligand system evolution technology (Systematic evolution of ligands by exponential enrichment, SELEX) , can bind to corresponding target molecules (proteins, viruses, bacteria, cells, heavy metal ions, etc.) with high affinity and strong specificity, providing a new efficient and rapid identification research platform for chemical biology and biomedicine .
  • RNA or DNA short structured oligonucleotide sequence obtained through in vitro screening technology-exponential enrichment ligand system evolution technology (Systematic evolution of ligands by exponential enrichment, SELEX) , can bind to corresponding target molecules (proteins, viruses, bacteria, cells, heavy metal ions, etc.) with high affinity and strong specificity, providing a new efficient and rapid identification research platform for chemical biology and biomedicine .
  • Colloidal gold lateral flow test paper based on nucleic acid aptamer labeling has the advantages of long-term stability, short detection time, low cost, simple operation, and fast speed. It is an ideal method for rapid on-site detection of various targets.
  • the nucleic acid chain-streptavidin complex is usually sprayed on the T line. With the change of the detection target, it is necessary to re-prepare specific Compounds and spraying to prepare nitrocellulose membranes lead to poor versatility of existing lateral flow test strips.
  • test strip requires the help of MMS, and the detection of kanamycin is transformed into the detection of cDNA, which is relatively complicated, which is not conducive to the stable production of the product and the repeatability of the application.
  • a lateral flow chromatography test strip based on nucleic acid aptamers was developed, which adopts a competitive binding mode for on-site rapid Detection of OTA in Astragalus membranaceus.
  • a competition reaction occurs between the DNA probe immobilized on the test strip (test zone) and the target in the sample (OTA), both of which compete for binding to the AuNPs-apt conjugate.
  • the color of the T-line of the test strip is negatively correlated with the concentration of OTA in the sample. Based on this method, the detection of OTA can be completed within 15 minutes, and the detection line is 1ng/mL.
  • the test strip couples the aptamer to AuNPs, which is not conducive to the competition of OTA in the sample for the aptamer, the product has poor stability, and the design is relatively complicated and the versatility is poor.
  • the probes on the test strips need to be repeatedly optimized for a specific target, which has poor versatility and detection sensitivity needs to be improved.
  • nucleic acid aptamer colloidal gold lateral flow chromatography test strips usually sprays gold-labeled nucleic acid aptamers on the gold-labeled pad, and usually streptavidin is sprayed on the detection line (T line).
  • the present invention uses polyA-DNA as a probe, and the probe uses polyA as an anchor block, and anchors the polyA-DNA on the gold nanoparticle to prepare an AuNPs@polyA-DNA conjugate.
  • the detection area (T line) of the nucleic acid test strip was sprayed with streptavidin, and the control area (C line) was sprayed with an oligonucleotide chain (DNAc) complementary to the polyA-DNA part of the AuNPs@polyA-DNA conjugate to construct a Universal test strips.
  • streptavidin sprayed on the T line of the test strip, and the streptavidin-biotin-oligo nucleic acid strand complex sprayed on the C line do not need to be changed, only the polyA-DNA complementary to the aptamer is changed. part, another substance can be detected, which provides convenience for future detection.
  • the nucleic acid aptamer used in the present invention adds 5 T bases on the basis of the base sequence of the reported aptamer, and these 5 nucleic acid bases are used for assisting hybridization. 15 bases on the probe are complementary to the nucleic acid aptamer. After the biotinylated nucleic acid aptamer is combined with the AuNPs@polyA-DNA conjugate anchored with the probe, the probe can further bind to the streptavidin on the T line.
  • the small molecular substance competes with the probe to bind to the nucleic acid aptamer, inhibiting the hybridization between the probe and the nucleic acid aptamer, thereby inhibiting the color development of the T line and realizing rapid detection.
  • the versatility of the nucleic acid aptamer colloidal gold lateral flow chromatography test strips provided by the present invention is reflected in the preparation of nitrocellulose membranes and the design of probes, that is, in the preparation of colloidal gold test strips for different small molecular substances , the T line and C line on the nitrocellulose membrane do not need to be changed, only the corresponding nucleic acid aptamer of the small molecule substance and the AuNPs@polyA-DNA conjugate on the gold standard pad need to be changed, that is, for different small molecule substances , the nucleic acid aptamer and the probe polyA-DNA make corresponding changes.
  • the present invention provides a universal colloidal gold lateral flow chromatography test strip for detecting kanamycin based on nucleic acid aptamers and AuNPs@polyA-DNA conjugates.
  • the test strip is generally composed of a sample pad, a binding pad ( Gold standard pad), nitrocellulose membrane (NC membrane), absorbent pad and PVC rubber sheet.
  • the detection principle of the test strip is as follows: the competition method is used to detect small molecular substances.
  • the biotinylated nucleic acid aptamer at the 5' end is coupled with AuNPs@polyA-DNA
  • the AuNPs@polyA-DNA-Apt complex was then captured by streptavidin on the T line. Due to the accumulation of AuNPs, a clear dark red band could be observed on the T line, and the test result was negative.
  • the sample solution to be tested contains small molecular substances
  • the small molecular substances bind to the biotinylated nucleic acid aptamer at the 5' end, and the nucleic acid aptamer cannot bind to the AuNPs@polyA-DNA probe, causing the probe to be on the T line
  • the accumulation in the area is reduced, the T line is lighter or not colored, and the test result is positive.
  • the color intensity on the T-line region is negatively correlated with the concentration of small molecular species.
  • AuNPs@polyA-DNA conjugates can be captured by DNAc in the C-line region to develop color.
  • the first object of the present invention is to provide a kind of universal colloidal gold lateral flow chromatography test strip, and said test strip contains nucleic acid aptamer, probe polyA-DNA and streptavidin-biotin-DNAc .
  • the probe polyA-DNA contains a polyA fragment, a fragment complementary to DNAc, and a fragment complementary to a nucleic acid aptamer of a small molecule substance.
  • the 5' end of the nucleic acid aptamer is labeled with biotin; the nucleic acid aptamer can specifically bind to the small molecule substance to be detected.
  • the streptavidin-biotin-DNAc is mixed with equal volumes of streptavidin and biotin-labeled DNAc at the 5' end, and incubated at 3-5°C for 0.8-1.2h prepared.
  • nucleotide sequence of the DNAc is shown in SEQ ID NO:5.
  • the streptavidin concentration is 2.5 mg/mL, and the DNAc concentration is 250 ⁇ M.
