CN102586429A - Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration - Google Patents
Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration Download PDFInfo
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- CN102586429A CN102586429A CN2012100183342A CN201210018334A CN102586429A CN 102586429 A CN102586429 A CN 102586429A CN 2012100183342 A CN2012100183342 A CN 2012100183342A CN 201210018334 A CN201210018334 A CN 201210018334A CN 102586429 A CN102586429 A CN 102586429A
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Abstract
The invention relates to the field of detection and analysis of lead ion concentration and discloses a lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe. The lead ion fluorescent DNA probe comprises double strands formed by hybridizing 17E deoxyribozyme with a substrate strand, wherein the tail end of the substrate strand is modified by FAM (Carboxyfluorescein). The invention also discloses a fluorescent determination method for the lead ion concentration by using the probe. The fluorescent detection of the lead ion concentration of a sample to be detected is carried out by taking the lead ion fluorescent DNA probe as an identification element of a lead ion, the FAM for modifying the tail end of the substrate strand as a fluorescent signal molecule and a colloidal gold nanoparticle as a fluorescence quencher according to the fluorescent signal difference generated when the FAM and the lead ion fluorescent DNA probe are subjected to single-double-strand combination. The fluorescent determination method disclosed by the invention has the advantages of high sensitivity, favorable selectivity for the lead ion, low cost, convenience in operation, quickness in detection and the like.
Description
Technical field
The present invention relates to the check and analysis field of plumbum ion concentration, relate to a kind of lead ion fluorescent DNA probe particularly and utilize this probe to carry out the fluorescence analysis of plumbum ion concentration.
Background technology
Lead is the environmental pollutant wide, that the property accumulated is arranged that distribute.Lead is the heavy metal element of stable in properties, and it is very harmful to human body, especially serious harm children's health.Along with developing rapidly of industry and other industry, people make Lead contamination expand to daily life environment such as water, atmosphere, food, medicine from occupational environment to a large amount of uses of lead.
In environment measuring, port rapid screening, consumer's goods safety, wastewater treatment and commercial run, to sensitivity, the quick screening and quantitatively very important accurately of lead.At present, the main measuring method for testing for trace lead concentrates on atom extinction spectrum method (Hong Ying, Chen Guosong, Zhang Hongman etc., " Guangdong trace element science " 2004,11 (4) 62 ~ 66 in the assay laboratory; Permitted wealth and admired " Chinese public health ", 2005,21 (3), 361), ICP-AES method (Chen Wenxin; Fang Yiwen, spectrographic laboratory, 2003 (20) 4,495 ~ 497), ICP-MS method (Chen Hong, Zhang Suzhen; Wu Xiaojun, " Anhui agricultural sciences ", 2010,38 (23), 12509 ~ 12513).At 10 μ g/kg ~ 1mg/kg, and these methods all need main equipment and burning gas to these methods to the detection limit of lead, and cost is higher, is difficult for microminiaturized.Dithizone is measured lead and is had better stability and highly sensitive (measurement range is 10 ~ 400mg/kg); Set up sophisticated national standard method (GB7470-87 " the mensuration dithizone spectrophotometry that water quality is plumbous "); But owing to relate to poisonous organic solvent in the process of the test, this method is unfavorable for environment protection.
