CN102031306B - Oligonucleotides chain-based mercury ion fluorescent detection chip, manufacturing method thereof and using method thereof - Google Patents
Oligonucleotides chain-based mercury ion fluorescent detection chip, manufacturing method thereof and using method thereof Download PDFInfo
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Abstract
The invention relates to an oligonucleotides chain-based mercury ion fluorescent detection chip, a manufacturing method thereof and a using method thereof. The detection chip can be in convalent binding with a T basic group on a T-enriched oligonucleotides chain specifically through Hg<2+> and mediates T-T pairing of two T-enriched oligonucleotides chains to form a stable intermolecular T-Hg Hg<2+>-T structure and thus induces the release of complementary strands which are crossed with the T-enriched oligonucleotides chain. The use of the detection chip comprises the following steps of: placing a sample to be tested on the chip, keeping a period of time, scanning the chip by using a chip signal analysis system and analyzing a fluorescent signal. The Hg<2+> detection is realized by the change of the fluorescent signal. Higher Hg<2+> concentration in the sample means the fluorescent signal weakens more. The Hg<2+> concentration range capable of being detected by the method is 1nM to 100mu M.
Description
Technical field
The present invention relates to a kind of mercury ion fluorescent detection chip, making and using method based on oligonucleotide chain, belong to the bioassay technique field.
Background technology
Mercury is the global environmental pollutant of high poison, especially it has the characteristics of high transport property, persistence, methylation, bioconcentration and food chain amplification, even if being present in environment of denier is also great threat to animals and plants and the mankind's health.Approximately 1.5 ten thousand tons of the global mercury quantity dischargeds of a year, be mainly derived from the burning of mercury ore, metallurgy, chlorine industry, electrical equipment industry and mineral fuel.Mercury exist in a variety of forms with environment in, water miscible dimercurion (Hg
2+) be one of common and the most stable form of mercury pollution.
How effectively to carry out the mensuration of mercury ion content in environment, become the problem of pendulum in face of vast analytical work person.Traditional mercury ion detecting method mainly contains at present: atom (absorbing emission, fluorescence) spectrography and Inductively coupled plasma mass spectrometry (Inductively coupled plasma mass spectrometry, ICP-MS).Although these methods can access more accurate detected result, but these technology rely on large-scale instrument and equipments, it is consuming time to expend, need carry out sample pretreatment, need special technician to be operated, testing cost is high, be difficult to meet the requirement of place of production field quick detection, and wherein some method also needs to use toxic reagent, be difficult to analyzed personnel and accept.Therefore, in a lot of important occasions, people are in the urgent need to easy, quick, economic, the method for analyzing and testing mercury ion accurately.The method of at present, both at home and abroad mercury ion being carried out to Site Detection can not meet the demands on sensitivity and specificity.
Recent research shows, a mercury ion can form stable T-Hg with two thymine alkali bases (T) covalent attachment specifically
2+-T structure.This special character based on mercury ion, developed various Hg
2+detection method: as fluorescent method (A.Ono, H.Togashi, Angew.Chem.Iht.Ed.Engl.2004,43,4300.), nanometer gold assembles colorimetry (J.S.Lee, M.S.Han, C.A.Mirkin, Angew.Chem.Iht.Ed.Engl.2007,46,4093.), electrochemical process (Z.Zhu, Y.Su, J.Li, D.Li, J.Zhang, S.Song, Y.Zhao, G.Li, C.Fan, Anal.Chem.2009,81,7660.).These methods generally have the characteristics such as selectivity is good, high specificity, but complex operation, and be not suitable for high-throughout detection.
Biochip is the new and high technology that this century, late nineteen eighties developed rapidly in life science, it mainly refers to the miniature organism chemical analysis system at solid chip surface construction by micro-processing technology and microelectronics, with realize to cell, protein, DNA and other materials accurately, fast, the detection of large information capacity.The principal feature of biochip is high-throughput, microminiaturization and automatization, can realize micro-example is detected fast and accurately.
The present inventor imagination is if form stable T-Hg with two thymine alkali bases (T) covalent attachment specifically by above-mentioned mercury ion
2+this characteristic of-T structure and the advantages of biochip are got up, and a kind of Hg based on oligonucleotide chain is provided
2+detection chip, to realize Hg
2 +low-cost, highly sensitive, detection accurately and rapidly.Thereby be guided out design of the present invention.
