CN103160504A - Nucleic acid fluorescent probe used for rapidly detecting heavy metal lead and detection method thereof - Google Patents
Nucleic acid fluorescent probe used for rapidly detecting heavy metal lead and detection method thereof Download PDFInfo
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- CN103160504A CN103160504A CN2013100661832A CN201310066183A CN103160504A CN 103160504 A CN103160504 A CN 103160504A CN 2013100661832 A CN2013100661832 A CN 2013100661832A CN 201310066183 A CN201310066183 A CN 201310066183A CN 103160504 A CN103160504 A CN 103160504A
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Abstract
The invention discloses a nucleic acid fluorescent probe used for rapidly detecting heavy metal lead and a detection method in the technical field of food safety. By comparing fluorescence intensity of five kinds of added metal ions which are Zn2+, Cd2+, Cu2+, Mg2 and Mn2 in the probe at 260 nm position, a specific result from the probe to lead ions is obtained, and Pb2+ qualitative detection is obtained through changing of the fluorescence intensity; due to the fact that Pb2+ with known concentration is added in the probe and the fluorescence intensity is scanned at the 260 nm position, a detection system between the Pb2+ concentration and a fluorescence intensity, and a linear relationship curve between the Pb2+ concentration and the fluorescence intensity are constructed; and then a fluorescence intensity value of a detected sample is scanned at the 260 nm position, the fluorescence intensity value of the detected sample is brought into the linear relationship curve, and therefore the Pb2+ concentration is obtained and the quantitative detection is achieved. The nucleic acid fluorescent probe used for rapidly detecting the heavy metal lead and the detection method are easy and fast in detection process, provide basis for that whether following accurate detection is needed, and provide technical supports for strengthening controlling and monitoring for environment and food safety in China.
Description
Technical field
What the present invention relates to is a kind of method of food safety technical field, specifically a kind ofly adopt fluorescent energy resonance effect monitoring heavy metal ion to make the structural changes after nucleic acid molecule generation hydrolysis reaction as cofactor and be used for the method for qualitative and quantitative detection metal ion, be applicable to the research of harmful heavy metal rapid detection.
Background technology
The heavy metal lead pollution has the characteristics such as disguise, the poly-property of table and non-reversibility, not only affect the growing of farm crop, output, quality etc., and by food chain, HUMAN HEALTH is caused serious harm, especially nearest for some time, China has in succession occured several and has polluted relevant public health event to heavy metal lead, allows people that Environmental Lead Exposure is paid close attention to more.Plumbous traditional detection technique or waste time and energy, or expensive all is difficult to satisfy the demand of environment and food security supervision, and therefore, foundation heavy metal detection technique easily and fast is very necessary.Method for nucleic acid analysis is used for the many of cause of disease detection, but the research of using it for the environmental pollutant context of detection is also rare.It is used in this research that nucleic acid-8-17 DNAzyme (DNAzyme8-17) is a kind of trans oligonucleotide, the DNA fragmentation of a strand, nonprotein, inorganic matter moiety, has molecular weight little, stability is high, easy synthetic, high with the accuracy of its target RNA pairing, but need specific metal ion could be hydrolyzed as cofactor.Find after deliberation at lead ion it is to make DNAzyme8-17 that the cofactor of hydrolysis occur, it is folding that other metal ion makes the duplex structure of DNAzyme8-17 produce, use fluorophor DNAzyme8-17, react the structural changes situation of nucleic acid according to the change in fluorescence of fluorophor, be used for detecting lead ion.
Find through the retrieval to prior art, (nucleic acid fluorescent probe detects the research of lead ion " nucleic acid fluorescent probe detects the research of lead ion " to hide the flourish Zhou Pei of cloud Xu Lu too, 1 phase in 2010) in, a kind of method is disclosed, 8-17DNAzyme is increased the structural modification that 2 " G-C " base pairs strengthen thermostability, and 1 fluorophor " FAM " and 2 quenching of fluorescence groups " Dabcyl " on mark, be designed to two quencher Pb
2+Fluorescent probe. studied this probe to Cd
2+, Zn
2+, Mg
2+, Cu
2+, Mn
2+, Pb
2+The response of 6 kinds of divalent-metal ions, result show that probe is to Pb
2+Having very strong specificity, is 2.5 * 10 in concentration and probe concentration
-7During mol/L, Pb
2+Concentration is 8.5 * 10
-8～7.5 * 10
-6Linear with the fluorescence intensity of probe in the mol/L scope, detect and be limited to 8.5 * 10
-8Mol/L. this probe can be used for Pb
2+Quantitative and qualitative analysis detect.But the stability of this probe can't reach the existing requirement that detects.