On the other hand, the fast development of nanometer biotechnology is that the detection technique change provides new direction.Along with can occurring one after another with Metal Ion Selective Electrode property bonded ionophore, people have been developed the detection method of contents of many kinds of heavy metal ion on this basis.DNAzyme (DNAzyme) is a kind of single strand dna that makes the RNA hydrolytic action that has, and is called catalytic DNA again, and it can obtain through in-vitro screening (SELEX) technology.The activity of part DNAzyme and some metals ion are closely related, and DNAzyme has and is easy to synthetic and modification, high stability and advantages of environment protection (Lu Y.Chem.Eur.J. [J], 2002,8 (20): 4588 ~ 4596; Li J., Zheng W., Kwon A.H., et a1..Nucleic Acids Res. [J], 2000,28 (2): 48l-488; Xiao Y., Rowe A.A., Plaxco K.W..J.Am.Chem.Soc [J], 2007,129 (2): 262-263).Wherein Yi Lu group utilizes Pb
2+Responsive 17E DNAzyme (DNAzyme) molecule has been developed a series of detection Pb
2+Fluorescence, colorimetric sensor (Li J. et. al, J. Am. Chem. Soc., 2000,122:10466-10467; Liu J. et.al., Anal. Chem., 2003,75:6666-6667).Mostly these transmitters are to utilize Pb at 17E DNAzyme and substrate chain marked fluorescent signal molecule and quencher molecule
2+The fluorescent signal that cutting back DNAzyme conformational change causes changes next quantitative Pb
2+Concentration.These transmitters are owing to the double-tagging that need carry out the DNA chain, even three marks, and cost is higher.Simultaneously, because at room temperature, the substrate chain after the cutting possibly hybridized with the 17E DNAzyme again, and in order to obtain lower fluorescence background, the test operation temperature must be strict controlled in below 4 ℃, and experiment condition is strict.Therefore, existing 17E DNAzyme and the substrate chain multiple labeling probe in detecting Pb of utilizing
2+Method exist deficiencies such as many, the difficult popularizations of operation difficult point.
The invention solves prior art utilizes the 17E DNAzyme to detect Pb
2+Method in requirement for experiment condition high, need dna probe shortcoming such as mark, complicated operation repeatedly; A kind of single mark, high-sensitive NEW Pb ion fluorescence dna probe of being made up of 17E DNAzyme and the fluorescein-labeled substrate chain of FAM proposed; With the quencher of nanometer gold size, carry out the fluoroscopic examination of plumbum ion concentration as the FAM resorcinolphthalein.The present invention has not only kept highly sensitive, the high specific of 17E DNAzyme, also greatly reduces the detection cost of lead ion.
Summary of the invention
The present invention proposes a kind of lead ion fluorescent DNA probe, comprise the two strands that the hybridization of 17E DNAzyme and substrate chain is formed, the 5' end of said substrate chain is modified with the FAM resorcinolphthalein;
Wherein, the sequence of said 17E DNAzyme is:
5'-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT?-3'；
The sequence of said substrate chain is:
5'-FAM-ACTCACTAT
rA GGAAGAGATG-3'；
Said FAM resorcinolphthalein is shown in following structural formula (II).
Lead ion fluorescent DNA probe among the present invention is shown in following structural formula (I).
The invention allows for and a kind ofly utilize said lead ion fluorescent DNA probe to carry out the fluorescence analysis of plumbum ion concentration; Hybridize the recognition component of the lead ion fluorescent DNA probe of composition with 17E DNAzyme and substrate chain as lead ion; With the end modified FAM resorcinolphthalein of substrate chain 5' as the fluorescent signal molecule; With the nanometer gold size as fluorescence quencher; Utilize the fluorescent signal difference when FAM resorcinolphthalein and said lead ion fluorescent DNA probe list are double-stranded to be combined, the plumbum ion concentration in the testing sample is carried out fluoroscopic examination.
Wherein, said lead ion fluorescent DNA probe is hybridized under 95 ℃, the annealing conditions of 15min by said 17E DNAzyme and substrate chain and is formed.
Wherein, under the situation that lead ion exists, open between the 9th and the tenth Nucleotide of the substrate chain in the said lead ion fluorescent DNA probe, said lead ion fluorescent DNA probe unwinds.
Wherein, the quencher of said FAM resorcinolphthalein is closed in substrate chain and the said nanometer gold gluing in the lead ion fluorescent DNA probe after said the unwinding.
Wherein, said fluoroscopic examination is 494nm in excitation wavelength, and emission wavelength 520nm carries out at the place.
Wherein, detecting of said plumbum ion concentration is limited to 2n mol/L.
