CN102031306A - 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, belong to the bioassay technique field based on oligonucleotide chain.
Background technology
Mercury is the global environmental pollutant of high poison, especially it has the characteristics of high transport property, persistence, methylation, biomagnification and food chain amplification, even if being present in the environment of denier also is great threat to animals and plants and human beings'health.About 1.5 ten thousand tons of the quantity discharged in 1 year of global mercury is 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 to carry out mercury ion Determination on content in the environment effectively, become the problem of pendulum in face of vast analytical work person.Traditional mercury ion detecting method mainly contains at present: and atom (absorbing emission, fluorescence) spectrography and inductively coupled plasma mass spectrograph (Inductively coupled plasma mass spectrometry, ICP-MS).Though 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 operate, detection cost height, be difficult to satisfy the requirement of place of production field quick detection, and wherein some method also needs to use toxic reagent, is difficult to analyzed personnel and accepts.Therefore, in a lot of important occasions, that people press for is easy, quick, economical, the method for analyzing and testing mercury ion accurately.At present, mercury ion being carried out the on-the-spot method that detects both at home and abroad can not meet the demands on sensitivity and specificity.
Recent studies show that a mercury ion can form stable T-Hg with two thymine alkali bases (T) covalent attachment specifically
2+-T structure.This special nature based on mercury ion has 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 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 is meant the miniature organism chemical analysis system that makes up at the solid chip surface by micro-processing technology and microelectronics, with realize pair 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 the micro-example accurate detection.
The present inventor imagination if can be with above-mentioned mercury ion form stable T-Hg with two thymine alkali bases (T) covalent attachment specifically
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 is 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 feature of the present invention is: 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, the rich T oligonucleotide chain of synthetic is fixed on the modified slide; Fluorescently-labeled then complementary strand and the rich T oligonucleotide chain hybridization that is fixed on the slide form duplex structure, prepare Hg
2+Detection chip; Add testing sample again, detect fluorescent signal.If contain Hg in the testing sample
2+The time, Hg then
2+Can be specifically with rich T oligonucleotide chain on T base covalent attachment, the T-T pairing that mediates on two rich T oligonucleotide chains forms stable intermolecular T-Hg
2+-T structure, thus the release of the complementary strand that has 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 among 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 finishes, and slide is placed certain humidity, fixes such as 48～72h under 70% humidity, the room temperature condition.With 0.2%SDS, deionization washing 2 times, each 2min uses aldehyde radical confining liquid (0.1g sodium borohydride, 30mL PBS, 10mL 99% ethanol) sealing 15min then respectively.Respectively wash 2 times with 0.2%SDS, deionized water successively, each 2min dries up, and is 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 that dilution is good is added in the spot place of chip, covered.Chip places 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, and is 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 is reacted 10-60min.10mM MOPS, 100mM NaNO
3, pH 7.2 washes 3 times, dries up.Chip signal analytical system Scanarray 3000 scanning and analytical resultss with General Scanning company.
In sum, the present invention has utilized at Hg
2+Under the situation about existing, Hg
2+Can be specifically with rich T oligonucleotide chain on T base covalent attachment, the T-T pairing that mediates on two rich T oligonucleotide chains forms stable intermolecular T-Hg
2+-T structure, thus release with the complementary strand of rich T oligonucleotide chain hybridization induced.If complementary strand is the mark fluorescent group in advance, release reduces fluorescent signal.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 that fluorescence is modified, then end modified rich T oligonucleotide chain is fixed on the modified slide, with the rich T oligonucleotide chain hybridization on fluorescently-labeled complementary strand and the slide, prepare the mercury ion detecting chip at last.The detection of using said chip to carry out mercury ion only need be added testing sample on the chip to, and keeps for some time, utilizes chip signal analytical system scanning chip then, and fluorescent signal is analyzed.By the variation of fluorescent signal, realize right Hg
2+Detect.Hg in the sample
2+Concentration is high more, and it is many more that fluorescent signal weakens.The Hg that this method can detect
2+Concentration range be 1nM-100 μ M.This mercury ion detecting chip also can use 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 height.
