CN104897585A - Preparation method of aptamer colorimetric sensor for MC-LR fast detection - Google Patents
Preparation method of aptamer colorimetric sensor for MC-LR fast detection Download PDFInfo
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- CN104897585A CN104897585A CN201510223809.5A CN201510223809A CN104897585A CN 104897585 A CN104897585 A CN 104897585A CN 201510223809 A CN201510223809 A CN 201510223809A CN 104897585 A CN104897585 A CN 104897585A
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Abstract
The invention belongs to the field of water pollution detection and treatment and relates to a preparation method of an aptamer colorimetric sensor for MC-LR fast detection. The preparation method comprises that a MC-LR aptamer with a specific base sequence and a common salt solution with a high concentration are orderly added into gold nanoparticle sol so that the aptamer colorimetric sensor for MC-LR fast detection is prepared. The preparation method utilizes the aptamer with a MC-LR specific recognition function as an identification element and greatly improves sensitivity and selectivity of colorimetric sensor-based MC-LR detection. The aptamer colorimetric sensor has a MC-LR detection linear range of 7.5*10<-6> to 5*10<-10> mol/L and a detection limit of 3.7*10<-10> mol/L. Compared with the existing colorimetric sensor, the aptamer colorimetric sensor has the advantages of operation simpleness, fast response and on-line detection easiness.
Description
Technical field
The invention belongs to water pollutions Tesing and Solution field, relate to the preparation method that one detects the aptamer colorimetric sensor of microcapsule algae toxin (MC-LR) fast.
Background technology
Microcapsule algae toxin (MC-LR) is a kind of ring-type seven peptide class water environment pollution thing of cyanobacterium release when breeding in the eutrophication water being rich in the elements such as N, P.MC-LR toxicity is huge, main is target organ with liver, and the MC-LR of low concentration can the activity of Profilin matter phosphate PP1 and PP2A, causes liver cell to decompose, break, cause hepatic dysfunction, strong hepatotoxicity wind agitation and tumor inducing effect are all shown to man and animal; MC-LR has stable chemical nature, is difficult to the feature of degrading under natural conditions.The freshwater lake such as Chaohu, Taihu Lake blue-green algae is frequently broken out in recent years, causes severe contamination to the Drinking Water of resident, therefore realizes having important practical significance to quick, the Sensitive Detection of MC-LR.
At present, the method measuring MC-LR mainly contains high performance liquid chromatography, mass spectroscopy, liquid-mass chromatography method etc., these methods show higher sensitivity when analyzing MC-LR, but method itself exist sample pre-treatments complexity, instrument price high, analyze the shortcomings such as consuming time.Relatively, colorimetric methods is easy and simple to handle, response is rapid, and does not need by complex large-sized instrument, therefore can be used as a kind of easy, practical analytical approach for testing environment pollutant.But the mixed system of environmental water sample multiple pollutant often, colorimetric methods cannot realize selectivity analysis and detect when analyzing pollutant.Therefore, construct the colorimetric sensor based on specific recognition element, in the water sample of complexity, realize the detection to MC-LR, there is important Significance for Environment.
Aptamer (aptamer) is DNA or the RNA sequence of a kind of strand obtained by aglucon phyletic evolution technology (SELEX) in-vitro screening of index concentration, to target molecules, there is high affinity, therefore impart the recognition capability high to target material.In addition, aptamer also have synthesis cycle short, synthesis cost low, stability is high, be easy to the advantages such as modification.Therefore in the present invention, the aptamer of specific recognition MC-LR is combined with colorimetric methods, achieves and the quick, sensitive of MC-LR and high selectivity are detected.Specific as follows: the sodium chloride solution of the aptamer solution and higher concentration with specific base sequence is joined in golden nanometer particle colloidal sol.Because aptamer is to the electrostatic screening effect of the protective effect of golden nanometer particle in conjunction with higher concentration sodium chloride, golden nanometer particle tends to disconnected from each other.After adding MC-LR solution to above-mentioned system, aptamer high-affinity specific binding MC-LR molecule, golden nanometer particle protective effect is cancelled.Under under the effect of higher concentration sodium chloride, golden nanometer particle is reunited, and feature plasma resonance absorption peak is by 521 nm red shift to 646 nm, and solution colour becomes blue by redness.Utilize absorbance ratio A
646/ A
521change and MC-LR concentration between relational implementation the analysis of MC-LR is detected.
Summary of the invention
The object of the invention is for instrument price costliness of existing detection MC-LR method, complicated operation, the analysis not enough and a kind of analytical approach of the adaptation of the nucleic acid based on particular sequence colorimetric sensor that provides such as consuming time.Utilize the recognition component of aptamer molecule as sensor of tool specific recognition MC-LR function, improve the selectivity of colorimetric sensor, add the sensitivity of colorimetric methods.Meanwhile, this colorimetric methods have preparation simple, respond the advantages such as rapid, easy and simple to handle.