Summary of the invention
The present invention is directed to the prior art above shortcomings, propose a kind of nucleic acid fluorescent probe and detection method thereof of the rapid detection for heavy metal lead, the foranalysis of nucleic acids method in conjunction with luminescent method, is used for the detection of heavy metal lead.Its testing process Simple fast provides foundation for whether needing to carry out follow-up accurate detection, provides technical support for strengthening China's environment and food safety control and supervision.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of nucleic acid fluorescent probe of the rapid detection for heavy metal lead, i.e. DNAzyme8-17 fluorescent probe, its DNA sequence dna is by following:
A) 5'-FAM-GCACTCACTAT
rAGGAAGA (Dabcy1) GATG-3', and
B) 3 '-Dabcy1---CGTGAGTGATA (AAGCTGGCCGAGCC) TCTTCTCTAC-5' forms.
The present invention relates to the preparation method of above-mentioned probe, by synthesizing respectively with the DNA/RNA synthesizer DNAzyme8-17 substrate chain and the enzyme chain that designs the fluorescent mark position, with enzyme: the mixed solution that the volume ratio of substrate: water=1:1:78 is made into is placed in 95 ℃ of annealing 15min of PCR instrument, the native gel electrophoresis separation and purification, downcut double-stranded band, cross ZipTipC after the sodium-chlor dissolving
18The microchromotography post reclaims double-stranded product, obtains double-stranded fluorescent probe.
The present invention relates to a kind of heavy metal lead Quick qualitative detection method based on above-mentioned probe, by adding other five metal ion species Zn in the contrast probe
2+, Cd
2+, Cu
2+, Mg
2+, Mn
2+Fluorescence intensity at the 260nm place obtains described probe for the specificity result of lead ion, and changes by fluorescence intensity and realize Pb
2+Qualitative detection.
The present invention relates to a kind of heavy metal lead fast quantitative measurement method for detecting based on above-mentioned probe, by to adding the Pb of concentration known in probe
2+, and in 260nm place scanning of fluorescent intensity, thereby construct Pb
2+Linear relationship curve between concentration and fluorescence intensity; The fluorescence intensity level that subsequently testing sample is scanned at the 260nm place is brought the linear relationship curve into, thereby obtains Pb
2+Concentration and realize detection by quantitative.
The principle of the invention is: based on DNAzyme8-17 at Pb
2+Booster action under, can be hydrolyzed site generation hydrolysis reaction from it, cause the duplex structure of DNAzyme8-17 to change, and other metal ion can not impel DNAzyme8-17 that hydrolysis reaction occurs as cofactor.
Technique effect
Compared with prior art, the present invention is by improved nucleic acid fluorescent probe, and not only not examined envrionment temperature must be controlled at 4 ℃ with interior constraint, at room temperature just can carry out; And realize detecting the lead ion of lower concentration by two cancellation design.Simple to operate, quick.
Description of drawings
Fig. 1 is the structural representation that the present invention improves probe.
Fig. 2 is fluorescence intensity variation tendency schematic diagram under the different Pb2+ concentration of the present invention.
Fig. 3 is the specificity schematic diagram of embodiment probe.
Fig. 4 is Pb
2+Linear relationship schematic diagram between concentration and fluorescence intensity.
Embodiment
The below elaborates to embodiments of the invention, and the present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The present embodiment comprises the following steps:
1) synthesize respectively with the DNA/RNA synthesizer DNAzyme8-17 substrate chain and the enzyme chain that designs the fluorescent mark position,
With enzyme: the mixed solution that the volume ratio of substrate: water=1:1:78 is made into is placed in 95 ℃ of annealing 15min of PCR instrument, and double-stranded band is downcut in the native gel electrophoresis separation and purification, crosses ZipTipC after the sodium-chlor dissolving
18The microchromotography post reclaims double-stranded product, obtains DNA sequence dna double-stranded fluorescent probe as shown in Figure 1; Then with the ultraviolet spectrophotometer 260nm OD=1.000 of place concentration, double-chain probe 1mL respectively at 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃ six temperature lucifuge placed 3 hours.
