CN101851677A - Nucleic acid nano-gold biosensor used for detecting Hg2<+> - Google Patents
Nucleic acid nano-gold biosensor used for detecting Hg2<+> Download PDFInfo
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- CN101851677A CN101851677A CN 201010169776 CN201010169776A CN101851677A CN 101851677 A CN101851677 A CN 101851677A CN 201010169776 CN201010169776 CN 201010169776 CN 201010169776 A CN201010169776 A CN 201010169776A CN 101851677 A CN101851677 A CN 101851677A
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Abstract
The invention relates to a nucleic acid nano-gold biosensor used for detecting Hg2<+>, which comprises a detection test paper strip, a detection nucleic acid reagent and sampling buffer solution, wherein the detection test paper strip consists of a sample pad, a glass fiber, a nitrocellulose membrane and absorbent paper which are fixed on a rubber plate in turn from left to right; the glass fiber is coated with nano-gold labeled first nucleic acid; the nitrocellulose membrane is provided with a quality control line and a detection line; third nucleic acid is fixed on the quality control line, a 5' end of the sequence of the third nucleic acid is labeled with biotin which is fixed on the quality control line of the nitrocellulose membrane after reacting with streptavidin; the streptavidin is fixed on the detection line; the sequence of the third nucleic acid is complementary with the sequence of the first nucleic acid in specificity; and the detection nucleic acid reagent comprises second nucleic acid, and a sixth basic group starting from the 5' end has a mismatch of T-T basic groups with the sequence of the first nucleic acid. The nucleic acid nano-gold biosensor can quickly detect the Hg2<+>, needs no instrument, is convenient to use, and has high sensibility.
Description
Technical field
The invention belongs to technological field of biochemistry, relate to a kind of rapid detection Hg
2+Biosensor.
Background technology
The main method of mercury detection at present is a spectrography, comprises atom (absorption, emission, fluorescence etc.) spectrographic technique and spectrophotometry etc.In recent years, the coupling of atomic spectrum and jigger coupling emmission spectrum has improved the sensitivity that detects, and widened the linearity range that detects, and inductivity coupled plasma mass spectrometry makes detection sensitive more, and data analysis is convenient.But this quasi-instrument is comparatively expensive, applying so being restricted in most of laboratories.Except that spectrography, be that the chemical sensitisation method of representative also has certain development with the crown ether-like small molecules, but weak points such as sensitivity is low, repeatable difference still arranged that the reliability of detection and accuracy are also not enough.
In recent years, developing into of nano material addresses this problem the thinking that provides new.Nm gold particles has excellent optical property, electrical properties, chemically reactive and bio-compatibility, is used widely at biological field.Studies show that the color of nano-Au solution is relevant with the size of the spacing of nm gold particles and nanometer gold aggregate.The spacing of nm gold particles is if obviously surpass its mean diameter, and nanometer gold is dispersion state, shows as redness on the macroscopic view; If this spacing is less than mean diameter, and then nanometer gold is easily reunited, and is state of aggregation, shows as purple on the macroscopic view to blue.Utilize this character of nanometer gold, can design a series of biochemical reactions, thereby realize detection the target material with the distance between the change nm gold particles.Recently, (Ono A, TogashIH.Angew Chem Int Ed, 2004,43 (33): 4300-4302) utilize Hg such as Ono
2+With the specific effect of DNA, designed a kind of new bio method for sensing, the dna probe by detecting the two ends mark is in conjunction with Hg
2+The change in fluorescence that the back produces realizes Hg
2+Specific detection.This method has higher specificity, can get rid of most of ionic interference in the actual sample system, but detection sensitivity is not high, and need carries out the dna double mark, also depends on fluorescent instrument, and it is still higher to detect cost.
Summary of the invention
The objective of the invention is to overcome existing Hg
2+The problem and shortage that detection technique exists provides a kind of Hg of being used for
2+The new tool of rapid detection, promptly nucleic acid nano-gold biosensor utilizes Hg
2+When existing, the principle that the T-T base can complementary pairing cooperates the Radioactive colloidal gold amplification system, is used for detecting Hg
2+, thereby can rapid detection Hg
2+, do not need instrument, easy to use, and highly sensitive.
