CN103940815A - Nucleic acid colorimetric sensor with Y-shaped structure and application of sensor - Google Patents
Nucleic acid colorimetric sensor with Y-shaped structure and application of sensor Download PDFInfo
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- CN103940815A CN103940815A CN201410184432.2A CN201410184432A CN103940815A CN 103940815 A CN103940815 A CN 103940815A CN 201410184432 A CN201410184432 A CN 201410184432A CN 103940815 A CN103940815 A CN 103940815A
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Abstract
The invention discloses a nucleic acid colorimetric sensor with a Y-shaped structure and an application of the sensor. The sensor comprises two parts, namely nanogold A asymmetrically modified by a probe DNA1 and nanogold B asymmetrically modified by a probe DNA2, wherein a target DNA can be respectively combined with a probe DNA1 and a probe DNA2 under the condition of existence of the target DNA, so as to form a DNA complex with the Y-shaped structure. Thus, independently dispersed nanogolds in a solution are close together. An absorption spectrum of a system greatly displaces due to an LSPR coupling effect, so that the existence of the target DNA can be judged by visual inspection. The nanogolds are subjected to asymmetric modification, so that a nanogold dimer is mainly formed in the solution under the existence of the target DNA while a plurality of nanogold aggregates are not formed. Compared with a conventional colloidal gold colorimetric method, for the sensor disclosed by the invention, the sensitivity of the sensor for detecting the target DNA can be improved by four orders of magnitudes, the linear range can be amplified by two orders of magnitudes, and meanwhile, the stability of the sensor is also greatly improved.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of preparation method and application thereof of y-type structure nucleic acid colorimetric sensor
.
Background technology
Nucleic acid is extensively present in animal, plant, microbial body.Nucleic acid is not only basic inhereditary material, and also accounts for critical positions in the biosynthesizing of protein, thereby plays conclusive effect in a series of great biological phenomenas such as biological growth, heredity, variation.Meanwhile, nucleic acid also has very important effect in clinical medicine application aspect, have now found that nearly 2000 kinds of genetic diseases all relevant with DNA structure (
nature Reviews Genetics,2012,
13, 679-692).Therefore, develop easy, fast, nucleic acid detection method all has very important meaning to the field such as life science and clinical medicine cheaply.
Different nucleic acid, the differences such as its chemical composition, nucleotidesequence, based on these characteristics, researcher has been developed nucleic acid detection method, and these methods comprise spectral analysis, red, orange, green, blue, yellow (ROGBY), electrochemical assay, Mass Spectrometer Method method etc.In these methods, collaurum colourimetry, because it is easy and simple to handle, without complex apparatus, even just can be carried out qualitative detection to object by range estimation, is therefore widely used in field quick detection and the medical clinic applications of environment and food object.But, traditional collaurum colorimetric sensor is to produce change color based on a large amount of collaurums reunions object is detected, there is following several respects problem in the colorimetric sensor based on this principle: is first that sensitivity is relatively low, only have in the time that the amount of object is much higher than the quantity of collaurum in solution, could produce change color; Next is that linear response range is little, conventionally only has one or two order of magnitude; In addition, reunite owing to producing a large amount of collaurums in course of reaction, can make the collaurum rapid subsidence in solution, thus affect result accurate judgement (
j. Am. Chem. Soc. 2013,135,12338-12345).
Summary of the invention
The object of the present invention is to provide a kind of y-type structure nucleic acid colorimetric sensor, by asymmetric modification is carried out in gold size surface, make under object existence condition, collaurum can only form dimer, and there will not be a large amount of clustering phenomenas.Meanwhile, we are by the design of target-probe, make probe be combined the Y shape DNA composite structure of rear formation with object and can farthest reduce the distance of two connected collaurums, thereby produce strong local surface plasma resonance coupling phenomenon.Can find out from absorption spectrum, collaurum, in the nanometer more than 60 that produced before and after dimer maximum absorption band position displacement, makes solution colour become purple from redness.Therefore, can whether qualitatively judge the existence of target dna sequence by the variation of range estimation solution colour, and can realize the quantitative detection to target gene by the uv-vis spectra of scanning solution.