Object of the present invention can be achieved through the following technical solutions:
By adding the aptamer solution of specific base sequence and the sodium chloride solution of higher concentration, incubated at room temperature 10 ~ 20min in the golden nanometer particle colloidal sol to preparation, construct the aptamer colorimetric sensor obtained based on specific base sequence.This aptamer colorimetric sensor take MC-LR as detected object, by the MC-LR solution of variable concentrations is joined in colorimetric sensor solution, incubated at room temperature, after 10 ~ 20 minutes, adopts ultraviolet-visible spectrophotometer to measure the uv-visible absorption spectra of above-mentioned mixed solution.By the absorbance ratio A measured
646/ A
521and the relation between MC-LR concentration can realize detecting the high sensitivity of MC-LR and high-selectivity analysis; Concrete steps are as follows:
(1) in the there-necked flask of cleaning, the gold chloride (HAuCl that 40 ~ 60 mL concentration are 0.03 ~ 0.06 mol/L is added
4) solution, continuous heating is to boiling; Under magnetic agitation, be the HAuCl that 0.03 ~ 0.04 mol/L sodium citrate solution joins boiling by 4 ~ 6 mL concentration
4in solution, continue to stop heating after 20 ~ 40 minutes; Treat that solution is cooled to room temperature, obtain golden nanometer particle colloidal sol with the membrane filtration of 0.22 μm;
(2) the golden nanometer particle colloidal sol that the step (1) adding 400 ~ 450 μ L to sample hose obtains, and add the aptamer solution of the MC-LR of 30 ~ 40 μ L and the NaCl solution of 20 ~ 30 μ L respectively, react after 10 ~ 20 minutes, obtain aptamer colorimetric sensor; Wherein, the base sequence of the aptamer of the MC-LR of use is:
5’-GGCGC-CAAAC-AGGAC-CACCA-TGACA-ATTAC
-CCATA-CCACC-TCATT-ATGCC-CCATC-TCCGC-3’。
The aptamer colorimetric sensor that preparation method of the present invention obtains is applied to the detection of MC-LR, and concrete steps are as follows:
Joined respectively by the MC-LR standard solution of variable concentrations in aptamer colorimetric sensor solution, incubated at room temperature 10 ~ 20 minutes, measures the uv-visible absorption spectra of different sample respectively with ultraviolet-visible spectrophotometer.According to absorbance A
646/ A
521ratio and the logarithm of MC-LR concentration between linear relationship set up working curve.
The aptamer colorimetric sensor that preparation method of the present invention obtains is applied to the selective enumeration method of MC-LR, and concrete steps are as follows:
In the solution containing aptamer colorimetric sensor, add the chaff interference solution of MC-LR solution and 50 times of MC-LR concentration respectively, adopt ultraviolet-visible spectrophotometer to measure the uv-visible absorption spectra of mixed liquor.According to the golden nanometer particle absorbance ratio A that the MC-LR added and chaff interference cause
646/ A
521change realize selectivity analysis to MC-LR; Wherein: described chaff interference is any one in Acetamiprid, glyphosate, metrifonate, clofentezine or Atrazine.
Easy and simple to handle, the colorimetric analysis technology rapidly that responds combine with aptamer technology MC-LR to specific recognition capability by the present invention, have constructed a novel MC-LR aptamer colorimetric sensor.Utilize the recognition component of aptamer molecule as sensor of specific recognition MC-LR function, improve the selectivity of colorimetric sensor, add the sensitivity of colorimetric methods.This sensor can realize detecting the high sensitivity of MC-LR and high-selectivity analysis simultaneously.Compared with the detection technique of existing MC-LR, the concrete following advantage of the present invention:
(1) with traditional analysis as compared with high performance liquid chromatography, mass spectroscopy, the aptamer colorimetric sensor constructed in the present invention do not need by complex large-sized instrument, simple to operate, highly sensitive, can realize to MC-LR quick detection;
(2) compared with existing biology sensor, aptamer solution directly mixes with golden nanometer particle colloidal sol by the present invention, prepares aptamer colorimetric sensor, and the preparation of this sensor is simple, has higher stability and reappearance; Choose synthesis cost low, stability is high, be easy to the recognition component of aptamer as sensor of the advantages such as modification, improves sensitivity and the selectivity of colorimetric methods.
Accompanying drawing explanation
The uv-visible absorption spectra of the aptamer colorimetric sensor solution of the MC-LR of Fig. 1 variable concentrations; Illustration is the aptamer base sequence of MC-LR.
The absorbance ratio A of the aptamer colorimetric sensor that Fig. 2 constructs
646/ A
521with the linear relationship chart of MC-LR log concentration.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment 1
A kind of aptamer colorimetric sensor based on specific base sequence detected fast for MC-LR, with golden nanometer particle colloidal sol for matrix, caused the change of golden nanometer particle aggregation extent by MC-LR and golden nanometer particle competition binding aptamer, realize the analysis to MC-LR.The concrete preparation process of aptamer colorimetric sensor is as follows:
(1) glass apparatus used all cleans up through chloroazotic acid immersion, high purity water and dries stand-by.During preparation, the concentration adding 50 mL in the there-necked flask of cleaning is the gold chloride (HAuCl of 0.05 mol/L
4) solution, continuous heating is to boiling; Under Keep agitation, be the HAuCl that 0.0388 mol/L sodium citrate solution joins boiling by 5 ml concentration
4in solution, reaction continues to stop heating after 30 minutes.Treat that solution is cooled to room temperature, obtain golden nanometer particle colloidal sol with the membrane filtration of 0.22 μm.