The present invention proposes a kind of NEW Pb ion fluorescence dna probe and utilize this probe and nanometer gold size fluorescence quencher carries out the plumbum ion concentration method for measuring.Said lead ion fluorescent DNA probe is made up of the dna double chain, and wherein a strand is 17E DNAzyme (17E DNAzyme) chain, and another is and its complementary substrate chain.The 17E DNAzyme is that sequence is the dna fragmentation of 5'-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3', at Pb
2+Existence under can to make sequence be 5'-FAM-ACTCACTAT
RA The phosphodiester bond hydrolysis of the substrate chain of GGAAGAGATG-3', thus reach the effect of cutting the substrate chain.Pb
2+When existing, the 17E DNAzyme is activated and cuts the substrate chain, causes the dna double chain to be opened, and the substrate chain under the cutting is wound to nanometer gold size surface, and quenching of fluorescence takes place the fluorescent signal molecule of substrate chain marked---6-Fluoresceincarboxylic acid (FAM resorcinolphthalein).Utilize the difference of fluorescence signal intensity when said resorcinolphthalein FAM and said probe list are double-stranded to be combined, realize fluoroscopic examination plumbum ion concentration in the testing sample.
The present invention utilizes in the method for dna probe fluorometric assay plumbum ion concentration; In two strands of said lead ion fluorescent DNA probe, add Tris-HCl damping fluid (tris-HCI buffer; Concentration is 50mmol/L); And with 50mmol/L NaCl adjusting ionic strength, gained solution is adjusted to pH7.0 with NaOH solution, and places 95 ℃ of annealing of PCR appearance 15min; Obtain hybridizing the probe of forming, put into 4 ℃ in refrigerator and keep in Dark Place subsequent use more than the 3h by 17E DNAzyme and substrate chain.Said probe solution concentration is 1 * 10
-4About mol/L.
The present invention utilizes lead ion fluorescent DNA fluorescence probe to measure in the method for plumbum ion concentration, and said nanometer gold size disperses with Tris-HCl solution (pH7.0), and working concentration is 10nmol/L.
The present invention utilizes lead ion fluorescent DNA fluorescence probe to measure in the method for plumbum ion concentration, and said testing sample filters before detection, and regulates acidity to neutral with NaOH solution.
The present invention utilizes lead ion fluorescent DNA fluorescence probe to measure in the method for plumbum ion concentration, and said fluoroscopic examination is 494nm in excitation wavelength, and emission wavelength 520nm carries out at the place.
The present invention utilizes lead ion fluorescent DNA fluorescence probe to measure in the method for plumbum ion concentration, and detecting of said plumbum ion concentration is limited to 2n mol/L.
The present invention utilizes lead ion fluorescent DNA fluorescence probe to measure in the method for plumbum ion concentration employed double chain DNA probe structure shown in structural formula (I).The underscore place in the substrate chain wherein promptly 5 ' has held that the position is a cleavage site between the 9th and the tenth Nucleotide.
When no lead ion existed, the FAM group of being modified on the substrate chain sent hyperfluorescence.When lead ion and the coexistence of said probe solution, said 17E DNAzyme is activated by lead ion, and the substrate chain is cut from cleavage site (between the 9th and the tenth Nucleotide of 5 ' end).After this substrate chain is from cutting the site hydrolysis, and the single stranded DNA fragment that has the FAM base group modification is released in the solution.In probe solution, add the nanometer gold size this moment, the single stranded DNA fragment of hydrolysis will be adsorbed on nanometer gold size surface owing to electrostatic interaction.After the FAM group was pressed close to nanometer gold size surface, the FAM fluorescent signal was by quencher.The concentration of the lead ion in fluorescence intensity and the solution is relevant, through setting up the typical curve of fluorescent signal molecule intensity and plumbum ion concentration linear relationship, and detects the fluorescence signal intensity of testing sample, can calculate the plumbum ion concentration in the testing sample.
Testing sample is carried out in the mensuration of plumbum ion concentration in the inventive method; At first with spectrophotofluorometer in the fluorescence intensity of 510～650nm wavelength region interscan probe solution at the 520nm place; The testing sample solution that will contain lead ion then is added drop-wise in the Tris-HCl damping fluid of lead ion fluorescent DNA probe of above-mentioned preparation, shakes up; Add certain amount of nano gold size solution again, shake up, the fluorescence intensity at record 520nm place is calculated the plumbum ion concentration in the solution to be measured according to the variation of fluorescence intensity.