A kind of Hg that is used to detect of the present invention
2+Nucleic acid nano-gold biosensor, comprise test strip, detect nucleic acid reagent and sample-loading buffer;
Described test strip from left to right is made up of the sample pad, glass fibre, nitrocellulose filter and the thieving paper that are fixed on the offset plate successively;
Scribble nano-gold labeled first nucleic acid on the described glass fibre, its sequence shown in SEQ ID NO.1,5 ' end labeling SH groups of this nucleotide sequence and with colloid gold particle coupling, 3 ' end mark vitamin H;
Described nitrocellulose filter is provided with nature controlling line and detection line; Be fixed with the 3rd nucleic acid on the nature controlling line, its sequence is shown in SEQ ID NO.3, and 5 ' end mark vitamin H of this nucleotide sequence after the Streptavidin reaction, is fixed on the nature controlling line of nitrocellulose filter; Be fixed with Streptavidin on the detection line;
Wherein, the 3rd nucleotide sequence and the first nucleotide sequence specificity complementation;
Described detection nucleic acid reagent contains second nucleic acid, and its sequence holds the 6th the base place and first nucleotide sequence that the mispairing of T-T base is arranged since 5 ' shown in SEQ ID NO.2; Described detection nucleic acid reagent drops on the described sample pad with solution to be detected and sample-loading buffer;
Described sample-loading buffer is a 4X nucleic acid hybridization liquid, contains: 0.6M sodium-chlor, 0.06M Trisodium Citrate.
According to the further feature of nucleic acid nano-gold biosensor of the present invention, described nature controlling line and detection line are at a distance of 3-10mm.
According to the further feature of nucleic acid nano-gold biosensor of the present invention, the described adjacent part that is fixed in sample pad, glass fibre, nitrocellulose filter and thieving paper on the offset plate 1-5mm that overlaps each other.
According to the further feature of nucleic acid nano-gold biosensor of the present invention, the particle diameter of described Radioactive colloidal gold is 8-100nm.
Further feature according to nucleic acid nano-gold biosensor of the present invention, the preparation method of described nanometer gold comprises: selecting particle diameter for use is the colloid gold particle of 8-100nm, Radioactive colloidal gold adopts conventional hydrochloro-auric acid to boil the reduction method preparation, controls grain diameter at 0.01%-0.05% and stirring velocity by the amount of control reductive agent between 1000-50000 changes.
Further feature according to nucleic acid nano-gold biosensor of the present invention, the coupling of described nanometer gold and nucleotide sequence comprises with sealing aged method: first nucleotide sequence of sulfydryl modification is joined in 5 times of spissated nano-Au solutions of volume, mix, 4-37 ℃ of reaction 10-24 hour, with the unnecessary avtive spot of 1% bovine serum albumin sealing, adding NaCl and SDS make its final concentration respectively to 0.15M and 0.01% after 30 minutes, aging after 10-20 hour, 10000-16000 rev/min centrifugal 20-60 minute, abandon supernatant, can obtain described nano gold mark oligonucleotide probe, this nano gold mark oligonucleotide probe precipitation is suspended 4 ℃ of preservations again with resuspended liquid.
Further feature according to nucleic acid nano-gold biosensor of the present invention, described sample pad is through following processing: adopt the mixed liquid dipping glass fibre that contains TritonX-100, boric acid, PEG4000, BSA, sucrose after 4 hours, 37 ℃ of oven dry are spent the night.
Know-why of the present invention is: according to working as Hg
2+When existing, the principle that the T-T base can complementary pairing designs special nucleotide sequence, is used for detecting Hg solution
2+Existence.The present invention designs 3 nucleotide sequences altogether: nucleotide sequence 1 has 36 bases, and the two ends complementary pairing forms a neck ring structure, is called Molecular Beacon (MB).This sequence 5 ' end labeling SH groups, 3 ' end mark vitamin H, 5 ' end sulfydryl can combine by static with colloid gold particle, is assemblied in the colloid gold particle surface.The colloid gold particle that assembles nucleic acid is sprayed on the gold mark pad.Nucleotide sequence 2 is target sequence DNA, and totally 24 bases, this sequence have the mispairing of T-T base since the 6th base place of 5 ' end and MB sequence.This dna solution drops on the sample pad with the solution that will detect, and does not have Hg in solution to be checked
2+When existing, target sequence DNA can not with the complete complementary pairing of MB sequence, thereby the neck ring of MB can not be opened, the vitamin H that is marked at MB one end can not be released, can not with the reaction of Streptavidin on the T line, thereby the T line does not develop the color, and has only the colour developing of C line; In detecting solution, Hg is arranged
2+When existing, Hg
2+Existence make that the T-T base of mispairing can complementary pairing, thereby the neck ring structure of MB is opened, hidden vitamin H on the colloid gold particle surface is exposed, combine with the Streptavidin on being marked at the T line, Radioactive colloidal gold is rested on the T line, the colour developing of T line, the C line also develops the color simultaneously.Nucleotide sequence 3 is a control sequence, and totally 30 bases are with the complete complementary pairing of MB sequence.This sequence 5 ' end mark vitamin H after the Streptavidin reaction, is drawn on the nature controlling line of nitrocellulose filter.When the colloid gold particle on the gold mark pad was released, the nucleotide sequence 1 of mark combined with nucleotide sequence 3 on the nature controlling line on the colloid gold particle, thereby makes nature controlling line develop the color.