  • the small molecular substances include but are not limited to kanamycin, OTA, aflatoxin, streptomycin, chloramphenicol, estradiol, bisphenol A, acetamiprid.
  • the nucleic acid aptamer is an oligonucleotide sequence designed for the target to be detected and obtained through an in vitro screening technique-exponential enrichment ligand system evolution technique.
  • the test strip includes a sample pad, a binding pad (gold standard pad), a nitrocellulose membrane (NC film), an absorbent pad, and a PVC rubber sheet; the sample pad is pasted sequentially on the PVC base plate , gold label pad, NC film and water-absorbent pad; the NC film is provided with a detection area and a control area in sequence, and the distance between the detection area and the control area is 4-6mm; there is streptavidin on the detection area Streptavidin-biotin-DNAc on the control region; AuNPs@polyA-DNA conjugates on the gold label pad.
  • the length of the overlapping part between the sample pad and the gold standard pad is 1-2 mm, the sample pad is placed above the gold standard pad; the length of the overlapping part between the gold standard pad and the NC film is 1-2 mm, and the gold standard pad The pad is placed above the NC membrane; the length of the overlapping part between the NC membrane and the absorbent pad is 1-3mm, and the absorbent pad is placed above the NC membrane.
  • the AuNPs@polyA-DNA conjugate is obtained by anchoring the probe polyA-DNA on gold nanoparticles.
  • the particle size of the gold nanoparticles is 13-17 nm; the concentration of the polyA-DNA is 80-120 ⁇ M.
  • the second object of the present invention is to provide a method for rapid detection of small molecular substances.
  • the method is to use the above-mentioned colloidal gold lateral flow chromatography test strips for testing, and mix the solution to be tested with the small molecular substance nucleic acid aptamer After incubation, pipette 50-100 ⁇ L dropwise onto the sample pad, incubate for 3-5 minutes, perform qualitative analysis with the naked eye or use a colloidal gold test paper quantitative analyzer for quantitative analysis based on the standard curve.
  • the preparation method of the standard curve is to dilute the standard solution of the small molecular substance to different concentrations, mix the diluents of different concentrations with the nucleic acid aptamer labeled with biotin at the 5' end, and then Add it dropwise to the sample pad, incubate for 3-5 minutes, perform quantitative analysis with a colloidal gold test paper quantitative analyzer, and fit the data to obtain a standard curve.
  • the mixing incubation time is 15-25 minutes.
  • the volume ratio of the nucleic acid aptamer to the solution to be tested is 1:99.
  • the initial concentration of the nucleic acid aptamer is 0.1-1.0 ⁇ M.
  • the third object of the present invention is to provide the preparation method of the above-mentioned universal colloidal gold lateral flow chromatography test strip, and the specific steps of the method are as follows:
  • the AuNPs@polyA-DNA conjugate is obtained by using the probe polyA-DNA as an anchor block, and anchoring the polyA-DNA on the gold nanoparticle;
  • the probe polyA-DNA Contains polyA fragments, fragments complementary to DNAc and fragments complementary to nucleic acid aptamers of small molecule substances;
  • the particle size of the gold nanoparticles is 13-17 nm.
  • the polyA-DNA concentration is 80-120 ⁇ M.
  • nucleotide sequence of the DNAc is shown in SEQ ID NO:5.
  • the length of the overlapping part between the sample pad and the gold standard pad is 1-2 mm, the sample pad is placed above the gold standard pad; the length of the overlapping part between the gold standard pad and the NC film is 1-2 mm, and the gold standard pad The pad is placed above the NC membrane; the length of the overlapping part between the NC membrane and the absorbent pad is 1-3mm, and the absorbent pad is placed above the NC membrane.
  • the present invention also provides the general colloidal gold lateral chromatography test strip or the preparation method of the universal colloidal gold lateral chromatography test strip in pesticide residues, veterinary drug residues, environmental monitoring, food safety monitoring and biological Applications in the field of medicine.
  • This paper invented a high-sensitivity, high-specificity aptamer-based universal colloidal gold lateral flow test strip.
  • the test strip is sensitive to kanamycin and OTA detection (the detection limit of naked eyes is 15ng/mL, 10ng/mL, and the detection limit of the reader is 0.3ng/mL, 0.18ng/mL), fast (20min), The test results are highly repeatable.
  • the detection method proposed by the present invention realizes the highly sensitive colorimetric detection of small molecular substances (kanamycin and OTA in the present invention).
  • small molecular substances kanamycin and OTA in the present invention.
  • the concentration range of 5-250ng/mL the T/C line showed a good linear relationship with the concentration of kanamycin, and the detection limit was 0.3ng/mL.
  • the concentration range of 1-250ng/mL the T/C line showed a good linear relationship with the concentration of kanamycin, and the detection limit was 0.18ng/mL.
  • the streptavidin and nucleic acid chain sprayed on the detection area and the control area do not need to be changed, and only need to change the colloidal gold-nucleic acid conjugate when preparing the test strip to detect another Substance, thereby improving the general type of preparation of nitrocellulose membrane by spraying, simplifying the preparation process, and reducing the cost of test strips.
  • the test strip can realize qualitative detection of small molecular substances by observing the color change of the detection area with the naked eye, and quantitative analysis can be realized by using a colloidal gold quantitative analyzer.
  • Fig. 1 is a structural schematic diagram and a detection principle diagram of a test strip.
  • Figure 2 is the characterization of gold nanoparticles: (a) TEM image of AuNPs; (b) absorption spectrum of AuNPs.
  • Figure 3 is a detection chart of different concentrations of kanamycin standard solutions with test strips; the concentrations from left to right are 0, 0.5, 5, 15, 25, 50, 150, 250 and 400 ng/mL.
  • Fig. 4 is in the standard detection solution, the relative signal intensity (T/C) standard curve figure of T line ratio C line when different concentrations of kanamycin; Concentrations from left to right are 0, 1, 10, 50, 100, 250 and 500ng/mL.
  • Fig. 5 is a detection diagram of different concentrations of OTA standard solutions with test strips.
  • Fig. 6 is a standard curve diagram of the relative signal intensity (T/C) of the T line to the C line at different concentrations of OTA in the standard detection solution.
  • Fig. 7 is a detection diagram of honey samples with different concentrations of kanamycin by test strips.
  • Figure 8 is the impact of nucleic acid strands of different sequences on the corresponding signal of kanamycin; (a) the impact of different aptamer complementary strands on the probe on the color development of T lines; (b) probe strand polyA-DNA (5+ 10) and polyA-DNA (10+5) on the kanamycin response signal.