3, use the Hg that the present invention is based on 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, do not need complicated expensive device, make on-the-spot more convenient, the easy row that detects with common scanner scanning observation and analysis.
Description of drawings
Fig. 1 is based on the schematic diagram of oligonucleotide chain fluoroscopic examination mercury ion on the chip.
Fig. 2 (A)-Fig. 2 (G) is based on the time dynamics of oligonucleotide chain fluoroscopic examination mercury ion in the one embodiment of the invention.In the fluorescent scanning photo fluorescence intensity on average refer to be successively: reaction is during 2min (A), 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 the detection chip based on the oligonucleotide chain fluoroscopic examination detects Hg in the one embodiment of the invention
2+Fluorescent scanning photo (A) and typical curve (B).In the fluorescent scanning photo fluorescence intensity on average refer to be 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), the detection chip of inventing among the embodiment based on the oligonucleotide chain fluoroscopic examination detects Hg
2+With other bivalent ions fluorescent scanning photo and result.In the fluorescent scanning photo fluorescence intensity on average refer to be 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 to detect Hg in the standard buffer solution of same concentrations based on the detection chip of oligonucleotide chain fluoroscopic examination in the one embodiment of the invention
2+With the Hg that mixes in the 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 obvious improvement.For clarity sake, the sequence of the nucleic acid probe that earlier the present invention is used is listed in table 1
Table 1: the nucleic acid probe sequence that uses among 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 the solution that concentration is 20 μ M, mixes with the Spotting Solution of equal volume then, the micro-array chip manufacturing system dot matrix of using Cartesian company is in aldehyde group modified slide surface, place under the room temperature, 70% relative humidity is preserved 48-72h and is fixed, and then, under the room temperature slide is immersed among the 0.2%SDS vibration several minutes, immerse again in the pure water and vibrated several minutes, immerse 0.2%SDS twice again, each 2min immerses in the pure water twice again, each 2min dries.With hybridization solution (10mM MOPS, 100mM NaNO
3, pH 7.2) and with probe B dilution, final concentration is 2-5 μ M, drips on chip covered, room temperature hybridization 12-16h.Use 0.2%SDS then successively, 2 * SSC, 0.2 * SSC washes 3min, dries up standby.
Embodiment 2: utilize the chip of probe A and probe B preparation to investigate 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+, with Hg
2+Solution is added in the chip spot place for preparing.Room temperature reaction took out chip in 1 hour, used 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) and analytical results (Fig. 2 G) with General Scanning company.
The 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+Afterreaction 2min, with respect to the damping fluid control group, fluorescence intensity has descended 63%, 45% respectively.20min has finished 95% reaction substantially.
Embodiment 3: the Hg that utilizes the chip detection different concns of probe A and B preparation
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 for preparing, and room temperature is reacted 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) and analytical results (Fig. 3 B) with General Scanning company.
The result shows, at Hg
2+Under the situation that ion exists, chip spot place fluorescence intensity weakens, and works as Hg
2+When concentration is 10nM, compare with the fluorescence intensity of damping fluid group, 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: the chip of investigating probe A and B preparation is to Hg
2+The specificity that detects.
Use 10mM MOPS, 100mM NaNO
3, the different divalent-metal ion of pH 7.2 dilution preparations 10 μ M adds different divalent-metal ion solution in the chip spot place for preparing, 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) and analytical results (Fig. 4 B) with General Scanning company.
The result shows, only at Hg
2+Under the situation about existing, chip spot place fluorescence intensity weakens Hg greatly
2+When being 10 μ M, compare with the fluorescence intensity of damping fluid group, fluorescence intensity has weakened 85%.And when adding 10 other divalent-metal ions of μ M, fluorescence intensity has only small weakening, about 0.9%～2.6%.Illustrate that this chip is to Hg
2+Detection has excellent specificity.