The invention has the advantages that: utilize molecular beacon can differentiate mononucleotide mispairing and Hg cleverly
2+Can make T-T base complementrity paired principle when existing, design a kind of highly sensitive, low expense, do not needed to use the colloidal gold strip biosensor of any instrument.Both solved the defective that needs large-scale instrument in the detection method in the past, and can guarantee detection sensitivity again, and prepare easy, detect rapidly, do not need the technical professional.
Description of drawings
Fig. 1 is that nucleic acid nano-gold biosensor of the present invention is for Hg
2+The test experience result of standardized solution gradient;
Fig. 2 is that nucleic acid nano-gold biosensor of the present invention is for different metal ionic test experience result.
Embodiment
Embodiment one: the preparation of nucleic acid nano-gold biosensor of the present invention
1, the design of three kinds of nucleotide sequences
According to know-why of the present invention, three sections nucleotide sequences of design are:
Nucleotide sequence 1:SEQ ID NO.1
5 '-sulfydryl modification-ACACGCTAATCAAGCTTTAACTCATAGTTAGCGTGT-3 ' biotin modification
Nucleotide sequence 2:SEQ ID NO.2
5’-ACGCTTACTATGAGTTAAAGCTTG-3’
Nucleotide sequence 3:SEQ ID NO.3
5 '-biotin modification-ACGCTAACTATGAGTTAAAGCTTGCTTAAG-3 '
2. the preparation of nanometer gold (Radioactive colloidal gold):
Take by weighing the HAuCL4 solution of 100g 0.01% in the round-bottomed flask of 250ML, magnetic agitation is heated to boiling; In above-mentioned solution, add the trisodium citrate of 4ml 1% then rapidly, after solution becomes redness, continue to boil 10min, stop heating and continue to stir until cooling; Colloidal gold solution keeps in Dark Place for 4 ℃, and nanometer gold is identified by 520nm maximum absorbance value.
The preparation of gold mark nucleic acid:
With 100 μ l deionized water dissolving 1OD nucleotide sequences 1, join in 5 times of spissated colloidal gold solutions of volume of 1ml, 4 ℃ 24 hours; 1% bovine serum albumin sealing is after 30 minutes, the SDS that adds NaCl and 1% is that 0.15M and 0.01%, 4 ℃ spend the night to final concentration respectively, 11500 rev/mins centrifugal 20 minutes, abandon supernatant, sink to the bottom with the resuspended liquid of 500ul (20mM Na3PO4,5%BSA, 0.25%Tween, with 10% sucrose) resuspended, repeating to give a baby a bath on the third day after its birth suspends again all over the resuspended liquid of back with 200ul, makes suspension.
3. the processing of sample pad
After glass fibre soaks and contains the solution of TritonX-100, boric acid, PEG4000, BSA, sucrose, 37 ℃ of dry for standby.
4. the preparation of gold mark pad
The gold mark nucleotide sequence 1 of the present invention preparation is applied on the glass fibre, and 37 ℃ of dryings 2 hours are made gold mark pad, and are standby.
5. the processing of nature controlling line and detection line on the nitrocellulose filter
With the biotin labeled nucleotide sequence 3 of 32 μ l deionized water dissolving 1OD, making its concentration is 100 μ M, get the nucleotide sequence 3 of 15 μ l, 100 μ M, add 15 μ l (1mg/ml) chain and sistomycocins, reaction is after 2 hours under the room temperature, adopt to draw a film metal spraying instrument and be applied on the nitrocellulose filter nature controlling line, 37 ℃ of dryings two hours.
With 30 μ l (1mg/ml) Streptavidins, adopt to draw a film metal spraying instrument and be applied on the nitrocellulose filter detection line, 37 ℃ of dryings two hours.