2) at room temperature with 5.0 * 10
-7The probe solution 10 μ L of mol/L, ultrapure sterilized water 490 μ L evenly mix, the background fluorescence intensity of scan-probe; Getting respectively subsequently 40 μ L concentration is 5.0 * 10
-6The solution of the cadmium of mol/L, zinc, magnesium, copper, manganese, plumbous 6 kinds of divalent metal salts is added drop-wise to 5.0 * 10
-7In the probe solution 10 μ L of mol/L, the ultrapure sterilized water 450 mixed uniformly probe solutions of μ L, scanning of fluorescent intensity; According to adding the later fluorescence intensity of different metal ions to change in probe, determine that probe is to Pb
2+Specificity, thereby realize Pb
2+Fast qualitative detect.
3) starting point concentration with fluorescent probe is designed to 2.5 * 10
-7Mol/L, 1.25 * 10
-7Two concentration gradients of mol/L, Pb
2+Starting point concentration be designed to 1.0 * 10
-5Mol/L, 7.5 * 10
-6Mol/L, 5.0 * 10
-6Mol/L, 2.5 * 10
-6Mol/L, 1.25 * 10
-6Mol/L, 1.0 * 10
-6Mol/L, 5.0 * 10
-7Mol/L, 2.5 * 10
-7Mol/L, 1.25 * 10
-7Mol/L, 1.0 * 10
-7Mol/L10 concentration gradient drips respectively the Pb of 40 each concentration of μ L in 10 μ L probes and the 450 μ L ultrapure water mixed solutions under room temperature
2+Solution detects fluorescence intensity corresponding to each concentration sample, and with Pb
2+μ M concentration be X-coordinate x, the fluorescence intensity that the 260nm place scans is ordinate zou y, the linear relationship curve that obtains as shown in Figure 4 is: the following curve of 1 μ M is y=118.11x+57.268; The above curve of 1 μ M is y=96.68x+82.37; Realize that thus probe is to Pb
2+Detection by quantitative.
4) add Pb(NO in tap water
3)
2Standardized solution, being mixed with concentration is 1.5 * 10
-7Mol/L, 7.5 * 10
-7Mol/L, 1.5 * 10
-6The sample solution of mol/L.The interference of impurity to measurement result in the tap water, the sample solution of preparation is first removed impurity with the filtering with microporous membrane of 0.45 μ m and 0.22 μ m.Then use 0.25 μ M probe in detecting, calculate corresponding concentration according to typical curve, by the Pb that calculates
2+Concentration mean value draws the interpolation rate of recovery divided by adding concentration, thus the accuracy of checking this method.Add the rate of recovery with flame atom spectrum measuring simultaneously, by with the contrast of conventional sense method, illustrate that this method is used for detecting Pb
2+The feasibility of concentration.It is as follows that Fluorescent Nucleic Acid Probe detects the interpolation rate of recovery plumbous in the interpolation rate of recovery plumbous in tap water and FAAS detection tap water:
Probe in detecting is the comparison of FAAS detected result as a result
Claims (7)
1. a nucleic acid fluorescent probe that is used for the rapid detection of heavy metal lead, is characterized in that, its DNA sequence dna is by following:
A) 5'-FAM-GCACTCACTAT
rAGGAAGA (Dabcy1) GATG-3', and
B) 3'-Dabcy1---CGTGAGTGATA (AAGCTGGCCGAGCC) TCTTCTCTAC-5' forms.
2. method for preparing the described probe of claim 1, it is characterized in that, synthesize respectively with the DNA/RNA synthesizer DNAzyme8-17 substrate chain and the enzyme chain that designs the fluorescent mark position, with enzyme: the mixed solution that the volume ratio of substrate: water=1:1:78 is made into is placed in 95 ℃ of annealing 15min of PCR instrument, the native gel electrophoresis separation and purification, downcut double-stranded band, cross ZipTipC after the sodium-chlor dissolving
18The microchromotography post reclaims double-stranded product, obtains double-stranded fluorescent probe.
3. the heavy metal lead Quick qualitative detection method based on the described probe of claim 1, add other five metal ion species Zn by contrasting in probe
2+, Cd
2+, Cu
2+, Mg
2+, Mn
2+Fluorescence intensity at the 260nm place obtains described probe for the specificity result of lead ion, and changes by fluorescence intensity and realize Pb
2+Qualitative detection.
