CN104655759B - A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A - Google Patents
A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A Download PDFInfo
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- CN104655759B CN104655759B CN201510078992.4A CN201510078992A CN104655759B CN 104655759 B CN104655759 B CN 104655759B CN 201510078992 A CN201510078992 A CN 201510078992A CN 104655759 B CN104655759 B CN 104655759B
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Abstract
A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A, belongs to field of detection of food safety, for the detection of content of bisphenol A in milk, bottled mineral water etc. Present invention bisphenol-A specific nucleic acid aptamers functional magnetic nano material, utilizes the enrichment effect of its Magneto separate sample, allows the object in sample can rapid concentration, it is possible to improve detection sensitivity. In combination with the highly sensitive feature of efficient liquid phase Yu fluoroscopic examination, by Magneto separate, the eluent after being adsorbed by magnetic nanometer is by its response rate of high-performance liquid chromatogram determination.
Description
Technical field
The invention belongs to the organic method of detection, be specifically related to a kind of DNA method as method a kind of aptamers functional magnetic nano material Magneto separate bisphenol-A of the detection bisphenol-A of absorption aptamers.
Background technology
Bisphenol-A [2,2-bis (4-hydroxyphenol) propane, BPA] is the primary raw material producing the multiple macromolecular material such as Merlon and epoxy resin, it is also possible in the production of the fine chemical products such as antioxidant, coating, pesticide. In food, bisphenol A residues is mainly by food material and packaging for foodstuff:
(1) bisphenol-A is difficult to degrade in the environment, is widely present in nature, is particularly present in Environmental Water and plastic, and middle enrichment in vivo, is entered in the middle of our food by food chain.
(2) owing to bisphenol-A is fat-soluble, therefore in the goods such as fatty higher canned food, bisphenol-A penetrates in food also by food container and plastic sheeting.
(3) article containing BPA through Reusability or are exposed to high thermal environment and also result in BPA and leach, and then are taken in by human body.
Therefore BPA is ubiquitous, from mineral water bottle, medical apparatus and instruments to packaging for foodstuff inside, has its figure.
Bisphenol-A can be absorbed and harmful to human by skin, respiratory tract, digestive tract, meeting cardiac trigger disease, cancer and growth problem, and body is produced wide influence, and human health in serious threat. The content of bisphenol-A in food and food contact material is all had strict restriction by present multiple country. In European Union, bisphenol-A is 3 �� g/g in the migration limitation of food contact material. China specifies that the human body daily intake of bisphenol-A not can exceed that 0.05 �� g/g.
At present, report that the detection method to bisphenol-A mainly includes UV-VIS spectrophotometry, fluorimetry, electrochemical analysis method, gas chromatography (GC), gas chromatographymass spectrum (GC-MS), liquid chromatography (LC), liquid-mass chromatography method (HPLC-MS) both at home and abroad, in addition with other the analytic process such as chemoluminescence method, euzymelinked immunosorbent assay (ELISA). Spectrophotography and Fluorescence spectrophotometer device equipment cost are low, and operating procedure is simple, but the sensitivity of detection method is relatively on the low side; It is low that electrochemical methods has cost, it is easy to the advantages such as operation. But BPA electrochemical response on bare electrode is poor, and cause electrode surface to be passivated, electrode need to be carried out modification and realize enhanced sensitivity and antipollution; The pretreatment process of sample is required strict by chromatography, analyze loaded down with trivial details time-consuming, conventional preprocess method has liquid-liquid extraction (LLE) and Solid-Phase Extraction (SPE) method, both approaches has advantages such as the response rate is high, reproducible, but there is also that organic reagent consumption is big, complex operation step, expends time in, bisphenol-A does not have the shortcomings such as slective extraction.And mostly adopt a large amount of volatile organic solvent to extract, not only pollute the environment, and affect experimenter's health.
Magnetic Nano material is a kind of new type functional material developed the 1950's, is generally magnetic element such as ferrum, cobalt and nickel and oxide thereof, wherein being most widely used of iron oxide magnetic nano material. Magnetic Nano material has skin effect, small-size effect, quantum effect, magnetic conductance tropism, biocompatibility and biological degradability etc. that coupling capacity is high, good, in combinations with multiple biologically functional molecule (enzyme, DNA, protein etc.). The superparamagnetism that magnetic Nano material is good makes it provide a great convenience in the separation of sample, purification and enrichment etc.
And the concept about aptamers was proposed first in nineteen ninety, refer to that the part evolution technology (systematicevolutionofligandsbyexponentialenrichment, SELEX) that utilization index is enriched with filters out the oligonucleotide (DNA or RNA) having specificity to interact target from specific oligonucleotide library. Aptamers has high-affinity, can quickly synthesize in a large number, can specific aim screening and the good characteristic such as stability is high. Therefore, it suffers from good application prospect in multiple fields such as medicament research and development, analysis detections.