The present invention utilizes 17E DNAzyme fluorescence probe to measure the method for plumbum ion concentration, and the mensuration of plumbum ion concentration is limited to 2n mol/L, can satisfy the requirement of limiting the quantity of international and domestic relevant criterion fully.The present invention has the following advantages: the inventive method adopts eco-friendly DNA as detection probes, and specificity is good, and can not cause second environmental pollution; Adopt lead ion specificity 17E DNAzyme, highly sensitive, selectivity good; Simultaneously; Because the dna probe in the detection method of the present invention has been removed in the art methods this operation of many fluorophors mark comparatively complicacy and the high step of cost from; Only need single mark to test, probe temperature is a normal temperature simultaneously, need not controlled temperature to avoid high fluorescence background.The present invention has advantage easy and simple to handle, with low cost.
Description of drawings
Fig. 1 is the principle of work synoptic diagram of the inventive method.
Fig. 2 is the gel chromatography electrophorogram that adds the lead ion front and back in the inventive method.
Fig. 3 is the lead ion solution that in dna probe solution, adds capacity concentration in the inventive method, add concentration again and be 10nmol/L gold size solution (curve from top to bottom, gold size solution add-on is respectively 0; 5; 10,15,20 μ L) the fluorescence spectrum figure of back acquisition.
Fig. 4 is that solution is fluorescent signal figure at the 520nm place after in probe solution, adding the different concns lead ion in the inventive method.
Fig. 5 is the canonical plotting of lead at different concentrations pair ion quenching of fluorescence rate in the inventive method.
Embodiment
In conjunction with following specific embodiment and accompanying drawing, the present invention is done further detailed description, protection content of the present invention is not limited to following examples.Under spirit that does not deviate from inventive concept and scope, variation and advantage that those skilled in the art can expect all are included among the present invention, and are protection domain with the appended claims.The process of embodiment of the present invention, condition, reagent, experimental technique etc. except that the following content of mentioning specially, are the universal knowledege and the common practise of this area, and the present invention does not have special limiting content.
Lead ion fluorescent DNA probe of the present invention comprises the two strands that the hybridization of 17E DNAzyme and substrate chain is formed, shown in the following structural formula (I).
The sequence of 17E DNAzyme and substrate chain is following:
17E DNAzyme: 5'-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3';
Substrate chain: 5'-FAM-ACTCACTAT
RA GGAAGAGATG-3'.
Wherein, substrate chain 5' end is modified with the FAM resorcinolphthalein; Shown in the following structural formula of FAM resorcinolphthalein (II).
The inventive method is hybridized the recognition component of the lead ion fluorescent DNA probe of composition as lead ion with 17E DNAzyme and substrate chain; With the end modified FAM resorcinolphthalein of substrate chain 5' as the fluorescent signal molecule; With the nanometer gold size as fluorescence quencher; Utilize the fluorescent signal difference when FAM resorcinolphthalein and said lead ion fluorescent DNA probe list are double-stranded to be combined, the plumbum ion concentration in the testing sample is carried out fluoroscopic examination, its principle is as shown in Figure 1.When lead ion did not exist, (pH7.0 50mMNaCl) existed with double chain form in the buffered soln lead ion fluorescent DNA probe of the present invention, and the FAM group of modifying on the substrate chain sends hyperfluorescence at 50mmol/L Tris-HCl; When having lead ion in the solution; The 17E DNAzyme is activated; The substrate chain is cut at the cleavage site place between the 9th and the tenth Nucleotide of 5 ' end, and the single stranded DNA fragment that has the modification of FAM fluorescein base group is released in the solution, and because electrostatic interaction is wound to nanometer gold size surface; The FAM group causes quenching of fluorescence owing to press close to the nanometer gold size at this moment.
Embodiment 1:
Adopt conventional gel electrophoresis experimental technique, the feasibility of lead ion fluorescent DNA probe test lead ion method of the present invention is assessed, experimental result is shown in the gel chromatography electrophorogram of Fig. 2.It is the band of lead ion fluorescent DNA probe that a left side is risen in first, second swimming lane, the disappearance that after lead ion adds, shoals, and the enzyme in the 3rd to six swimming lane after the new electrophoretic band that the position occurred representative adds lead ion is cut product.The adding of lead ion is the sole cause that causes that band shown in Figure 2 changes, and therefore, lead ion fluorescent DNA probe of the present invention and method are applicable to the detection of lead ion.