A kind of y-type structure nucleic acid colorimetric sensor, described sensor is:
(1) sensor is mixed by the nm of gold A of DNA probe 1 asymmetric modification and the nm of gold B equimolar amounts of DNA probe 2 asymmetric modifications, is dissolved in DNA hybridization buffer liquid;
(2) when adding after target dna in sensing system, target dna can be hybridized with DNA probe 1 and DNA probe 2, form Y shape composite structure, thereby make nm of gold A and nm of gold B close, due to the coupling effect of surface plasma resonance, observe the significant change of solution colour, thereby the existence that judges target dna is whether, and the number of content;
(3) because nm of gold A and B have carried out asymmetric modification, adding of target dna can not make nm of gold form large aggregation, and only can make nm of gold form dimer.
Described nm of gold is spherical or class is spherical, and diameter is between 5 nanometer to 100 nanometers.
Described nm of gold, for will carry out asymmetric modification before being combined with DNA probe, is modified and is strengthened the dispersiveness of nm of gold in aqueous solution at nm of gold surface 80%-95% area, and the compound not reacting with DNA probe.
It is complementary that described DNA probe 1 has partial sequence with DNA probe 2, forms DNA double chain in hybridization solution.
Described DNA probe 1 and DNA2 respectively have partial sequence and target dna complementation, form DNA double chain in hybridization solution.
One terminal modified upper nm of gold of described DNA probe and an other DNA probe complementation, and with the complementary one end of target dna away from nm of gold surface.
A kind of y-type structure nucleic acid colorimetric sensor detects the application on relevant field in clinical diagnosis, environmental contaminants detection and food security to DNA.
Compared with traditional collaurum colorimetric method,
remarkable advantage of the present invention is:
(1) sensitivity is higher.Y-type structure nucleic acid colorimetric sensor of the present invention is in the time that target dna exists, DNA probe and target dna form Y type DNA compound, thereby the distance of two nano Au particles that DNA probe is connected infinitely furthers, produce great LSPR coupling effect, therefore only need nm of gold dimer just can produce significant change color; And traditional collaurum colorimetric sensor closes in object combination, because the distance between gold size is near not, need a large amount of nm of gold aggregations could produce significant change color.From we contrast experiment's result, y-type structure nucleic acid colorimetric sensor of the present invention is than highly sensitive four orders of magnitude of traditional collaurum colorimetric sensor.
(2) range of linearity is wider.Just can observe change color because traditional collaurum colorimetric sensor need to form large nm of gold aggregation, and colloidal gold aggregation spends when high, can not stable existence in solution, thus cause the range of linearity of sensor narrower; And sensor of the present invention exists in situation at object and only can form dimer, and the nm of gold dimer obtaining also can stable existence in solution, therefore this sensor has the wider range of linearity.Result by contrast experiment shows, y-type structure nucleic acid colorimetric sensor of the present invention is than wide two orders of magnitude of traditional collaurum colorimetric sensor range of linearity.
(3) stability is better.Y-type structure nucleic acid colorimetric sensor of the present invention has improved the stability of sensor aspect following two: be first by asymmetric modification, modify a kind of organic molecule that can make nm of gold stable dispersion in solution in 80%-98% position, nm of gold surface, thereby improved and be not combined the stability of front sensor with target dna; Next is due to asymmetric modification, can form nm of gold dimer, and this dimer equally can stable dispersion in solution with object in conjunction with rear.Therefore, this sensor is all improved being combined the stability of front and back with target dna.
Brief description of the drawings
Fig. 1 is the schematic diagram that y-type structure nucleic acid colorimetric sensor of the present invention detects DNA.
Fig. 2 is DNA probe and the target dna sequence that application y-type structure nucleic acid colorimetric sensor of the present invention detects cholera vibrio gene group DNA.