  • Figure 9 is the structure of polyA-DNA.
  • nucleic acid probe strands of different sequences (polyA-DNA ( 20) , polyA-DNA (15) , polyA-DNA (5+10) , polyA-DNA (5+5) , polyA-DNA (10+5) ) coupled with AuNPs (as shown in Table 1), suitable The concentration of ligand was 0.5 ⁇ M.
  • the probe chains polyA-DNA (5+10) and polyA-DNA (10+5) are used to detect kanamycin, as shown in Figure 8(b), adding During the same concentration of kanamycin (150ng/mL), when using the nucleic acid probe chain polyA-DNA (10+5) of nucleic acid sequence such as SEQ ID NO:2, to the corresponding signal intensity of kanamycin The change ( ⁇ T/C value) is the largest. Therefore, the probe used in the following examples is polyA-DNA (10+5) whose nucleotide sequence is shown in SEQ ID NO:2.
  • polyA-DNA has three functional regions.
  • the first functional region is polyA, which acts as an anchor block to anchor polyA-DNA on gold nanoparticles;
  • the second functional region is complementary to DNAc;
  • the third functional region is complementary to nucleic acid aptamers area ( Figure 9).
  • AuNPs were prepared by sodium citrate reduction method. Specific steps are as follows:
  • step (1) 1) Add 5-15 ⁇ L of 100 ⁇ M polyA-DNA to 1 mL of AuNPs (10 nM) prepared in step (1) and mix well, then add 20 ⁇ L of 500 mM citrate buffer (pH 3.0). After mixing evenly, incubate at room temperature for 3 min.
  • the AuNPs prepared in step (1) and the AuNPs@polyA-DNA prepared in step (2) were characterized by transmission electron microscopy. The results are shown in Figure 2.
  • the prepared AuNPs with a particle size of 15nm have a single characteristic absorption at 520nm peak, when combined with polyA-DNA, the maximum absorption wavelength shifted to 530nm, preliminarily proving that polyA-DNA successfully modified AuNPs.
  • composition of the resuspension 20 mM Na 3 PO 4 , 5% BSA, 10% sucrose, 0.25% Tween-20.
  • step 2 Spray streptavidin and streptavidin-biotin-DNAc prepared in step 2 evenly on the NC membrane with a three-dimensional spraying device at a speed of 0.9 ⁇ L/cm, respectively as the detection area (T line) And the control area (C line), the distance between the detection area and the control area is fixed at 5mm, and dried at 37°C for 2h.
  • Embodiment two with test strip to the mensuration of kanamycin standard solution
  • Kanamycin standard solution was diluted with Running buffer (4 ⁇ SSC, pH 7) to a final concentration of 0.5, 5, 15, 25, 50, 150, 250 and 400 ng/mL.
  • the kanamycin aptamer whose nucleotide sequence is shown in SEQ ID NO:1 was diluted to 0.5 ⁇ M with ultrapure water.
  • step (1) 99 ⁇ L of kanamycin standard solution of different concentrations and 1 ⁇ L of kanamycin aptamer solution were mixed and incubated for 20 min. After the mixed reaction, the mixed liquid was added to the sample pad for detection. After 3 min of reaction, the (T /C) relative signal intensity, and establish a standard curve of the corresponding relationship between relative signal intensity (T/C) and different kanamycin concentrations.
  • the colloidal gold test paper quantitative analyzer reads the relative signal intensity (T/C) of different concentrations of kanamycin, and obtains the relative signal intensity (T/C) and the change relation curve of kanamycin concentration as shown in Figure 4, the lowest The detection line is 0.3ng/mL.
  • Embodiment three use test strip to the mensuration of OTA standard solution
  • the OTA standard solution was diluted with Running buffer (4 ⁇ SSC, pH7) to a final concentration of 1, 10, 50, 100, 250 and 500 ng/mL. Dilute the OTA aptamer whose nucleotide sequence is shown in SEQ ID NO:1 to 0.5 ⁇ M with ultrapure water.
  • step (1) 99 ⁇ L of OTA standard solutions of different concentrations and 1 ⁇ L of OTA aptamer solution were mixed and incubated for 20 minutes. After the mixed reaction, the mixed droplets were added to the sample pad for detection, and the relative signal intensity (T/C) was measured after 3 minutes of reaction. , to establish a standard curve of the corresponding relationship between relative signal intensity (T/C) and different OTA concentrations.
  • the color of the T line on the test strip can be significantly different from the color of the T line on the test strip of the solution (0ng/mL OTA) without OTA. Difference; when the OTA concentration is 1-250ng/mL, the color of the T line weakens with the increase of the OTA concentration, and the color of the T line basically does not change when the OTA concentration is 250ng/mL, so the lower limit of naked eye detection is 10ng/mL, and the upper limit is 250ng /mL.
  • the colloidal gold test paper quantitative analyzer reads the relative signal intensity (T/C) of different concentrations of OTA, and obtains the change relationship curve between relative signal intensity (T/C) and OTA concentration as shown in Figure 6, and the lowest detection line is 0.18ng/ mL.
  • T/C relative signal intensity
  • y the relative signal intensity
  • x the Log function (ng/mL) of the concentration of OTA.
  • the nucleic acid aptamer test strip prepared in Example 1 was used to detect the content of kanamycin in milk, and the results are shown in Table 4 and FIG. 7 .