For confirming the optimum add-on of nanometer gold size, in many parts of dna probe solution, add the lead ion solution of capacity concentration, again with 0; 5,10,15; It is 10nmol/L gold size solution that the add-on of 20 μ L adds concentration respectively, and the scanning back obtains fluorescence spectrum figure, and is as shown in Figure 3.This tests used probe solution concentration is 1.06 * 10
-6Mol/L, Pb
2+Concentration is 5.0 * 10
-9Mol/L, medium are the Tris-HCl solution of pH7.0.Along with the adding gradually (0-20 μ L) of gold size, the fluorescent signal that the FAM group is sent progressively reduces, and quencher efficient is more and more obvious.When the gold size add-on was 15 ~ 20 μ L, quencher efficient began near platform.Under the prerequisite that guarantees the quencher rate, in order to prevent false signal, thereby reduce the gold size add-on as far as possible, confirm that finally the gold size add-on is 20 μ L.
After above confirmatory experiment and optimization experiment, utilize the lead ion fluorescent DNA probe that contains 17E DNAzyme sequence, use the nanometer gold size as quencher, measure the concentration of lead ion in the testing sample.As the lead ion recognition component, FAM is the fluorescent signal molecule with the 6-Fluoresceincarboxylic acid with 17E DNAzyme sequence, utilize the fluorescent signal difference of said probe when lead ion exists and do not exist to testing sample in plumbum ion concentration detect.Specifically may further comprise the steps:
(1) preparation of probe solution: 17E DNAzyme chain mixed being placed in the PCR appearance 95 ℃ of annealing 15min with substrate chain solution, (concentration is 1 * 10 to obtain probe solution
-4About mol/L).Putting into 4 ℃ in refrigerator keeps in Dark Place subsequent use more than the 3h; Article two, single stranded DNA is synthetic, and the FAM mark is after the HPLC purifying, available from be precious biology (TaKaRa, Dalian).
(2) foundation of typical curve:
2.1 in the fragrant doffers' pipe of the graduated dust of 7 2.0mL, (ultimate density is 1.06 * 10 to add the probe solution that step (1) prepares respectively
-6Mol/L).It is mixed with 1.5 mL 50mM Tris-HCl damping fluids (tris-HCI buffer), use 50mM NaNO
3Solution is regulated ionic strength, and regulates the Tris-HCl damping fluid to pH7.0 with 0.1mol/L NaOH solution.(concentration is 2,5,10,50,100 to prepare a series of lead ion standardized solution; 500,1000,2000nmol/L), add probe solution and at 20～25 ℃ of following mixings, cultivate 2min after; Add 20 μ L, the gold size solution of 10nmol/L, mixing leaves standstill 2min. once more; Wherein, and nanometer gold sol solution reference literature (Zhang Conghui, Lanzhou-Xinjiang wise, " chemical reduction method prepares the research of nano gold sol method "; " rare metal ", 2006,30 (4); 549 ~ 551) process, use 50mM, the Tris-HCl damping fluid dissolving back of pH7.0 stores.
2.2 the method according to step 2.1 does not add lead ion, preparation blank solution;
2.3 the solution with 2.1 and 2.2 preparations places quartz colorimetric utensil respectively, uses spectrophotofluorometer, slit 5nm, and excitation wavelength 494nm in the interscan of 510～650nm wavelength region, writes down the fluorescence intensity (I at 520nm place respectively
520nm), obtain fluorescent signal figure as shown in Figure 4, and record blank value (I
b), calculate fluorescence intensity Δ I
520nm=I
520nm-I
bAnd calculate quenching of fluorescence rate (Quenching Yeild) (%)=(Δ I
The 520nm probe-Δ I
The 520nm sample)/Δ I
The 520nm probe* 100;
2.4 be respectively 0.01 * 10 with concentration
-6Mol/L, 0.1 * 10
-6Mol/L, 1.0 * 10
-6Mol/L and 10 * 10
-6The lead ion of mol/L is mapped to the quenching of fluorescence rate, and the drawing standard curve is as shown in Figure 5;
(3) pre-treatment of testing sample:, filter the back and use 1mol/L NaOH to regulate acidity to neutral for liquid sample; For solid sample, use nitric acid-hydrogen peroxide (7mL+1.5mL) micro-wave digestion, catch up with acid and filtration back to use 1mol/LNaOH to regulate acidity to neutrality.Get a certain amount of gained sample solution replacement lead ion standardized solution, Δ I is asked in 2.1 and 2.2 operations set by step
Sample
(4) test testing sample: the Δ I that records according to testing sample
Sample, calculate the quenching of fluorescence rate, look into typical curve, try to achieve the lead content of testing sample.