Fig. 3 is that the solution colour that y-type structure nucleic acid colorimetric sensor detects before and after cholera vibrio gene group DNA described in application invention changes photo.
Fig. 4 is the working curve that y-type structure nucleic acid colorimetric sensor detects cholera vibrio gene group DNA described in application invention.
Embodiment
Narrate the specific embodiment of the present invention below in conjunction with accompanying drawing:
The schematic diagram of y-type structure nucleic acid colorimetric sensor detection target dna of the present invention as shown in Figure 1, when being modified with after the nm of gold of DNA probe 1 and DNA probe 2 is combined with target dna, can form Y type DNA composite structure as shown in Figure 1, thereby make solution colour change purple into by redness.Therefore, by the variation of range estimation solution colour, whether can qualitatively judge the existence of target dna, and by measuring the uv-vis spectra of solution, can quantitatively detect the content of target dna in solution.
Below the detailed protocol of preparation y-type structure nucleic acid colorimetric sensor of the present invention:
(1) preparation of nm of gold: adopt traditional sodium citrate reducing process to prepare the colloidal gold solution of sodium citrate protection, concrete method of modifying referring to but be not limited to Publication about Document: Nature Phys. Sci. 1973,241:20-22;
(2) the asymmetric modification of nm of gold: in the surperficial 80%-95% modification of solid phase modification side preparation that adopts CTAB to mediate, can strengthen the dispersiveness of nm of gold in aqueous solution, and the compound not reacting with DNA probe.Concrete method of modifying referring to but be not limited to the Science with Publication about Document Journal of Colloid & Interface, 2013,409,32-37;
(3) DNA probe 1 is fixing: adopt TCEP that the end modified DNA probe 1 that has sulfydryl is activated, then the DNA probe after activation is mixed with the nm of gold of the asymmetric modification obtaining in step (2), in the phosphate buffer of pH=7.4, react and spend the night, centrifuging is fallen not to be attached to after the DNA probe in nm of gold, the nm of gold that is modified with DNA probe 1 is re-dispersed in the hybridization buffer of 2 times of concentration for subsequent use;
(4) DNA probe 2 is fixing: method is identical with step (3), just DNA probe 1 is changed to DNA probe 2;
(5) before using, isoconcentration, isopyknic DNA probe 1 and DNA probe 2 are mixed.
The using method of y-type structure nucleic acid colorimetric sensor of the present invention is as follows:
(1) DNA probe 1 that in the preparation scheme of above-mentioned y-type structure nucleic acid colorimetric sensor, step (5) prepares is mixed with DNA solution 1:1 to be measured with DNA probe 2 mixed solutions, be placed in DNA hybridization reaction device and carry out hybridization reaction.
(2) quilitative method of target dna: by visual observations solution in the change color of carrying out before and after hybridization reaction, whether qualitatively judge the existence of target target dna sequence, concrete criterion is as follows: if solution does not change in the color of carrying out hybridization reaction front and back, illustrate in solution to be measured containing target dna sequence; If solution changes purple in the color of carrying out hybridization reaction front and back into by pale red, illustrate in solution to be measured and contain target dna sequence.
(3) quantivative approach of target dna:
A. the drafting of working curve: the target dna standard solution by measuring a series of concentration known with sensor hybridization reaction before and after the ultraviolet-visible spectrogram of solution, adopting the concentration of target dna is horizontal ordinate, measured spectrogram is ordinate in the spectral absorption value of the maximum absorption wave strong point at dimer place, drawing curve;
B. the mensuration of target dna content in testing sample: first measure testing sample with sensor hybridization reaction before and after the ultraviolet-visible spectrogram of solution, obtain its spectral absorption value in the maximum absorption wave strong point at dimer place, in the working curve that this value substitution said method is tried to achieve, can accurately calculate the content of target dna in testing sample.