Abstract

本发明公开了一种检测小分子物质的通用型适配体胶体金侧向层析试纸，属于分析化学、医药、环境、食品安全检测和纳米生物传感等领域。利用探针AuNPs@poly-DNA快速、灵敏地捕获适配体，且在检测区和控制区喷涂的链霉亲和素和链霉亲和素-生物素-DNAc无需改变，仅改变探针AuNPs@poly-DNA上的核酸链部分即可检测另一种物质。开发出用于快速、灵敏、低成本的通用型胶体金侧向层析试纸。试纸条法检测小分子物质简便快速，可随时检测，只需将样品口中加入测试溶液，5min后试纸条显色完全，即可观察实验结果，可以大幅度的提高检测效率。用肉眼进行定性分析，使用胶体金试纸定量分析仪进行定量分析。

Description

一种检测小分子物质的通用型适配体胶体金侧向层析试纸 技术领域
本发明涉及一种检测小分子物质的通用型适配体胶体金侧向层析试纸，属于分析化学、医药、环境、食品安全检测和纳米生物传感等领域。
背景技术
侧流层析分析技术是一种基于纸张的检测平台，可以用于检测多种靶标物质，由于其在几分钟内提供结果的潜力引起研究者极大地兴趣。因为其具有低成本和易于开发生产的特点，侧流层析分析技术已被大量用于现场检测，并广泛应用于各种领域，包括生物医学、食品安全、质量控制以及环境卫生。侧流层析分析技术可以应用一系列生物样本，包括尿液、唾液、汗液、血清、血浆和血液。因此，侧流层析分析技术在多种领域中具有较高的应用价值。
侧流层析分析技术有两种模式，即竞争型和三明治型，对于低分子量的化合物(如真菌毒素等)的检测中通常采用的是竞争型。在采用竞争型胶体金试纸条的制备过程中，与靶标特异性结合的人工抗原和物种特异性抗免疫球蛋白抗体通常喷洒在消化纤维素膜(NC)上分别作为检测线(T线)和质控线(C线)，或，可以将针对靶标的适配体互补链和人工设计的核酸链喷洒在消化纤维素膜(NC)上分别作为检测线(T线)和质控线(C线)。其中，T线的信号强度与待检样本中的检测目标物的浓度成反比。无论待检样本中是否含有检测目标物，C线都具有可见的独立信号。近年来，越来越多的研究人员重视通过侧流层析分析技术检测小分子物质。
核酸适配体是一种经过体外筛选技术-指数富集的配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)，得到的一小段结构化的寡核苷酸序列(RNA或DNA)，可以与相应的靶标分子(蛋白质，病毒，细菌，细胞，重金属离子等)进行高亲和力和强特异性的结合，为化学生物学界和生物医学界提供了一种新的高效快速识别的研究平台。
基于核酸适配体标记的胶体金侧向层析试纸，具有长期稳定、检测时间短、成本低、操作简单、快速等优点，是现场快速检测多种目标物的理想方法。现有核酸适配体胶体金侧向层析试纸条的设计，通常在T线上喷涂的是核酸链-链霉亲和素复合物，随着检测目标物的改变，需要重新制备特定的复合物及喷涂制备硝酸纤维素膜，导致现有的侧向层析试纸条的通用性差。
目前，基于适配体识别小分子化合物的胶体金侧向层析试纸研究较少。在文献(Biosensors&Bioelectronics,2015,71(15):230-242.)中，采用了DNA功能化的金纳米球(AuNPs-DNA) 作为探针，采用磁性微球(MMS)快速分离卡那霉素适配体的互补链(cDNA)，将cDNA作为检测目标，应用于试纸条上，根据cDNA的检测浓度转换为卡那霉素的检测浓度，以此来完成卡那霉素的定性以及定量检测，检测限达到4.96nmol/L(S/N＝3)。该试纸条需要借助MMS，且对卡那霉素的检测转化为对cDNA的检测，较为复杂，不利于产品的稳定生产和应用的重复性。在文献(Journal of Chromatography B,2016,1022:102-108.)中，开发了一种基于核酸适配体的侧向层析试纸条，该试纸条采用竞争结合模式，用于现场快速检测黄芪中的OTA。竞争反应发生在固定在试纸条上的DNA探针(测试区)和样品中的靶标(OTA)之间，两者竞争结合AuNPs-apt偶联物。该试纸条T线的颜色与样品中OTA的浓度成负相关。基于该方法，可在15min内完成OTA的检测，检测线为1ng/mL。该试纸条将适配体偶联到AuNPs上，不利于样品中OTA对适配体的竞争，产品稳定性差，且设计较为复杂通用性差。
目前，已开发的核酸适配体胶体金侧向层析试纸，针对某个特定的目标物，试纸条上的探针需要反复优化设计，通用性差，检测灵敏度有待提高。
发明内容
[技术问题]
现有技术中，核酸适配体胶体金侧向层析试纸条的设计，通常是在金标垫上喷涂金标核酸适配体，通常在检测线(T线)上喷涂的是链霉亲和素-核酸链复合物或带有荧光标记的核酸适配体或核酸适配体的互补链，针对某个特定的检测目标物，需要重新制备链霉亲和素-核酸链复合物，及重新喷涂制备硝酸纤维素膜，导致现有的侧向层析试纸条通用性不佳，试纸条的制备成本高。
[技术方案]
本发明采用polyA-DNA作为探针，该探针采用polyA作为锚定块，将polyA-DNA锚定在纳米金粒子上制备得到AuNPs@polyA-DNA偶联物。核酸试纸条的检测区域(T线)喷涂链霉亲和素，控制区域(C线)喷涂与AuNPs@polyA-DNA偶联物的polyA-DNA部分互补的寡核酸链(DNAc)，构建了通用型的试纸条。该试纸条在T线喷涂的链霉亲和素，以及在C线喷涂的链霉亲和素-生物素-寡核酸链复合物无需改变，仅改变polyA-DNA上与适配体互补的部分，即可检测另一种物质，为以后的检测提供了方便。
本发明所使用的核酸适配体，是在已报道的适配体的碱基序列的基础上增加5个T碱基，这5个核酸碱基用于辅助杂交。探针上有15个碱基与核酸适配体互补。生物素化的核酸适配体与锚定有探针的AuNPs@polyA-DNA偶联物结合后，探针才能进一步与T线上的链霉亲和素结合。当存在小分子物质时，小分子物质与探针竞争结合核酸适配体，抑制了探针与核酸 适配的杂交，从而抑制了T线的显色，实现了快速检测。
本发明提供的核酸适配体胶体金侧向层析试纸条的通用性体现在硝酸纤维素膜的制备和探针的设计上，即在制备针对不同的小分子物质的胶体金试纸条时，硝酸纤维素膜上的T线和C线无需变动，仅需变更小分子物质相应的核酸适配体和金标垫上的AuNPs@polyA-DNA偶联物，即，针对不同的小分子物质，核酸适配体与探针polyA-DNA做相应的变动。