The fluoroscopic examination of testing sample is to carry out the fluoroscopic examination of lead ion at 50mM Tris-HCl (pH7.0,50mM NaCl) damping fluid.
The mensuration of plumbum ion concentration is limited to 2n mol/L in the inventive method, can satisfy the requirement of limiting the quantity of international and domestic relevant criterion fully.
Embodiment 2: dissoluble lead ion determination in the toy sample
Reference standard: EN71-3:1994+A1:2001
Situation when present embodiment is depicted as testing sample and is toy is all tested all the touched parts on the toy sample.
One, sample preparation
For the toy of types such as weaving face fabric, plastics or rubber, the fritter that material is cut into 5mm * 5mm with scissors or pliers is as testing sample.
For toy, will scrape with clean blade and to get part as testing sample with paint, printing ink or similar coatings.
For the toy that is liquid paint or printing ink, get part and be applied on the clean sheet glass after the seasoning at room temperature, scrape as testing sample.Can not be at room temperature dry like testing sample, then press use temperature in drying in oven, scrape the acquisition testing sample.
All powdery testing samples must use the aperture to sieve as the stainless steel metal of 0.5mm and sieve.
For the toy that contains grease, oils, wax or analogous material, sample is wrapped in the toughened filter paper, use the normal heptane will contained grease through dissolution extraction and after analogous components removes, the acquisition testing sample.
Two, the pre-treatment before the acid extraction of testing sample and the sample detection
With load weighted testing sample be equivalent to 50 times of testing sample quality, temperature is 37 ± 2 ℃, concentration is the combined of 0.07 mol/L.Mixture is shaken 1min, and check its acidity.If the pH value is greater than 1.5, shake mixture, and meanwhile dropwise add 2 mol/L hydrochloric acid up to the pH value between 1.0 ~ 1.5.Make the mixture lucifuge, stir 1h down, under 37 ± 2 ℃, leave standstill 1h again at 37 ± 2 ℃.
Use filter membrane (aperture is 0.45 μ m) to filter, gained filtrating is measured the fluorescence signal intensity of testing sample with 1 mol/L NaOH neutralization in the Tris-HCL damping fluid.
Three, measure
In containing the 1.5mL Tris-HCl buffered soln of lead ion fluorescent DNA probe, add the testing sample of 20 μ L, mixing is also cultivated 2min; Add 20 μ L nanometer gold sol solutions again, detect in the scanning of 510-650nm scope with spectrophotofluorometer behind the 2min.
After detect, adding testing sample, the fluorescence intensity of solution at the 520nm place is 150, and probe solution is 243 in the fluorescence intensity at 520nm place, the typical curve from Fig. 5, and in conjunction with dilution factor, the content that calculates lead ion in the sample solution is 4.6 * 10
-6Mol/L.
Embodiment 3: total lead content is measured in the plastic sample
One, the pre-treatment before the sample detection
Plastic sample is pulverized, taken by weighing 0.5g, be accurate to 0.1mg to counteracting tank, add 7mLHNO
3And 1.5mLH
2O
2With the counteracting tank sealing, place microwave digestion system to clear up.Heating schedule (reference instrument, Anton Paar Microwave 3000) as shown in table 1 below.
The gained digestion solution catches up with acid to the solution 1mL in low-grade fever on the hot plate, adds ultrapure water then, is settled to about 50mL.Measure solution acidity, greater than 7, then use 1 mol/LNaOH to be neutralized to pH7.0, in the Tris-HCl damping fluid, carry out fluorometric assay like the pH value.
Two, measure
In the 50mmol/L Tris-HCl that contains lead ion fluorescent DNA probe (pH7.0,50mM NaCl) buffered soln, add the testing sample of 20 μ L, mixing is also cultivated 2min; Add 20 μ L nanometer gold sol solutions again, detect in the scanning of 510-650nm scope with spectrophotofluorometer behind the 2min.