Summary of the invention
The object of the present invention is to provide a kind of mercury ion detecting chip of oligonucleotide chain.Mercury ion detecting chip of the present invention is characterised in that: utilize Hg
2+can be specifically and two thymine alkali bases (T) covalent attachment of DNA, mediation T-T pairing forms stable T-Hg
2+-T structure.Specifically, synthetic rich T oligonucleotide chain is fixed on modified slide; Then fluorescently-labeled complementary strand and the rich T oligonucleotide chain hybridization be fixed on slide, form duplex structure, prepares Hg
2+detection chip; Add again testing sample, detect fluorescent signal.If contain Hg in testing sample
2+the time, Hg
2+can be specifically with rich T oligonucleotide chain on T base covalent attachment, mediate two T-T pairings on rich T oligonucleotide chain and form stable intermolecular T-Hg
2+-T structure, thus the release with the complementary strand of fluorophor induced, cause chip spot place fluorescent weakening.Whether mercury ion exists, and can pass through the quantitative analysis of fluorescent scanning instrument.
Hg based on oligonucleotide chain of the present invention
2+the making method of detection chip and application method thereof is characterized in that:
Hg in the present invention
2+the making method of detection chip comprises the steps:
(1) the rich T oligonucleotide chain of chemically modified is fixed on the slide of chemically modified
The rich T oligonucleotide chain of 2-20 μ M chemically modified adds 2 * point sample damping fluid in the ratio of 1: 1 (V/V).By with contact Cartesian microarray manufacturing system dot matrix in the slide glass of chemically modified.Point sample is complete, and slide is placed in to certain humidity, such as 48～72h under 70% humidity, room temperature condition is fixed.With 0.2%SDS, deionization washing 2 times, each 2min, then use aldehyde radical confining liquid (0.1g sodium borohydride, 30mL PBS, 10mL 99% ethanol) sealing 15min respectively.With 0.2%SDS, deionized water, respectively wash 2 times successively, each 2min, dry up, standby again.
(2) hybridization of complementary strand and rich T oligonucleotide chain
Mark the complementary strand of fluorophor be diluted to 1-10 μ M with hybridization solution.The complementary strand solution diluted is added in to the spot place of chip, covered.Chip is placed in 25 ℃ of wet boxes, after 12-16 hour, with 10mM MOPS[(3-(N-morpholino) propanesulfonic acid], 100mM NaNO
3, pH 7.2 washes 5-10min, dries up, standby.
Described Hg
2+the using method of detection chip comprises the steps:
Prepare the Hg of different concns with the hybridization solution dilution
2+, add the Hg of different concns
2+solution is in chip spot place, and room temperature, react 10-60min.10mM MOPS, 100mM NaNO
3, pH 7.2 washes 3 times, dries up.Chip signal analytical system Scanarray 3000 scanning analytical resultss with General Scanning company.
In sum, the present invention has utilized at Hg
2+in situation about existing, Hg
2+can be specifically with rich T oligonucleotide chain on T base covalent attachment, mediate two T-T pairings on rich T oligonucleotide chain and form stable intermolecular T-Hg
2+-T structure, thus the release with the complementary strand of rich T oligonucleotide chain hybridization induced.If complementary strand is the mark fluorescent group in advance, discharge fluorescent signal is reduced.The preparation of mercury ion fluorescent detection chip comprises three steps: at first design the rich T oligonucleotide chain of corresponding chemically modified and the complementary strand of fluorescent decoration, then end modified rich T oligonucleotide chain is fixed on modified slide, finally, by the rich T oligonucleotide chain hybridization on fluorescently-labeled complementary strand and slide, prepare the mercury ion detecting chip.The detection of using said chip to carry out mercury ion only need to be added testing sample on chip to, and keeps for some time, then utilizes chip signal analytical system scanning chip, and fluorescent signal is analyzed.By the variation of fluorescent signal, realize right Hg
2+detect.Hg in sample
2+concentration is higher, and it is more that fluorescent signal weakens.The Hg that the method can detect
2+concentration range be 1nM-100 μ M.This mercury ion detecting chip also can be used other various signal marks, such as radio-labeling, quantum dot, enzyme or nanometer gold etc.
Technique effect of the present invention:
1, the present invention is based on the Hg of oligonucleotide chain
2+simple, the easy row of the making method of detection chip.