6. the assembling of colloidal gold strip
Be fixed on the offset plate successively with being fixed with nitrocellulose filter, the thieving paper of oligonucleotide probe, the glass fibre that scribbles the nanoparticle labeled oligonucleotide probe, sample pad, the adjacent part 2mm that overlaps each other promptly obtains nucleic acid nano-gold biosensor of the present invention after cutting into wide 4mm.
Embodiment two: the quality control of nucleic acid nano-gold biosensor of the present invention and detection effect
Adopt the made nucleic acid nano-gold biosensor of embodiment one, carry out following experiment, verify that it detects effect.
1. prepare Hg
2+Standardized solution gradient, concentration are respectively 1 μ M, 500nM, 100nM, 50nM, 20nM, 10nM.
2. prepare the Hg of 100nM
2+, Mn
2+, Cd
2+, Pb
2+, Mg
2+, Zn
2+, Fe
2+, Ca
2+, Fe
3+, Al
3+, Ag
+Solution.
3. with deionized water dissolving 1OD nucleotide sequence 2, making its concentration is 100nM.
4. get above-mentioned nucleotide sequence 2 of 10 μ l and 90 μ l Hg to be checked
2+Solution mixes, drips on the sample pad of above-mentioned nano-gold biosensor, but 10 minutes judged results.(see figure 1)
The result shows: along with Hg
2+The increase of concentration, the detection line band is more and more darker, and naked eyes visible (Fig. 1 left side) are also consistent with the result of gold test strip bar readout instrument, along with Hg
2+The increase of concentration, the curve of detection line also increase (Fig. 1 right side), nature controlling line colour developing gradually.
5. get the above-mentioned nucleotide sequence 2 of 10 μ l and 90 μ l other solions to be checked and mix, drip on the sample pad of above-mentioned nano-gold biosensor, but 10 minutes judged results.(see figure 2)
The result shows: under 100nM concentration, have only Hg
2+When existing, the detection line colour developing, other ions all do not develop the color, and naked eyes visible (Fig. 2 left side) are also consistent with the result of gold test strip bar readout instrument, have only Hg
2+When existing, detection line has curve, and when other ions existed, detection line did not have curve (Fig. 2 right side), the nature controlling line colour developing.
Quality control standard:
(1) it is effective that red line proof nano-gold biosensor appears in C line (nature controlling line).
(2) whether T line (detection line) red line occurs, is the positive negative standard of differentiating.
Criterion as a result:
(1) red line appears in the C line, and red line appears in the T line simultaneously, and Hg in the test sample is described
2+Content overproof;
(2) red line appears in the C line, and red line does not appear in the T line simultaneously, and Hg in the test sample is described
2+Content does not exceed standard;
(3) red line does not appear in the C line, illustrates that nano-gold biosensor lost efficacy.
Sequence table (SEQUENCE LISTING)
＜110〉Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
＜120〉be used to detect the nucleic acid nano-gold biosensor of Hg2+
<130>
<160>3
<170>PatentIn?version?3.4
<210>1
<211>36
<212>DNA
＜213〉synthetic
<400>1
acacgctaat?caagctttaa?ctcatagtta?gcgtgt 36
<210>2
<211>24
<212>DNA
＜213〉synthetic
<400>2
acgcttacta?tgagttaaag?cttg 24
<210>3
<211>30
<212>DNA
＜213〉synthetic
<400>3
acgctaacta?tgagttaaag?cttgcttaag 30
Claims (7)
1. one kind is used to detect Hg
2+Nucleic acid nano-gold biosensor, it is characterized in that: comprise the detection reagent bar, detect nucleic acid reagent and sample-loading buffer;
Described test strip from left to right is made up of the sample pad, glass fibre, nitrocellulose filter and the thieving paper that are fixed on the offset plate successively;
Scribble nano-gold labeled first nucleic acid on the described glass fibre, its sequence shown in SEQ ID NO.1,5 ' end labeling SH groups of this nucleotide sequence and with colloid gold particle coupling, 3 ' end mark vitamin H;
Described nitrocellulose filter is provided with nature controlling line and detection line; Be fixed with the 3rd nucleic acid on the nature controlling line, its sequence is shown in SEQ ID NO.3, and 5 ' end mark vitamin H of this nucleotide sequence after the Streptavidin reaction, is fixed on the nature controlling line of nitrocellulose filter; Be fixed with Streptavidin on the detection line;
Wherein, the 3rd nucleotide sequence and the first nucleotide sequence specificity complementation;
Described detection nucleic acid reagent contains second nucleic acid, and its sequence holds the 6th the base place and first nucleotide sequence that the mispairing of T-T base is arranged since 5 ' shown in SEQ ID NO.2; Described detection nucleic acid reagent drops on the described sample pad with solution to be detected and sample-loading buffer;
Described sample-loading buffer is a 4X nucleic acid hybridization liquid, contains: 0.6M sodium-chlor, 0.06M Trisodium Citrate.