Owing to bisphenol-A content in actual sample is relatively low, analysis process can be produced serious interference by complicated sample substrate, and therefore before being analyzed, pretreatment is a critically important process. And preprocess method conventional at present is also complex, time-consuming. The magnetic nanometer superparamagnetism according to its uniqueness, by magnetic nanometer functionalization, i.e. coupling DNA aptamers, form magnetic nanometer probe, with this probe, the tested substance in sample is easily separated and is enriched with, it is possible to the effective pre-treatment flow process that simplifies, shortening detection time.
Summary of the invention
It is an object of the invention to provide a kind of aptamers functional magnetic nano-material biosensor detecting bisphenol-A, build the novel sensor utilizing high-efficient liquid phase signals to detect.
Technical scheme: a kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A, it is characterised in that: the synthesis of magnetic nanometer, the magnetic nanometer of aptamers functionalization, aptamers functionalization magnetic biosensor adsorbed target thing bisphenol-A after by efficient Liquid Detection.
Present invention:
A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A, it is characterized in that: the synthesis of magnetic nanometer, the magnetic nanometer of aptamers functionalization, aptamers functionalization magnetic biosensor adsorbed target thing bisphenol-A, and by efficient Liquid Detection after eluting; Concretely comprise the following steps:
(1) synthesis of 100nm magnetic nanometer
Magnetic Nano material (the Fe of 100nm3O4) adopt water heat transfer; Accurately weigh 2g hexahydrate iron chloride and 4.8g sodium acetate is dissolved in 60mL ethylene glycol, it is subsequently adding 1.5g Polyethylene Glycol and is sufficiently stirred for 2h under mechanical agitator effect, yellow liquid is fully transferred in the airtight rustless steel hydrothermal reaction kettle with polytetrafluoroethylliner liner (100mL), reacting by heating 12h in 210 DEG C of oil bath pans; Repeat to wash 6 times by the atrament dehydrated alcohol obtained after having reacted, be placed in ambient temperature in vacuum and dry;
(2) preparation of aptamers functionalization magnetic nanometer
Accurately weigh 0.3gFe3O4It is dissolved in 0.1M hydrochloric acid (100mL), ultrasonic 10 minutes, then with deionized water wash 5 times, Magnet separates, by Fe3O4It is dispersed in 150mL solvent (ethanol: ultra-pure water, 4:1), and adds 1.5mL ammonia (mass fraction 28%), be subsequently added silester 3mL, quickly stir 16 hours at 20 DEG C, with ethanol and water washing Fe3O4SiO2, vacuum drying;Take the magnetic nanometer of 5mL Silica-coated, add 30mL dehydrated alcohol, 5mL3-aminopropyl 3-Ethoxysilane (APTES); 60 DEG C of heated and stirred, react 6h; After being cooled to room temperature, Magnet separates, and is finally scattered in 5mL dehydrated alcohol standby after absolute ethanol washing 3 times; By the Fe after amination3O4SiO2Silicon bag magnetic nanometer is resuspended in ultra-pure water (5mg/mL), at room temperature hatches 30min with excessive EDC (200mM) and NHS (100mM), then passes through Magneto separate and removes EDC and the NHS of residual; The silicon bag magnetic nanometer taking 10 ��Ms of DNA and activation reacts 6h; Then by MNP-DNA deionized water wash 4 times, it is resuspended in ultra-pure water to be stored at 4 DEG C standby;
(3) aptamers absorption goal object
200 �� LMNP-DNA probe solutions are inserted in 2mL centrifuge tube, are subsequently adding the standard specimen of 800 �� L (10pg/mL-100ng/mL), incubated at room temperature 2 hours, and Magnet separates;
(4) object eluting
By above-mentioned aptamers functionalization magnetic nanometer after Magneto separate in methanol solvate at 60 DEG C eluting 1 hour, retain eluent; Aptamers functionalization magnetic nanometer after regeneration is repeated step (3) and (4) 3-5 time, collects eluent;
(5) efficient Liquid Detection
By eluent isolated in above-mentioned steps by efficient Liquid Detection; High-efficient liquid phase chromatogram determining condition: chromatographic column: C18Post; Mobile phase: methanol-water (70+30); Flow velocity: 1mL/min; Column temperature: room temperature; Fluorescence detector: excitation wavelength 227nm, launches wavelength 313nm. The present invention, when detecting actual sample, adds the DNA of 10 ��Ms.