本发明提供一种基于核酸适配体和AuNPs@polyA-DNA偶联物的通用型胶体金侧向层析试纸条检测卡那霉素，该试纸条一般是由样品垫、结合垫(金标垫)、硝酸纤维素膜(NC膜)、吸水垫和PVC胶板组成。
所述试纸条的检测原理为：采用竞争法检测小分子物质，当待测样品溶液中不含小分子物质时，5’端生物素化的核酸适配体与AuNPs@polyA-DNA偶联物结合，进而在T线上被链霉亲和素捕获形成AuNPs@polyA-DNA-Apt复合物，由于AuNPs的积累，在T线可以观察到一个清晰地深红色带，检测结果为阴性。当待测样品溶液中含有小分子物质时，小分子物质与5’端生物素化的核酸适配体结合，核酸适配体不能与AuNPs@polyA-DNA探针结合，导致探针在T线区域的积累降低，T线较浅或不显色，检测结果为阳性。T线区域上的颜色强度与小分子物质的浓度呈负相关。无论样品溶液中有没有小分子物质，AuNPs@polyA-DNA偶联物都可以在C线区域被DNAc捕获进而显色。
本发明的第一个目的是提供一种通用型胶体金侧向层析试纸条，所述试纸条含有核酸适配体、探针polyA-DNA和链霉亲和素-生物素-DNAc。
在一种实施方式中，所述探针polyA-DNA含有polyA片段、与DNAc互补的片段和与小分子物质的核酸适配体互补的片段。
在一种实施方式中，所述核酸适配体的5’端经生物素标记；所述核酸适配体可与待测的小分子物质特异性结合。
在一种实施方式中，所述链霉亲和素-生物素-DNAc是将链霉亲和素与5’端经生物素标记的DNAc等体积混合，在3～5℃孵育0.8～1.2h制备得到的。
在一种实施方式中，所述DNAc的核苷酸序列如SEQ ID NO:5所示。
在一种实施方式中，所述链霉亲和素浓度为2.5mg/mL，所述DNAc浓度为250μM。
在一种实施方式中，所述小分子物质包括但不限于卡那霉素、OTA、黄曲霉毒素、链霉素、氯霉素、雌二醇、双酚A、啶虫脒。
在一种实施方式中，所述核酸适配体为针对待检测目标物设计的经过体外筛选技术-指数富集的配体系统进化技术得到的寡核苷酸序列。
在一种实施方式中，所述试纸条包括样品垫、结合垫(金标垫)、硝酸纤维素膜(NC膜)、吸水垫和PVC胶板；所述PVC底板上依次黏贴样品垫、金标垫、NC膜和吸水垫；所述NC膜上依次设有检测区和控制区，且检测区和控制区之间的距离为4～6mm；所述检测区上有链霉亲和素，控制区上有链霉亲和素-生物素-DNAc；所述金标垫上含有AuNPs@polyA-DNA偶联物。
在一种实施方式中，所述样品垫与金标垫间重叠部位长度为1～2mm，样品垫置于金标垫上方；金标垫与NC膜间重叠部位长度为1～2mm，金标垫置于NC膜上方；NC膜与吸水垫间重叠部位长度为1～3mm，吸水垫置于NC膜上方。
在一种实施方式中，所述AuNPs@polyA-DNA偶联物是将探针polyA-DNA锚定在纳米金粒子上得到的。
在一种实施方式中，所述纳米金粒子的粒径为13～17nm；所述polyA-DNA的浓度为80～120μM。
本发明的第二个目的是提供一种快速检测小分子物质的方法，所述方法是采用上述胶体金侧向层析试纸条进行测试，将待测溶液与小分子物质核酸适配体混合孵育后吸取50-100μL滴加到样品垫上，孵育3～5min，用肉眼进行定性分析或使用胶体金试纸定量分析仪根据标准曲线进行定量分析。
在一种实施方式中，所述标准曲线的制备方法为将小分子物质的标准溶液稀释至不同浓度后，将不同浓度的稀释液与5’端经生物素标记的核酸适配体混合，再滴加到样品垫上，孵育3～5min，使用胶体金试纸定量分析仪进行定量分析，将数据进行拟合得到标准曲线。
在一种实施方式中，所述混合孵育的时间为15～25min。
在一种实施方式中，所述核酸适配体与待测溶液的体积比为1:99。
在一种实施方式中，所述核酸适配体的初始浓度为0.1～1.0μM。
本发明的第三个目的是提供上述通用型胶体金侧向层析试纸条的制备方法，所述方法具体步骤如下：
(1)将样品垫和金标垫切割后加入PBS浸泡并干燥。
(2)将探针AuNPs@polyA-DNA喷涂在金标垫上并干燥。
(3)在NC膜的检测区喷涂链霉亲和素，控制区喷涂链霉亲和素-生物素-DNAc，检测区和喷涂区之间距离固定在5mm，35～39℃干燥2h。
(4)将步骤(1)～(3)制备完成的样品垫、金标垫、NC膜和吸水垫依次粘贴在PVC板上即可得到检测卡那霉素的胶体金侧向层析试纸条。
在一种实施方式中，所述AuNPs@polyA-DNA偶联物是将探针polyA-DNA作为锚定块，将polyA-DNA锚定在纳米金粒子上得到的；所述探针polyA-DNA含有polyA片段、与DNAc互补的片段和与小分子物质的核酸适配体互补的片段；
在一种实施方式中，所述纳米金粒子的粒径为13～17nm。
在一种实施方式中，所述polyA-DNA的浓度为80～120μM。
在一种实施方式中，所述DNAc的核苷酸序列如SEQ ID NO:5所示。
在一种实施方式中，所述样品垫与金标垫间重叠部位长度为1～2mm，样品垫置于金标垫上方；金标垫与NC膜间重叠部位长度为1～2mm，金标垫置于NC膜上方；NC膜与吸水垫间重叠部位长度为1～3mm，吸水垫置于NC膜上方。
本发明还提供了所述通用型胶体金侧向层析试纸条或所述通用型胶体金侧向层析试纸条的制备方法在农药残留、兽药残留、环境监测、食品安全监测和生物医药领域中的应用。
本发明的有益效果：
(1)本文发明制备了高灵敏度、高特异性的一种基于适配体的通用型胶体金侧向层析试纸条。该试纸条对卡那霉素、OTA检测反应灵敏(肉眼检测限分别为15ng/mL、10ng/mL，读数仪检测限分别为0.3ng/mL、0.18ng/mL)、快速(20min)、检测结果重复性高。
(2)本发明所提出的检测方法实现了小分子物质(本发明为卡那霉素和OTA)的高灵敏比色检测。在5-250ng/mL的浓度范围内，T/C线与卡那霉素的浓度呈现良好的线性关系，检出限为0.3ng/mL。在1-250ng/mL的浓度范围内，T/C线与卡那霉素的浓度呈现良好的线性关系，检出限为0.18ng/mL。
(3)本发明构建的试纸条，检测区和控制区喷涂的链霉亲和素和核酸链无需改变，制备试纸条时仅仅需要改变胶体金-核酸偶联物即可检测另一种物质，从而提高了喷涂制备硝酸纤维素膜的通用型，简化制备过程，降低试纸条成本。