2, the present invention is based on the Hg of oligonucleotide chain
2+detection chip has detection sensitivity and specificity is high.
3, application the present invention is based on the Hg of oligonucleotide chain
2+detection chip detects Hg
2+the time, sample, reagent consumption are few, and cost is low.
4, use the result of detection of the present invention to get final product with common scanner scanning observation and analysis, do not need complicated expensive device, make more convenient, the easy row of Site Detection.
The accompanying drawing explanation
Fig. 1 is the schematic diagram based on oligonucleotide chain fluoroscopic examination mercury ion on chip.
Fig. 2 (A)-Fig. 2 (G) is the time dynamics based on oligonucleotide chain fluoroscopic examination mercury ion in one embodiment of the invention.In the fluorescent scanning photo, fluorescence intensity on average refers to successively: during reaction 2min (A), and damping fluid group 61356,10 μ M Hg
2+group 33296,100 μ M Hg
2+group 22273.During reaction 5min (B), damping fluid group 59859,10 μ M Hg
2+group 28697,100 μ M Hg
2+group 15833.During reaction 10min (C), damping fluid group 60761,10 μ M Hg
2+group 18440,100 μ MHg
2+group 10121.During reaction 20min (D), damping fluid group 662187,10 μ M Hg
2+group 12726,100 μ M Hg
2+group 6755.During reaction 40min (E), damping fluid group 59893,10 μ M Hg
2+group 9441,100 μ M Hg
2+group 5369.During reaction 60min (F), damping fluid group 61789,10 μ M Hg
2+group 7882,100 μ M Hg
2+group 3612.G is the 100 μ M Hg that prepare
2+(curve 1) and 10 μ MHg
2+(curve 2) fluorescence intensity curve in time weakens.
Fig. 3 (A)-Fig. 3 (B) is that in one embodiment of the invention, the detection chip based on the oligonucleotide chain fluoroscopic examination detects Hg
2+fluorescent scanning photo (A) and typical curve (B).In the fluorescent scanning photo, fluorescence intensity on average refers to successively: damping fluid group 61789,10nM Hg
2+group 49411,100nM Hg
2+group 35127,1 μ M Hg
2+group 20666,10 μ M Hg
2+group 7882,100 μ M Hg
2+group 3612.
Fig. 4 (A)-Fig. 4 (B), invent the detection chip based on the oligonucleotide chain fluoroscopic examination in an embodiment and detect Hg
2+with other bivalent ions fluorescent scanning photo and result.In the fluorescent scanning photo, fluorescence intensity on average refers to successively: damping fluid group 60961, Hg
2+group 8996, Pb
2+group 59408, Zn
2+group 59753, Ca
2+group 59945, Cu
2+group 59630, Ni
2+group 59370, Mg
2+group 60433.
Fig. 5 (A)-Fig. 5 (C) is that in one embodiment of the invention, the detection chip based on the oligonucleotide chain fluoroscopic examination detects Hg in the standard buffer solution of same concentrations
2+with the Hg mixed in tap water
2+fluorescent scanning photo and result.
Embodiment
Below, by the description of embodiment, further illustrate substantive distinguishing features of the present invention and significant progressive.The sequence of the nucleic acid probe for clarity sake, first the present invention used is listed in table 1
Table 1: the nucleic acid probe sequence used in the present invention.
| The nucleic acid probe title | Sequence |
| Probe A | 5’-NH 2-C 12-TTTTTTTTTTTTTT-3’ |
| Probe B | 5’-Cy5-AAAAAAAAAAAAAA-3’ |
Embodiment 1: utilize probe A and probe B to prepare Hg
2+detection chip.
Probe A is made into to the solution that concentration is 20 μ M, then mixes with the Spotting Solution of equal volume, use the micro-array chip manufacturing system dot matrix of Cartesian company in aldehyde group modified slide surface, be placed under room temperature, 70% relative humidity is preserved 48-72h and is fixed, and then, under room temperature, slide is immersed in 0.2%SDS and vibrates several minutes, immerse again in pure water and vibrate several minutes, immerse again 0.2%SDS twice, each 2min, then immerse in pure water twice, each 2min, dry.With hybridization solution (10mM MOPS, 100mM NaNO
3, pH 7.2) and by probe B dilution, final concentration is 2-5 μ M, drips on chip covered, room temperature hybridization 12-16h.Then use successively 0.2%SDS, 2 * SSC, 0.2 * SSC washes 3min, dries up standby.