Present invention have the advantage that 1, detection probe provided by the invention is highly sensitive, selectivity good, and detection limit is low; 2, object can be enriched with within the time quickly; 3, the inventive method is simply, quickly, easily operate.
Accompanying drawing explanation
Fig. 1 .1 (A) Fe3O4Transmission electron microscope photo.
Fig. 1 .2 (B) Fe3O4SiO2Transmission electron microscope photo.
Fig. 2 Fe3O4SiO2-aptamer Solid-Phase Extraction reuses research.
Detailed description of the invention
A kind of method of aptamers functional magnetic nano material Magneto separate bisphenol-A, the synthesis of magnetic nanometer, the magnetic nanometer of aptamers functionalization, aptamers functionalization magnetic biosensor adsorbed target thing bisphenol-A, and by efficient Liquid Detection after eluting; Concretely comprise the following steps:
(1) synthesis of 100nm magnetic nanometer
Magnetic Nano material (the Fe of 100nm3O4) adopt water heat transfer. Accurately weigh 2g hexahydrate iron chloride and 4.8g sodium acetate is dissolved in 60mL ethylene glycol, it is subsequently adding 1.5g Polyethylene Glycol and is sufficiently stirred for 2h under mechanical agitator effect, yellow liquid is fully transferred in the airtight rustless steel hydrothermal reaction kettle with polytetrafluoroethylliner liner (100mL), reacting by heating 12h in 210 DEG C of oil bath pans. Repeat to wash 6 times by the atrament dehydrated alcohol obtained after having reacted, be placed in ambient temperature in vacuum and dry.
(2) preparation of aptamers functionalization magnetic nanometer
Accurately weigh 0.3gFe3O4It is dissolved in 0.1M hydrochloric acid (100mL), ultrasonic 10 minutes, then with deionized water wash 5 times, Magnet separates, by Fe3O4It is dispersed in 150mL solvent (ethanol: ultra-pure water, 4:1), and adds 1.5mL ammonia (mass fraction 28%), be subsequently added silester 3mL, quickly stir 16 hours at 20 DEG C, with ethanol and water washing Fe3O4SiO2, vacuum drying. Take the magnetic nanometer of 5mL Silica-coated, add 30mL dehydrated alcohol, 5mL3-aminopropyl 3-Ethoxysilane (APTES).60 DEG C of heated and stirred, react 6h. After being cooled to room temperature, Magnet separates, and is finally scattered in 5mL dehydrated alcohol standby after absolute ethanol washing 3 times. By the Fe after amination3O4SiO2Silicon bag magnetic nanometer is resuspended in ultra-pure water (5mg/mL), at room temperature hatches 30min with excessive EDC (200mM) and NHS (100mM), then passes through Magneto separate and removes EDC and the NHS of residual. The silicon bag magnetic nanometer taking 10 ��Ms of DNA and activation reacts 6h. Then by MNP-DNA deionized water wash 4 times, it is resuspended in ultra-pure water to be stored at 4 DEG C standby.
(3) aptamers absorption goal object
200 �� LMNP-DNA probe solutions are inserted in 2mL centrifuge tube, are subsequently adding the standard specimen of 800 �� L (10pg/mL-100ng/mL), incubated at room temperature 2 hours, and Magnet separates.
(4) object eluting
By above-mentioned aptamers functionalization magnetic nanometer after Magneto separate in methanol solvate at 60 DEG C eluting 1 hour, retain eluent.
(5) regeneration of aptamers functionalization magnetic nanometer
Magnetic nanometer after eluting adsorbs bisphenol-A again, and operation is with (3), then is placed in methanol solvate and carries out eluting, and operation, with (4), retains eluent. Repeat this step 3-5 time, collect all eluents.
(6) efficient Liquid Detection
Eluent isolated in above-mentioned steps is obtained spectrogram by efficient Liquid Detection, record the peak area at bisphenol-A peak in its solution, contrast with the peak area of bisphenol-A standard specimen, then obtain the aptamers functionalization magnetic nanometer biosensor response rate to bisphenol-A. High-efficient liquid phase chromatogram determining condition: chromatographic column: C18Post; Mobile phase: methanol-water (70+30); Flow velocity: 1mL/min; Column temperature: room temperature; Fluorescence detector: excitation wavelength 227nm, launches wavelength 313nm.