(4)该试纸条可通过观察检测区的颜色变化，肉眼可以实现小分子物质的定性检测，使用胶体金定量分析仪可实现定量分析。
(5)该试纸条的核酸适配体的设计中，通过在3’端增加多个T碱基以增加适配体与探针的结合效率，提高灵敏度，降低了核酸适配体的用量，减少成本。
附图说明
图1是试纸条的结构示意图和检测原理图。
图2是金纳米颗粒的表征：(a)AuNPs的TEM图像；(b)AuNPs的吸收光谱。
图3是用试纸条对不同浓度卡那霉素标准溶液的检测图；从左到右的浓度依次为0、0.5、5、15、25、50、150、250和400ng/mL。
图4是在标准检测液中，不同浓度卡那霉素时T线比C线的相对信号强度(T/C)标准曲线图；从左到右的浓度依次为0、1、10、50、100、250和500ng/mL。
图5是用试纸条对不同浓度OTA标准溶液的检测图。
图6是在标准检测液中，不同浓度OTA时T线比C线的相对信号强度(T/C)标准曲线图。
图7是用试纸条对不同浓度卡那霉素的蜂蜜样品的检测图。
图8是不同序列的核酸链对卡那霉素相应信号的影响；(a)探针上不同适配体互补链对T线显色的影响；(b)探针链polyA-DNA(5+10)和polyA-DNA(10+5)对卡那霉素响应信号的影响。
图9是polyA-DNA的结构。
具体实施方式
实施例一：基于核酸适配体的卡那霉素快速检测试纸条的制备
具体步骤为：
1.适配体序列和探针的设计
为了确保5’端生物素化的核酸适配体能与AuNPs@polyA-DNA结合并且在T线区域能被链霉亲和素(SA)捕获，选择不同序列的核酸探针链(polyA-DNA (20)，polyA-DNA (15)，polyA-DNA (5+10)，polyA-DNA (5+5)，polyA-DNA (10+5))与AuNPs偶联(如表1所示)，适配体的浓度为0.5μM。
结果如图8(a)所示，，当核酸探针链为核酸序列如SEQ ID NO:2所示的polyA-DNA (10+5)和SEQ ID NO:10所示的polyA-DNA (5+10)时，与3’端不增加T碱基的核酸探针链polyA-DNA (20)和polyA-DNA (15)相比，相对信号强度(T/C)显著增强。因此，将探针链为polyA-DNA (5+10)和polyA-DNA (10+5)应用于卡那霉素的检测，检验检测的灵敏性。选用适配体的浓度为0.5μM时，应用探针链polyA-DNA (5+10)和polyA-DNA (10+5)对卡那霉素进行检测，如图8(b)所示，加入相同浓度的卡那霉素(150ng/mL)时，当应用核苷酸序列如SEQ ID NO:2的核酸探针链polyA-DNA (10+5)时，对卡那霉素相应信号强度的变化(ΔT/C值)最大。因此，下述实施例中应用的探针为核苷酸序列如SEQ ID NO:2所示的polyA-DNA (10+5)。
表1卡那霉素适配体通用型LFS所用核酸序列
Figure PCTCN2021118498-appb-000001
Figure PCTCN2021118498-appb-000002
(带下划线或加粗的序列表示互补序列；加粗斜体序列表示适配体部分序列或其互补序列，加粗序列表示适配体延伸序列或其互补序列。)
polyA-DNA的设计：polyA-DNA有三个功能区。第一个功能区是polyA，其作为锚定块，将polyA-DNA锚定在金纳米粒子上；第二个功能区是与DNAc互补的区域；第三个功能区是与核酸适配体互补的区域(图9)。
寡核苷酸DNAc的设计：如表1、表2中所示，DNAc序列划线处与polyA-DNA互补，并在3’端连有5个A碱基。
表2卡那霉素快速检测试纸条的DNA序列
Figure PCTCN2021118498-appb-000003
(带下划线或加粗的序列表示互补序列；加粗序列表示适配体部分序列或其互补序列，加粗斜体序列表示适配体延伸序列或其互补序列。)
表3 OTA快速检测试纸条的DNA序列
Figure PCTCN2021118498-appb-000004
(带下划线或加粗的序列表示互补序列；加粗序列表示适配体部分序列或其互补序列；加粗斜体序列表示 适配体延伸序列或其互补序列。)
2.金纳米粒子(AuNPs)的制备及功能化
(1)AuNPs的制备
实验用于合成和储备纳米材料的玻璃器皿均用王水(盐酸：硝酸＝3:1)浸泡12h，用超纯水洗涤干净后使用。
AuNPs的制备采用的是柠檬酸钠还原法。具体步骤如下：
1)将100mL 0.01％的HAuCl 4加入250mL锥形瓶中，加热搅拌至溶液暴沸，保持1～2min。
2)向锥形瓶中迅速加入2mL 1％的柠檬酸三钠溶液，继续加热搅拌。溶液的颜色由浅黄色逐渐变为深紫色最后变成酒红色，保持加热10min，制备得到粒径15nm的AuNPs，将其冷却至室温后，4℃冷藏备用。
(2)AuNPs功能化
1)将5-15μL 100μM的polyA-DNA加入1mL步骤(1)中制备得到的AuNPs(10nM)中混合均匀，然后加入20μL 500mM的柠檬酸缓冲液(pH 3.0)。混合均匀后，在室温下孵育3min。
2)孵育完成后，加入60μL pH 7.6的500mM HEPES缓冲液，调整AuNPs溶液的pH为中性，然后在室温下孵育5-10min。
3)孵育完成后，10000r/min离心20min，去上清，向沉淀物中加入重悬液复溶，10000r/min、20min重复离心三次除去未反应的核酸，最终加入400μL重悬液获得功能化的AuNPs，即探针AuNPs@polyA-DNA，4℃冷藏备用。
用透射电子显微镜分别对步骤(1)制备的AuNPs和步骤(2)制备的AuNPs@polyA-DNA进行表征，结果如图2所示，制备的粒径15nm的AuNPs在520nm处有单一的特征吸收峰，当与polyA-DNA结合时，最大吸收波长移至530nm，初步证明polyA-DNA成功修饰了AuNPs。
重悬液的组成：20mM Na 3PO 4，5％BSA，10％蔗糖，0.25％吐温-20。
3.链霉亲和素-生物素-DNAc的制备
(1)100μL 2.5mg/mL的链霉亲和素与100μL 250μM 5’端经生物素标记的DNAc混合并在4℃下孵育1h，获得混合液。