Embodiment 2: the chip that utilizes probe A and probe B to prepare is investigated Hg
2+reaction times kinetics.
Use 10mM MOPS, 100mM NaNO
3, pH 7.2 dilutions prepare the Hg of 100 μ M, 10 μ M
2+, by Hg
2+solution is added in the chip spot place prepared.Room temperature reaction takes out chip in 1 hour, uses 10mM MOPS, 100mM NaNO
3, pH 7.2 damping fluids are washed 3 times, dry up.Chip signal analytical system Scanarray 3000 scanned photograph (Fig. 2 A-F) analytical results (Fig. 2 G) with General Scanning company.
Result shows, Hg
2+the release of inducing probe B is individual process fast, adds the Hg of 100 μ M, 10 μ M
2+rear reaction 2min, with respect to the damping fluid control group, fluorescence intensity has descended respectively 63%, 45%.20min has completed 95% reaction substantially.
Embodiment 3: the Hg of the chip detection different concns that utilizes probe A and B to prepare
2+.
Use 10mM MOPS, 100mM NaNO
3, pH 7.2 dilutions prepare the Hg of different concns
2+, add the Hg of different concns
2+solution is in the chip spot place prepared, and room temperature, react 1h.Use 10mMMOPS, 100mM NaNO
3, pH 7.2 damping fluids are washed 3 times, dry up.Chip signal analytical system Scanarray 3000 scanned photograph (Fig. 3 A) analytical results (Fig. 3 B) with General Scanning company.
Result shows, at Hg
2+in the situation that ion exists, chip spot place fluorescence intensity weakens, and works as Hg
2+when concentration is 10nM, with the fluorescence intensity of damping fluid group, compare, spot place fluorescence intensity weakens 20%, along with Hg
2+concentration increases, and fluorescent signal weakens gradually.According to three times of calculating of zero standard deviation, this chip detection Hg
2+detection be limited to 1nM.
Embodiment 4: chip prepared by investigation probe A and B is to Hg
2+the specificity detected.
Use 10mM MOPS, 100mM NaNO
3, the different divalent-metal ion of pH 7.2 dilution preparation 10 μ M, add different divalent-metal ion solution in the chip spot place prepared, room temperature, reaction 1h.Use 10mM MOPS, 100mM NaNO
3, pH 7.2 damping fluids are washed 3 times, dry up.Chip signal analytical system Scanarray 3000 scanned photograph (Fig. 4 A) analytical results (Fig. 4 B) with General Scanning company.
Result shows, only at Hg
2+in situation about existing, chip spot place fluorescence intensity weakens greatly, Hg
2+while being 10 μ M, with the fluorescence intensity of damping fluid group, compare, fluorescence intensity has weakened 85%.And when adding 10 other divalent-metal ions of μ M, fluorescence intensity to only have small weakening, in 0.9%～2.6% left and right.Illustrate that this chip is to Hg
2+detection has good specificity.
Embodiment 5: the chip detection prepared with probe A and B is incorporated in the Hg in tap water
2+.
With the tap water preparation, containing 10mM MOPS, pH 7.2,100mM NaNO
3damping fluid, then mix wherein the mercury storage liquid of certain volume, the concentration that makes mercury ion is 5 μ M, 500nM.The mercury ion solution prepared is added to chip spot place, set up the contrast of tap water damping fluid simultaneously.And the mercury ion of same concentrations in the examination criteria damping fluid simultaneously.Room temperature reaction 1h, use 10mM MOPS, pH7.2,100mM NaNO
3damping fluid is washed 3 times, and each 2min, dry up.Also use Signal Analysis System Scanarray 3000 analytical resultss (Fig. 5 C) by laser confocal scanning instrument scanned photograph (Fig. 5 A, 5B).
Result shows, with the Hg in tap water that is incorporated in of this chip detection same concentrations
2+and the Hg in standard buffer solution
2+the time, the degree that fluorescent signal weakens, without significant difference, illustrates with this chip and can detect the Hg be incorporated in tap water
2+.