Embodiment 5: be incorporated in Hg in the tap water with the chip detection of probe A and B preparation
2+
Contain 10mM MOPS with the tap water preparation, pH 7.2,100mM NaNO
3Damping fluid, to the mercury storage liquid that wherein mixes certain volume, the concentration that makes mercury ion is 5 μ M, 500nM again.The mercury ion solution for preparing is added 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 uses 10mM MOPS, pH7.2,100mM NaNO
3Damping fluid is washed 3 times, and each 2min dries up.Also use Signal Analysis System Scanarray 3000 analytical resultss (Fig. 5 C) with laser confocal scanning instrument scanned photograph (Fig. 5 A, 5B).
The result shows, with the Hg in the tap water of being incorporated in of this chip detection same concentrations
2+And the Hg in the standard buffer solution
2+The time, the degree that fluorescent signal weakens does not have significant difference, illustrates with this chip to detect the Hg that is incorporated in the tap water
2+
Claims (9)
1. mercury ion fluorescent detection chip is characterized in that described detection chip based on oligonucleotide chain, utilizes Hg
2+With two thymus pyrimidine alkali T covalent attachment of DNA, mediation T-T pairing forms stable T-Hg specifically
2+-T structure.
2. make the method for detection chip as claimed in claim 1, it is characterized in that the rich T oligonucleotide chain of synthetic is fixed on the modified slide; Fluorescently-labeled then complementary strand and the rich T oligonucleotide chain hybridization that is fixed on the slide form duplex structure.
3. by the described making method of claim 2, it is characterized in that described method may further comprise 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 by 1: 1 volume ratio, by with contact Cartesian microarray manufacturing system dot matrix in the slide surface of chemically modified; Point sample finishes, and slide is placed 70% humidity, and 48～72h fixes under the room temperature condition; With 0.2%SDS, deionization washing 2 times, each 2min is then with aldehyde radical confining liquid sealing 15min respectively; Respectively wash 2 times with 0.2%SDS, deionized water successively, each 2min dries 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 that dilution is good is added in the spot place of chip, covered.Chip places 25 ℃ of wet boxes, uses 10mM MOPS behind the 12-16h, 100mM NaNO
3, pH 7.2 washes 5-10min, dries up.
4. by the described making method of claim 3, it is characterized in that the confining liquid described in the step (1) is that 99% ethanol is formed by 0.1g sodium borohydride, 30mlPB S and 10ml mass percent.
5. use the method for chip as claimed in claim 1, it is characterized in that preparing 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 is reacted 10-60min; Use 10mM NaNO
3, pH7.2 washes 3 times, dries up; Chip signal analytical system Scanarray 3000 with General Scanning company scans and analytical resultss then.
6. by the described using method of claim 5, it is characterized in that
1. the Hg that adds 100 μ M, 10 μ M
2+Afterreaction 2min, with respect to the damping fluid control group, fluorescence intensity has descended 63%, 45% respectively; 20min has finished 95% reaction;
2. at Hg
2+Under the situation that ion exists, chip spot place fluorescence intensity weakens, and works as Hg
2+When concentration is 10nM, compare with the fluorescence intensity of damping fluid group, spot place fluorescence intensity weakens 20%, along with Hg
2+Concentration increases, and fluorescent signal weakens gradually.
7. by the described using method of claim 5, it is characterized in that using 10mM MOPS, 100mM NaNO
3, the different divalent-metal ion of pH 7.2 dilution preparations 10 μ M adds different divalent-metal ion solution in the chip spot place for preparing, 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 with General Scanning company; Only at Hg
2+Under the situation about existing, chip spot place fluorescence intensity weakens Hg greatly
2+When being 10 μ M, compare with the fluorescence intensity of damping fluid group, 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.
8. by the described using method of claim 5, it is characterized in that described chip detection Hg
2+Limit of detection be 1nM, the scope of detection is 1nM-10 μ M.
9. by the application of the described fluoroscopic examination chip of claim 1, it is characterized in that described detection chip is used for detecting the Hg that is incorporated in tap water
2+
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