embodiment 1
Below prepare y-type structure nucleic acid colorimetric sensor for applying the method for the invention, and be applied to the specific embodiment of the discriminating of cholera vibrio gene group DNA:
(1) preparation of nm of gold: the HAuCl4 aqueous solution that is 0.01% by 50 milliliters of massfractions is heated to reflux, then add the sodium citrate aqueous solution that 0.5 milliliter of massfraction is 1%, mixed solution continues to reflux and within 30 minutes, obtains the nm of gold sol solution that mean grain size is 41 nanometers;
(2) the asymmetric modification of nm of gold: first glass sheet being immersed to 12 milliliters of concentrated sulphuric acids is to react in 30% mixed solution of hydrogen peroxide 10 minutes with 3 milliliters of massfractions, then beaker is put into Ultrasound Instrument water-bath ultrasonic 10 minutes; Next the cover glass of processing is taken out, with after intermediate water flushing three times, immerse containing in the DTAB solution of 0.05 M hold over night; Surface-assembled is had the cover glass of DTAB to take out, with after intermediate water flushing three times, the mean grain size that immerses preparation in 10 ml above-mentioned steps (1) is in the beaker of colloidal gold solution of 41 nanometers, leaves standstill and reacts 60 minutes again; Then absorption there is is the cover glass of individual layer nm of gold to take out, rinse and immerse afterwards for three times in the surface plate of 2-sulfydryl-1, 4-succinic acid solution that 2 ml volume fractions are 1% with intermediate water, leave standstill reaction 24 hours.Finally, the nm of gold of the above-mentioned 2-of being modified with sulfydryl-1, 4-succinic acid is taken out together with cover glass, with after intermediate water cleaning three times, immerse and be equipped with in the surface plate of 2 ml intermediate waters, put into Ultrasound Instrument water-bath ultrasonic 2 minutes.The nm of gold that is modified with 2-sulfydryl-1, 4-succinic acid will come off and enter intermediate water from cover glass surface, is the colloidal gold solution of 41 nanometers thereby obtain the mean grain size that homodisperse 2-sulfydryl-1, 4-succinic acid part modifies;
(3) DNA probe 1 is fixing: this test DNA probe used and the target dna sequence identified as shown in Figure 3.The DNA probe 1 that is 1.0 mM by 10 microlitre concentration joins the Tris-HCl(pH7.4 of 90 microlitre 50mM) in solution, leave standstill reaction after 1 hour, add 2-sulfydryl-1 preparing in 1 milliliter of above-mentioned steps (2), the colloidal gold solution (collaurum concentration 100 pM) that 4-succinic acid part is modified, standing reaction is spent the night.Solution is transferred to 6000 rpms of centrifugings of centrifuge tube 10 minutes, abandoning supernatant, (the composition: 20 mM pH 7.4 Tris-HCl damping fluids of 1 milliliter of hybridization buffer for sediment, 15% 4 acid amides, 10% dextran sulfate, 0.3 M NaCl, 3.75 mM MgCl2) dispersion is rear for subsequent use again.
(4) DNA probe 2 is fixing: method is identical with step (3), just DNA probe 1 is changed to DNA probe 2;
(5) before using, isoconcentration, isopyknic nm of gold colloidal solution that is modified with DNA probe 1 and DNA probe 2 are mixed, then with DNA(cholera vibrio gene group DNA to be measured) solution 1:1 mixes, be placed in DNA hybridization reaction device and carry out hybridization reaction, temperature is controlled at 68 DEG C, 60 seconds, then slowly cools to room temperature.
(6) qualitative detection of target dna: Fig. 3 is solution at the photo carrying out before and after hybridization reaction, and as can be seen from the figure, solution changes purple in the color of carrying out before and after hybridization reaction into by pale red, illustrates in solution to be measured and contains target dna sequence.