After detect, adding testing sample, the fluorescence intensity of solution at the 520nm place is 80, and probe solution is 250 in the fluorescence intensity at 520nm place, the typical curve from Fig. 5, and in conjunction with dilution factor, the content that calculates lead ion in the Specimen eliminating liquid is 1.8 * 10
-4Mol/L.
Claims (8)
1. a lead ion fluorescent DNA probe is characterized in that, said probe comprises the two strands that 17E DNAzyme and the hybridization of substrate chain are formed, and the 5' end of said substrate chain is modified with the FAM resorcinolphthalein;
Wherein, the sequence of said 17E DNAzyme is:
5'-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT?-3'；
The sequence of said substrate chain is:
5'-FAM-ACTCACTAT
rA GGAAGAGATG-3'；
Said FAM resorcinolphthalein is shown in following structural formula (II).
2. lead ion fluorescent DNA probe as claimed in claim 1 is characterized in that said probe is shown in following structural formula (I).
3. fluorescence analysis that utilizes lead ion fluorescent DNA probe according to claim 1 to carry out plumbum ion concentration; It is characterized in that; Hybridize the recognition component of the lead ion fluorescent DNA probe of composition with 17E DNAzyme and substrate chain as lead ion; With the end modified FAM resorcinolphthalein of substrate chain 5' as the fluorescent signal molecule; As fluorescence quencher, utilize the fluorescent signal difference when FAM resorcinolphthalein and said lead ion fluorescent DNA probe list are double-stranded to be combined with the nanometer gold size, the plumbum ion concentration in the testing sample is carried out fluoroscopic examination.
4. method as claimed in claim 3 is characterized in that, said lead ion fluorescent DNA probe is hybridized under 95 ℃, the annealing conditions of 15min by said 17E DNAzyme and substrate chain and formed.
5. method as claimed in claim 3 is characterized in that, under the situation that lead ion exists, opens between the 9th and the tenth Nucleotide of the substrate chain in the said lead ion fluorescent DNA probe, and said lead ion fluorescent DNA probe unwinds.
6. method as claimed in claim 5 is characterized in that, the quencher of said FAM resorcinolphthalein is closed in substrate chain and said nanometer gold gluing in the lead ion fluorescent DNA probe after said the unwinding.
7. method as claimed in claim 3 is characterized in that, said fluoroscopic examination is 494nm in excitation wavelength, and emission wavelength 520nm carries out at the place.
8. method as claimed in claim 3 is characterized in that, detecting of said plumbum ion concentration is limited to 2n mol/L.
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| CN103901033A (en) * | 2014-04-23 | 2014-07-02 | 常熟理工学院 | Method for detecting lead ion concentration in sample |
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| CN108318479A (en) * | 2017-12-23 | 2018-07-24 | 张睿 | A kind of lead ion detection method of high sensitivity |
| CN111705113A (en) * | 2020-06-24 | 2020-09-25 | 上海海洋大学 | Functional nucleic acid fluorescence sensor and application thereof in lead ion detection |
| CN111705113B (en) * | 2020-06-24 | 2023-12-05 | 上海海洋大学 | Functional nucleic acid fluorescence sensor and application thereof in lead ion detection |
| CN114058678A (en) * | 2021-11-01 | 2022-02-18 | 大连理工大学 | Application of deoxyribozyme probe in high-throughput sensing of escherichia coli drug-resistant phenotype |
| CN114414337A (en) * | 2022-01-21 | 2022-04-29 | 山东大学 | DNA gel-based lead ion rapid detection method |
| CN114414337B (en) * | 2022-01-21 | 2023-11-10 | 山东大学 | Lead ion rapid detection method based on DNA gel |
| CN114703256A (en) * | 2022-04-18 | 2022-07-05 | 中国农业科学院农业资源与农业区划研究所 | Detecting plant Pb2+DNAzyme fluorescent sensor |
| CN114703256B (en) * | 2022-04-18 | 2023-08-11 | 中国农业科学院农业资源与农业区划研究所 | Detection of plant Pb 2+ DNAzyme fluorescence sensor of (C) |
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