Claims (2)
1. the method for an aptamers functional magnetic nano material Magneto separate bisphenol-A, it is characterized in that: the synthesis of magnetic nanometer, the magnetic nanometer of aptamers functionalization, aptamers functionalization magnetic biosensor adsorbed target thing bisphenol-A, and by efficient Liquid Detection after eluting; Concretely comprise the following steps:
(1) synthesis of 100nm magnetic nanometer
Magnetic Nano material (the Fe of 100nm3O4) adopt water heat transfer; Accurately weigh 2g hexahydrate iron chloride and 4.8g sodium acetate is dissolved in 60mL ethylene glycol, it is subsequently adding 1.5g Polyethylene Glycol and is sufficiently stirred for 2h under mechanical agitator effect, yellow liquid is fully transferred in the airtight rustless steel hydrothermal reaction kettle with polytetrafluoroethylliner liner 100mL, reacting by heating 12h in 210 DEG C of oil bath pans; Repeat to wash 6 times by the atrament dehydrated alcohol obtained after having reacted, be placed in ambient temperature in vacuum and dry;
(2) preparation of aptamers functionalization magnetic nanometer
Accurately weigh 0.3gFe3O4It is dissolved in 0.1M hydrochloric acid 100mL, ultrasonic 10 minutes, then with deionized water wash 5 times, Magnet separates, by Fe3O4It is dispersed in 150mL solvent, this etoh solvent: ultra-pure water, 4:1, and add 1.5mL mass fraction 28% ammonia, it is subsequently added silester 3mL, quickly stirs 16 hours at 20 DEG C, with ethanol and water washing Fe3O4SiO2, vacuum drying; Take the magnetic nanometer of 5mL Silica-coated, add 30mL dehydrated alcohol, 5mL3-aminopropyl 3-Ethoxysilane (APTES); 60 DEG C of heated and stirred, react 6h; After being cooled to room temperature, Magnet separates, and is finally scattered in 5mL dehydrated alcohol standby after absolute ethanol washing 3 times; By the Fe after amination3O4SiO2Silicon bag magnetic nanometer is resuspended in ultra-pure water 5mg/mL, at room temperature hatches 30min with excessive EDC200mM and NHS100mM, then passes through Magneto separate and removes EDC and the NHS of residual;The silicon bag magnetic nanometer taking 10 ��Ms of DNA and activation reacts 6h; Then by MNP-DNA deionized water wash 4 times, it is resuspended in ultra-pure water to be stored at 4 DEG C standby;
(3) aptamers absorption goal object
200 �� LMNP-DNA probe solutions are inserted in 2mL centrifuge tube, are subsequently adding the 800 �� L standard specimens of 10pg/mL-100ng/mL, incubated at room temperature 2 hours, and Magnet separates;
(4) object eluting
By above-mentioned aptamers functionalization magnetic nanometer after Magneto separate in methanol solvate at 60 DEG C eluting 1 hour, retain eluent; Aptamers functionalization magnetic nanometer after regeneration is repeated step (3) and (4) 3-5 time, collects eluent;
(5) efficient Liquid Detection
By eluent isolated in above-mentioned steps by efficient Liquid Detection; High-efficient liquid phase chromatogram determining condition: chromatographic column: C18Post; Mobile phase: methanol-water, its methanol 70+ water 30; Flow velocity: 1mL/min; Column temperature: room temperature; Fluorescence detector: excitation wavelength 227nm, launches wavelength 313nm.
2. the method for a kind of aptamers functional magnetic nano material Magneto separate bisphenol-A according to claim 1, it is characterised in that: the present invention, when detecting actual sample, adds the DNA of 10 ��Ms.
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| KR101646610B1 (en) | 2015-05-28 | 2016-08-09 | (주)바이오니아 | High active magnetic silica nano particles for separating biomaterial and method for preparing the same |
| CN105675759B (en) * | 2016-01-28 | 2018-01-23 | 华南师范大学 | A kind of method for separating and detecting of bisphenol-A |
| CN109609507B (en) * | 2016-10-26 | 2022-04-26 | 首都师范大学 | Single-stranded DNA aptamer of phthalate plasticizer, screening and characterization method of single-stranded DNA aptamer and electrochemical sensor |
| CN107841527B (en) * | 2017-11-07 | 2022-01-18 | 天津科技大学 | Fluorescence detection method for detecting thrombin by using aptamer and magnetic material |
| CN109632985B (en) * | 2018-12-13 | 2021-07-06 | 温州医科大学 | Method for detecting bisphenol compounds and derivatives thereof based on extraction technology of metal organic framework nano materials |
| CN109894081A (en) * | 2019-02-21 | 2019-06-18 | 浙江大学 | A kind of magnetic solid phase extraction material and its method of preparation and detection incretion interferent |
| CN110068561B (en) * | 2019-04-29 | 2021-10-01 | 河南中医药大学 | Bisphenol A fluorescence detection method based on atom transfer radical polymerization reaction and truncated aptamer |
| CN110016146B (en) * | 2019-05-10 | 2021-08-06 | 华东理工大学 | Preparation method and application of magnetic functionalized rare earth fluorescent probe solution |
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