(2)使用超滤管(MWCO 30kDa)处理混合液，6000r/min 20min离心三次，重悬于300μL 10mM PBS中，获得链霉亲和素-生物素-DNAc，4℃储存备用。
4.核酸适配体试纸条的组装
(1)将样品垫和结合垫(金标垫)切成适当尺寸，加入10mM PBS浸泡30min，然后在45℃下干燥。
(2)将步骤1中制备得到的探针AuNPs@polyA-DNA均匀的喷涂在结合垫上，37℃干燥2h。
(3)在NC膜上用三维喷点仪器以0.9μL/cm的速度均匀喷涂链霉亲和素和步骤2制备的链霉亲和素-生物素-DNAc，分别作为检测区(T线)和控制区(C线)，检测区和控制区之间的距离固定在5mm，37℃干燥2h。
(4)将步骤(1)～(3)制备完成的样品垫、结合垫、NC膜、吸水垫按图1依次粘贴在PVC板上，将组装好的试纸条均匀切割为4mm的宽度，放入分装袋中，密封保存。
实施例二：用试纸条对卡那霉素标准溶液的测定
(1)卡那霉素标准溶液的制备
卡那霉素标准溶液用Running buffer(4×SSC，pH 7)分别稀释终浓度为0.5、5、15、25、50、150、250和400ng/mL。用超纯水将核苷酸序列如SEQ ID NO:1所示的卡那霉素适配体稀释至0.5μM。
(2)卡那霉素核酸试纸条检测标准曲线的建立：
将步骤(1)中99μL不同浓度的卡那霉素标准溶液与1μL卡那霉素适配体溶液混合孵育20min，混合反应后将混合液滴加入到样品垫进行检测，反应3min后测定(T/C)相对信号强度，建立相对信号强度(T/C)与不同卡那霉素浓度之间对应关系的标准曲线。
结果如图3所示，当卡那霉素浓度为15ng/mL时，试纸条上T线的颜色能与不含卡那霉素的溶液(0ng/mL卡那霉素)的试纸条上的T线的颜色有明显的差异；在卡那霉素浓度为5～250ng/mL时T线颜色随卡那霉素浓度的增高而减弱，在卡那霉素浓度为250ng/mL时T线颜色基本没有变化，因此肉眼检测下限为15ng/mL，上限为250ng/mL。
胶体金试纸定量分析仪读取不同浓度卡那霉素的相对信号强度(T/C)，得到如图4所示相对信号强度(T/C)与卡那霉素浓度的变化关系曲线，最低检测线为0.3ng/mL。其中在5～250ng/mL浓度范围内，相对信号强度(T/C)与浓度存在线性关系，线性回归方程为y＝-0.1637x+0.4341，R 2＝0.9819，式中y为相对信号强度(T/C)，x为卡那霉素的浓度的Log函数(ng/mL)。
实施例三：用试纸条对OTA标准溶液的测定
(1)OTA标准溶液的制备
OTA标准溶液用Running buffer(4×SSC，pH7)分别稀释终浓度为1、10、50、100、 250和500ng/mL。用超纯水将核苷酸序列如SEQ ID NO:1所示的OTA适配体稀释至0.5μM。
(2)OTA核酸试纸条检测标准曲线的建立：
将步骤(1)中99μL不同浓度的OTA标准溶液与1μL OTA适配体溶液混合孵育20min，混合反应后将混合液滴加入到样品垫进行检测，反应3min后测定相对信号强度(T/C)，建立相对信号强度(T/C)与不同OTA浓度之间对应关系的标准曲线。
结果如图5所示，当OTA浓度为10ng/mL时，试纸条上T线的颜色能与不含OTA的溶液(0ng/mL OTA)的试纸条上的T线的颜色有明显的差异；在OTA浓度为1～250ng/mL时T线颜色随OTA浓度的增高而减弱，在OTA浓度为250ng/mL时T线颜色基本没有变化，因此肉眼检测下限为10ng/mL，上限为250ng/mL。
胶体金试纸定量分析仪读取不同浓度OTA的相对信号强度(T/C)，得到如图6所示相对信号强度(T/C)与OTA浓度的变化关系曲线，最低检测线为0.18ng/mL。其中在1～250ng/mL浓度范围内，相对信号强度(T/C)与浓度存在线性关系，线性回归方程为y＝-0.151x+0.4371，R 2＝0.9794，式中y为相对信号强度(T/C)，x为OTA的浓度的Log函数(ng/mL)。
实施例四：蜂蜜样品中卡那霉素残留的检测
利用蜂蜜模拟样品来测试回收率，步骤为：
(1)AuNPs@polyA-DNA溶液预处理：将制备储藏的AuNPs@polyA-DNA添加0.8～1.4μL体积至试纸条金标垫上，存储于4℃。
(2)样品预处理：将蜂蜜样本稀释10倍，0.22μm微孔滤膜过滤。在蜂蜜中加入不同浓度的卡那霉素(50、150、250ng/mL)；
(3)蜂蜜中卡那霉素的回收率测定：取实施例1中核苷酸序列如SEQ ID NO:1所述的卡那霉素适配体(0.5μM)1μL与步骤(2)中含不同浓度卡那霉素的蜂蜜溶液99μL混合孵育20min，混合反应后再采用试纸条进行检测。
应用实施例一制备得到的核酸适配体试纸条检测牛奶中卡那霉素的含量，结果如表4和图7所示。
表4蜂蜜样品中卡那霉素残留的检测
Figure PCTCN2021118498-appb-000005
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以 权利要求书所界定的为准。

Claims (15)

  1. 一种通用型胶体金侧向层析试纸条，其特征在于，含有核酸适配体、探针polyA-DNA和链霉亲和素-生物素-DNAc；所述探针polyA-DNA含有polyA片段、与DNAc互补的片段和与核酸适配体互补的片段；所述核酸适配体的5’端经生物素标记；所述核酸适配体可与待测的小分子物质特异性结合。
  2. 根据权利要求1所述的通用型胶体金侧向层析试纸条，其特征在于，所述链霉亲和素-生物素-DNAc是将链霉亲和素与5’端经生物素标记的DNAc等体积混合，在3～5℃孵育0.8～1.2h制备得到的；所述DNAc的核苷酸序列如SEQ ID NO:5所示。
  3. 根据权利要求2所述的通用型胶体金侧向层析试纸条，所述链霉亲和素浓度为2.5mg/mL，所述DNAc浓度为250μM。
  4. 根据权利要求1所述的通用型胶体金侧向层析试纸条，其特征在于，所述小分子物质包括但不限于卡那霉素、OTA、黄曲霉毒素、链霉素、氯霉素、雌二醇、双酚A、啶虫脒。