4. method according to claim 3, is characterized in that, described qualitative detection refers to: at room temperature with 5.0 * 10
-7The probe solution 10 μ L of mol/L, ultrapure sterilized water 490 μ L evenly mix, the background fluorescence intensity of scan-probe; Getting respectively subsequently 40 μ L concentration is 5.0 * 10
-6The solution of the cadmium of mol/L, zinc, magnesium, copper, manganese, plumbous 6 kinds of divalent metal salts is added drop-wise to 5.0 * 10
-7In the probe solution 10 μ L of mol/L, the ultrapure sterilized water 450 mixed uniformly probe solutions of μ L, scanning of fluorescent intensity; According to adding the later fluorescence intensity of different metal ions to change in probe, determine that probe is to Pb
2+Specificity, thereby realize Pb
2+Fast qualitative detect.
5. heavy metal lead fast quantitative measurement method for detecting based on the described probe of claim 1 is by to adding the Pb of concentration known in probe
2+, and in 260nm place scanning of fluorescent intensity, thereby construct Pb
2+Detection system and linear relationship curve thereof between concentration and fluorescence intensity; The fluorescence intensity level that subsequently testing sample is scanned at the 260nm place is brought the linear relationship curve into, thereby obtains Pb
2+Concentration and realize detection by quantitative.
6. method according to claim 5, is characterized in that, the specific Pb that contain different concns of described probe to different concns
2+The detection system of ion refers to: the starting point concentration of fluorescent probe is designed to 2.5 * 10
-7Mol/L, 1.25 * 10
-7Two concentration gradients of mol/L, Pb
2+Starting point concentration be designed to 1.0 * 10
-5Mol/L, 7.5 * 10
-6Mol/L, 5.0 * 10
-6Mol/L, 2.5 * 10
-6Mol/L, 1.25 * 10
-6Mol/L, 1.0 * 10
-6Mol/L, 5.0 * 10
-7Mol/L, 2.5 * 10
-7Mol/L, 1.25 * 10
-7Mol/L, 1.0 * 10
-7Mol/L10 concentration gradient drips respectively the Pb of 40 each concentration of μ L in 10 μ L probes and the 450 μ L ultrapure water mixed solutions under room temperature
2+Solution.
7. method according to claim 6, is characterized in that, described linear relationship curve refers to: with Pb
2+μ M concentration be X-coordinate x, the fluorescence intensity that the 260nm place scans is ordinate zou y, obtaining the following curve of 1 μ M is y=118.11x+57.268; The above curve of 1 μ M is y=96.68x+82.37.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849247A (en) * | 2015-04-15 | 2015-08-19 | 刘骁勇 | Method for detecting heavy metal ion based on DNA and heavy metal ion mismatch principle |
| CN105548109A (en) * | 2015-12-22 | 2016-05-04 | 北京农业质量标准与检测技术研究中心 | A fluorescence detecting system for heavy metal cadmium and a fluorescence detecting method |
| CN106153587A (en) * | 2016-06-21 | 2016-11-23 | 何文 | A kind of detection method of soil activation state cadmium |
| CN106841130A (en) * | 2016-12-28 | 2017-06-13 | 成都理工大学 | A kind of method of uranyl ion content in unmarked fluoroscopic examination water sample |
| CN107192711A (en) * | 2017-06-01 | 2017-09-22 | 扬州大学 | A kind of silver sol modified by fluorescence molecule detects Cd2+、Pb2+The method of ion |
| CN107764785A (en) * | 2017-09-18 | 2018-03-06 | 鲁东大学 | A kind of fluorescence detection method of lead ion |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849247A (en) * | 2015-04-15 | 2015-08-19 | 刘骁勇 | Method for detecting heavy metal ion based on DNA and heavy metal ion mismatch principle |
| CN105548109A (en) * | 2015-12-22 | 2016-05-04 | 北京农业质量标准与检测技术研究中心 | A fluorescence detecting system for heavy metal cadmium and a fluorescence detecting method |
| CN106153587A (en) * | 2016-06-21 | 2016-11-23 | 何文 | A kind of detection method of soil activation state cadmium |
| CN106841130A (en) * | 2016-12-28 | 2017-06-13 | 成都理工大学 | A kind of method of uranyl ion content in unmarked fluoroscopic examination water sample |
| CN107192711A (en) * | 2017-06-01 | 2017-09-22 | 扬州大学 | A kind of silver sol modified by fluorescence molecule detects Cd2+、Pb2+The method of ion |
| CN107764785A (en) * | 2017-09-18 | 2018-03-06 | 鲁东大学 | A kind of fluorescence detection method of lead ion |
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Application publication date: 20130619 |