2. nucleic acid nano-gold biosensor according to claim 1 is characterized in that: described nature controlling line and detection line are at a distance of 3-10mm.
3. nucleic acid nano-gold biosensor according to claim 1 is characterized in that: the described adjacent part that is fixed in sample pad, glass fibre, nitrocellulose filter and thieving paper on the offset plate 1-5mm that overlaps each other.
4. nucleic acid nano-gold biosensor according to claim 1 is characterized in that: the particle diameter of described Radioactive colloidal gold is 8-100nm.
5. nucleic acid nano-gold biosensor according to claim 1, it is characterized in that, the preparation method of described nanometer gold comprises: selecting particle diameter for use is the colloid gold particle of 8-100nm, Radioactive colloidal gold adopts conventional hydrochloro-auric acid to boil the reduction method preparation, controls grain diameter at 0.01%-0.05% and stirring velocity by the amount of control reductive agent between 1000-50000 changes.
6. nucleic acid nano-gold biosensor according to claim 1, it is characterized in that, the coupling of described nanometer gold and nucleotide sequence comprises with sealing aged method: first nucleotide sequence of sulfydryl modification is joined in 5 times of spissated nano-Au solutions of volume, mix, 4-37 ℃ of reaction 10-24 hour, with the unnecessary avtive spot of 1% bovine serum albumin sealing, adding NaCl and SDS make its final concentration respectively to 0.15M and 0.01% after 30 minutes, aging after 10-20 hour, 10000-16000 rev/min centrifugal 20-60 minute, abandon supernatant, can obtain described nano gold mark oligonucleotide probe, this nano gold mark oligonucleotide probe precipitation is suspended 4 ℃ of preservations again with resuspended liquid.
7. nucleic acid nano-gold biosensor according to claim 1, it is characterized in that, described sample pad is through following processing: adopt the mixed liquid dipping glass fibre that contains TritonX-100, boric acid, PEG4000, BSA, sucrose after 4 hours, 37 ℃ of oven dry are spent the night.
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| CN102183433A (en) * | 2011-02-25 | 2011-09-14 | 中国科学院化学研究所 | Method for detecting mercury ion concentration of water sample |
| CN102207502A (en) * | 2011-03-25 | 2011-10-05 | 宁波大学 | Mercury ion test paper and preparation method thereof |
| CN102608108A (en) * | 2012-02-21 | 2012-07-25 | 温州医学院 | Nano biosensor method and kit for rapid detection of Hg ions |
| CN102706940A (en) * | 2012-06-15 | 2012-10-03 | 湖南大学 | Electrochemical sensor capable of detecting trace mercury in water body, and preparation method and application thereof |
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| CN102183433A (en) * | 2011-02-25 | 2011-09-14 | 中国科学院化学研究所 | Method for detecting mercury ion concentration of water sample |
| CN102207502A (en) * | 2011-03-25 | 2011-10-05 | 宁波大学 | Mercury ion test paper and preparation method thereof |
| CN102207502B (en) * | 2011-03-25 | 2014-01-29 | 宁波大学 | Mercury ion test paper and preparation method thereof |
| CN102608108A (en) * | 2012-02-21 | 2012-07-25 | 温州医学院 | Nano biosensor method and kit for rapid detection of Hg ions |
| CN103048463A (en) * | 2012-05-02 | 2013-04-17 | 中国科学院广州生物医药与健康研究院 | Microwell plate nucleic acid hybridization ELISA (enzyme-linked immuno sorbent assay) method for detecting DNA-binding proteins based on different rigors |
| CN102706940A (en) * | 2012-06-15 | 2012-10-03 | 湖南大学 | Electrochemical sensor capable of detecting trace mercury in water body, and preparation method and application thereof |
| CN102706940B (en) * | 2012-06-15 | 2014-05-14 | 湖南大学 | Electrochemical sensor capable of detecting trace mercury in water body, and preparation method and application thereof |
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| CN104155289B (en) * | 2014-06-25 | 2016-06-08 | 宁波大学 | A kind of solid-state electrochemistry illumination sensor detecting mercury ion and its preparation method and application |
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