  5. 根据权利要求1～4任一所述的通用型胶体金侧向层析试纸条，其特征在于，所述试纸条包括样品垫、金标垫、硝酸纤维素膜、吸水垫和PVC胶板；所述PVC底板上依次黏贴样品垫、金标垫、硝酸纤维素膜和吸水垫；所述NC膜上依次设有检测区和控制区，且检测区和控制区之间的距离为4～6mm；所述检测区上有链霉亲和素，控制区上有链霉亲和素-生物素-DNAc；所述金标垫上含有AuNPs@polyA-DNA偶联物。
  6. 根据权利要求5所述的通用型胶体金侧向层析试纸条，其特征在于，所述样品垫与金标垫间重叠部位长度为1～2mm，样品垫置于金标垫上方；金标垫与NC膜间重叠部位长度为1～2mm，金标垫置于NC膜上方；NC膜与吸水垫间重叠部位长度为1～3mm，吸水垫置于NC膜上方。
  7. 根据权利要求5所述的通用型胶体金侧向层析试纸条，其特征在于，所述AuNPs@polyA-DNA偶联物是将权利要求1中所述探针polyA-DNA锚定在纳米金粒子上得到的。
  8. 根据权利要求7所述的通用型胶体金侧向层析试纸条，其特征在于，所述纳米金粒子的粒径为13～17nm；所述polyA-DNA的浓度为80～120μM。
  9. 一种快速检测卡那霉素的方法，其特征在于，所述方法是采用权利要求1～8任一所述的通用型胶体金侧向层析试纸条进行测试，将待测溶液与小分子物质的核酸适配体混合孵育后滴加到样品垫上，用肉眼进行定性分析或使用胶体金试纸定量分析仪根据标准曲线进行定量分析。
  10. 根据权利要求9所述的方法，其特征在于，所述标准曲线的制备方法为将小分子物质的标准溶液稀释至不同浓度后，将不同浓度的稀释液分别与5’端经生物素标记的核酸适 配体混合，再滴加到样品垫上，使用胶体金试纸定量分析仪进行定量分析，将数据进行拟合得到标准曲线。
  11. 根据权利要求9或10所述的方法，其特征在于，所述待测溶液与核酸适配体混合孵育的时间为15～25min；所述待测溶液与核酸适配体混合的体积比为99:1；所述核酸适配体的初始浓度为0.1～1.0μM。
  12. 权利要求1～8任一所述通用型胶体金侧向层析试纸条的制备方法，其特征在于，所述方法具体步骤如下：
    (1)将样品垫和金标垫切割后加入PBS浸泡并干燥；
    (2)将探针AuNPs@polyA-DNA喷涂在金标垫上并干燥；
    (3)在NC膜的检测区喷涂链霉亲和素，控制区喷涂链霉亲和素-生物素-DNAc，检测区和喷涂区之间距离固定在5mm，35～39℃干燥2h；
    (4)将步骤(1)～(3)制备完成的样品垫、金标垫、NC膜和吸水垫依次粘贴在PVC板上即可得到检测卡那霉素的胶体金侧向层析试纸条。
  13. 根据权利要求12所述的制备方法，其特征在于，所述AuNPs@polyA-DNA偶联物是将探针polyA-DNA作为锚定块，将polyA-DNA锚定在纳米金粒子上得到的；所述探针polyA-DNA含有polyA片段、与DNAc互补的片段和与小分子物质的核酸适配体互补的片段。
  14. 根据权利要求12所述的制备方法，其特征在于，所述纳米金粒子的粒径为13～17nm，所述polyA-DNA的浓度为80～120μM。
  15. 权利要求1～8任一所述的通用型侧向层析试纸条或权利要求12～14任一所述的通用型胶体金侧向层析试纸条的制备方法在农药残留、兽药残留、环境监测、食品安全监测和生物医药领域中的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004042084A1 (en) * 2002-11-07 2004-05-21 Automated Analysers And Diagnostic Reagents Medicon Hellas S.A. Dry reagent strip and nucleic acid detection
CN106990237A (zh) * 2017-05-10 2017-07-28 江南大学 一种基于核酸适配体的卡那霉素快速检测试纸及其制备方法和应用
CN108008128A (zh) * 2017-12-21 2018-05-08 江南大学 核酸适配体识别和功能化磁性微球分离预处理的卡那霉素快速检测试纸及其制备和应用
CN108303415A (zh) * 2018-01-18 2018-07-20 北京勤邦生物技术有限公司 一种检测黄曲霉毒素b1的核酸适配体试纸条及其制备方法和应用
CN111621598A (zh) * 2020-06-12 2020-09-04 中国人民解放军军事科学院军事医学研究院 一种消线法免疫层析试纸及其在crispr核酸检测中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180024989A (ko) * 2016-08-31 2018-03-08 성균관대학교산학협력단 형광 자성 나노입자를 이용한 엔도톡신 바이오센싱 키트
CN110646610B (zh) * 2019-06-28 2023-04-14 上海海洋大学 一种基于TdT信号放大技术的试纸条及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004042084A1 (en) * 2002-11-07 2004-05-21 Automated Analysers And Diagnostic Reagents Medicon Hellas S.A. Dry reagent strip and nucleic acid detection
CN106990237A (zh) * 2017-05-10 2017-07-28 江南大学 一种基于核酸适配体的卡那霉素快速检测试纸及其制备方法和应用
CN108008128A (zh) * 2017-12-21 2018-05-08 江南大学 核酸适配体识别和功能化磁性微球分离预处理的卡那霉素快速检测试纸及其制备和应用
CN108303415A (zh) * 2018-01-18 2018-07-20 北京勤邦生物技术有限公司 一种检测黄曲霉毒素b1的核酸适配体试纸条及其制备方法和应用
CN111621598A (zh) * 2020-06-12 2020-09-04 中国人民解放军军事科学院军事医学研究院 一种消线法免疫层析试纸及其在crispr核酸检测中的应用

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