Claims (5)
1. the making method of a mercury ion fluorescent detection chip, is characterized in that synthetic rich T oligonucleotide chain is fixed on modified slide; Then fluorescently-labeled complementary strand and the rich T oligonucleotide chain hybridization be fixed on slide, form duplex structure, comprises the following steps:
(1) the rich T oligonucleotide chain of chemically modified is fixed on the slide of chemically modified
The rich T oligonucleotide chain of 2-20 μ M chemically modified adds 2 * point sample damping fluid by the 1:1 volume ratio, by with contact Cartesian microarray manufacturing system dot matrix in the slide surface of chemically modified; Point sample is complete, and slide is placed in to 70% humidity, and under room temperature condition, 48～72 h are fixed; With 0.2% SDS, deionization washing 2 times, each 2 min, then seal 15 min with the aldehyde radical confining liquid respectively; With 0.2% SDS, deionized water, respectively wash 2 times successively, each 2 min, dry up again;
(2) hybridization of complementary strand and rich T oligonucleotide chain
Mark the complementary strand of fluorophor be diluted to 1-10 μ M with hybridization solution, the complementary strand solution diluted is added in to the spot place of chip, covered; Chip is placed in 25 ℃ of wet boxes, after 12-16h with 10 mM MOPS, 100 mM NaNO
3, pH 7.2 washes 5-10 min, dries up;
The sequence of the rich T oligonucleotide chain of described chemically modified is 5 '-NH
2-C
12-TTTTTTTTTTTTTT-3 '; The sequence of described complementary strand is 5 '-Cy5-AAAAAAAAAAAAAA-3 '.
2. by making method claimed in claim 1, it is characterized in that the ethanol that the confining liquid described in step (1) is 99% by 0.1g sodium borohydride, 30mlPBS and 10ml mass percent forms.
3. use the method for the mercury ion fluorescent detection chip of being made by the method for claim 1, it is characterized in that preparing with the hybridization solution dilution Hg of different concns
2+, add the Hg of different concns
2+solution is in chip spot place, and room temperature, react 10-60min; Use 10mM MOPS, 100mM NaNO
3, pH7.2 washes 3 times, dries up; Then with the chip signal analytical system Scanarray 3000 of General Scanning company, scan and analytical resultss.
4. by method claimed in claim 3, it is characterized in that:
1. the Hg that adds 100 μ M, 10 μ M
2+rear reaction 2 min, with respect to the damping fluid control group, fluorescence intensity has descended respectively 63%, 45%; 20 min have completed 95% reaction;
2. at Hg
2+in the situation that ion exists, chip spot place fluorescence intensity weakens, and works as Hg
2+when concentration is 10 nM, with the fluorescence intensity of damping fluid group, compare, spot place fluorescence intensity weakens 20%, along with Hg
2+concentration increases, and fluorescent signal weakens gradually.
5. by method claimed in claim 3, it is characterized in that with 10 mM MOPS 100 mM NaNO
3, the different divalent-metal ion of pH 7.2 dilution preparation 10 μ M, add different divalent-metal ion solution in the chip spot place prepared, and room temperature, react 1 h; With 10 mM MOPS, 100 mM NaNO
3, pH 7.2 damping fluids are washed 3 times, dry up; Chip signal analytical system Scanarray 3000 scanned photograph with General Scanning company; Only at Hg
2+in situation about existing, chip spot place fluorescence intensity weakens greatly, Hg
2+while being 10 μ M, with the fluorescence intensity of damping fluid group, compare, fluorescence intensity has weakened 85%; And, when adding 10 other divalent-metal ions of μ M, fluorescence intensity is only in small the weakening of 0.9%～2.6% scope.
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| CN102621120B (en) * | 2012-03-31 | 2014-04-23 | 江苏省农业科学院 | Method for detecting mercury ion residue of fluorescent signal conversion mechanism based on nucleic acid aptamer structure |
| CN102759526B (en) * | 2012-06-28 | 2014-08-27 | 宁波大学 | Method for quantitative detection of mercury ions through gold label silver stain and kit thereof |
| CN102778492B (en) * | 2012-07-13 | 2014-07-02 | 首都师范大学 | Electrochemical transducer for mercury ion detection and manufacturing method and detection method thereof |
| CN102912011A (en) * | 2012-08-24 | 2013-02-06 | 中国科学院上海微系统与信息技术研究所 | Fluorescence-enhanced Hg<2+> detection chip based on oligonucleotide chains and method thereof |
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