(7) the quantitative detection of target dna:
A. the drafting of working curve: by measuring a series of concentration known (0,1 pM, 10 pM, 10 pM, 100 pM, 1 nM, 10 nM) target dna standard solution with sensor hybridization reaction before and after the ultraviolet-visible spectrogram of solution, adopting the concentration of target dna is horizontal ordinate, and measured spectrogram is ordinate in the spectral absorption value at the 600 nm places at dimer place, draws the working curve obtaining as shown in Figure 4;
B. the mensuration of target dna content in testing sample: first measure testing sample with sensor hybridization reaction before and after the ultraviolet-visible spectrogram of solution, trying to achieve its spectral absorption value at 600 nm places is 0.13, by in the above-mentioned working curve of this value substitution, the content that calculates target dna in testing sample is 0.73 nM.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University of Fuzhou
<120> y-type structure nucleic acid colorimetric sensor and application thereof
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> DNA probe 1
<400> 1
ctgttactgt actaccgagg 20
<210> 2
<211> 20
<212> DNA
<213> DNA probe 2
<400> 2
ctgtcattag gcagtaacag 20
<210> 3
<211> 22
<212> DNA
<213> target dna
<400> 3
cctcggtagt acctaatgac ag 22
Claims (8)
1. a y-type structure nucleic acid colorimetric sensor, is characterized in that: described sensor is:
(1) sensor is mixed by the nm of gold A of DNA probe 1 asymmetric modification and the nm of gold B equimolar amounts of DNA probe 2 asymmetric modifications, is dissolved in DNA hybridization buffer liquid;
(2) when adding after target dna in sensing system, target dna can be hybridized with DNA probe 1 and DNA probe 2, form Y shape composite structure, thereby make nm of gold A and nm of gold B close, due to the coupling effect of surface plasma resonance, observe the significant change of solution colour, thereby the existence that judges target dna is whether, and the number of content.
2. y-type structure nucleic acid colorimetric sensor according to claim 1, is characterized in that: described DNA probe 1 sequence is as shown in SEQ ID NO.1; DNA probe 2 sequences are as shown in SEQ ID NO.2.
3. y-type structure nucleic acid colorimetric sensor according to claim 1, is characterized in that: described nm of gold is spherical or class is spherical, diameter is between 5 nanometer to 100 nanometers.
4. y-type structure nucleic acid colorimetric sensor according to claim 1, it is characterized in that: described nm of gold for carrying out asymmetric modification before being combined with DNA probe, modify and strengthen the dispersiveness of nm of gold in aqueous solution at nm of gold surface 80%-95% area, and the compound not reacting with DNA probe.
5. y-type structure nucleic acid colorimetric sensor according to claim 1, is characterized in that: it is complementary that described DNA probe 1 has partial sequence with DNA probe 2, forms DNA double chain in hybridization solution.
6. y-type structure nucleic acid colorimetric sensor according to claim 1, is characterized in that: described DNA probe 1 and DNA2 respectively have partial sequence and target dna complementation, form DNA double chain in hybridization solution.
7. y-type structure nucleic acid colorimetric sensor according to claim 1, is characterized in that: a terminal modified upper nm of gold of described DNA probe and an other DNA probe complementation, and with the complementary one end of target dna away from nm of gold surface.
8. a y-type structure nucleic acid colorimetric sensor as claimed in claim 1 detects the application on relevant field in clinical diagnosis, environmental contaminants detection and food security to DNA.
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| CN106370868A (en) * | 2016-09-23 | 2017-02-01 | 中国科学院重庆绿色智能技术研究院 | SPR sensor for detecting microcystic toxin based on nucleic acid aptamer signal amplification strategy and preparation method and application thereof |
| CN111122677A (en) * | 2020-01-07 | 2020-05-08 | 安徽科技学院 | Electrochemical aptamer sensor for quantitatively detecting ochratoxin A and application thereof |
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| CN106370868A (en) * | 2016-09-23 | 2017-02-01 | 中国科学院重庆绿色智能技术研究院 | SPR sensor for detecting microcystic toxin based on nucleic acid aptamer signal amplification strategy and preparation method and application thereof |
| CN111122677A (en) * | 2020-01-07 | 2020-05-08 | 安徽科技学院 | Electrochemical aptamer sensor for quantitatively detecting ochratoxin A and application thereof |
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Application publication date: 20140723 |