(2) in sample hose, add 433 μ L(1) in the golden nanometer particle colloidal sol for preparing, add the aptamer solution of 32 μ L and the NaCl solution of 24 μ L subsequently respectively, after 25 ° of C effect 15min, obtain the aptamer colorimetric sensor of specific recognition MC-LR.The pitch sequence of the aptamer of the MC-LR used in experiment is:
5’-GGCGC-CAAAC-AGGAC-CACCA-TGACA-ATTAC
-CCATA-CCACC-TCATT-ATGCC-CCATC-TCCGC-3’。
Embodiment 2
Two concentration parameters in aptamer colorimetric sensor preparation process: the optimal concentration of aptamer and sodium chloride is determined.Be specially: get a series of golden nanometer particle colloidal sol, respectively to the aptamers solution and the sodium chloride solution that wherein add variable concentrations, the concentration that preparation obtains aptamer is positioned at 1.40 × 10
-8~ 1.40 × 10
-7the scope of mol/L, the concentration of sodium chloride are positioned at a series of aptamer colorimetric sensor solution of 0.01 ~ 0.04 mol/L scope.The A of above-mentioned serial solution is measured respectively with ultraviolet-visible spectrophotometer
646/ A
521.Result of study shows, when the concentration of aptamers is 1.28 × 10
-7mol/L, when the concentration of sodium chloride is 0.024 mol/L, A
646/ A
521ratio maximum.Therefore, 1.28 × 10 are respectively in the optimal concentration of constructing aptamer and the sodium chloride adopted in process of aptamer colorimetric sensor
-7mol/L, 0.024 mol/L.
Embodiment 3
Get MC-LR solid sample, first obtain 1.0 × 10 with high purity water preparation
-3the MC-LR mother liquor of mol/L, then with the high purity water of different volumes, above-mentioned MC-LR mother liquor is diluted respectively, preparation obtains the standard solution of the MC-LR of a series of variable concentrations.The aptamer colorimetric sensor solution prepared in Example 1, respectively to the MC-LR solution wherein adding variable concentrations, after acting on 15min under 25 ° of C, measures the uv-visible absorption spectra of solution with ultraviolet-visible spectrophotometer.Result of study shows, along with the increase of MC-LR concentration in test system, the feature plasma resonance absorption peak of golden nanometer particle is by 521 nm red shift to 646 nm.The concentration of MC-LR is higher, and golden nanometer particle is positioned at the absorbance A at 646 nm places
646larger, be positioned at the absorbance A at 521 nm places
521more and more less, and the ratio A of the two
646/ A
521larger.Utilize A
646/ A
521and the linear relationship between the logarithm of MC-LR concentration sets up working curve, as shown in Figure 1.In the present invention, the range of linearity that aptamer colorimetric sensor measures MC-LR is 7.5 × 10
-6~ 5.0 × 10
-10mol/L, detects and is limited to 3.7 × 10
-10mol/L.
Embodiment 4
The aptamer colorimetric sensor solution prepared in Example 1, respectively to wherein adding 1 × 10
-9the MC-LR standard solution of mol/L and 5 × 10
-8the Atrazine standard solution of mol/L, after acting on 15min, measures the uv-visible absorption spectra of the aptamer colorimetric sensor solution adding MC-LR and Atrazine under 25 ° of C respectively with ultraviolet-visible spectrophotometer.Result of study shows, adds 1 × 10
-9the golden nanometer particle absorptance A that the MC-LR standard solution of mol/L causes
646/ A
521be 0.273, and add the golden nanometer particle absorptance A that 50 times of concentration cause in the Atrazine of MC-LR
646/ A
521be 0.0292, much smaller than the changing value of the absorbance that MC-LR causes.Visible, prepared aptamer colorimetric sensor shows higher selectivity when measuring MC-LR.After the pollutant Atrazine coexisted with MC-LR in environment adds system to be measured, MC-LR is not measured to aptamer colorimetric sensor and cause interference.
Embodiment 5
Get actual lake water sample, first through Filter paper filtering with the wherein contained particle of removing and suspension, carry out multiple times of filtration with the filter membrane of 0.22 μm subsequently, lake water sample be further purified.In the lake water sample after purifying, add MC-LR standard solution, make the content of MC-LR in lake water sample be 1 × 10
-9mol/L.During test, above-mentioned pretreated water sample is joined in aptamer colorimetric sensor solution, after acting on 15min at 25 ° of C temperature, measure the uv-visible absorption spectra of above-mentioned solution with ultraviolet-visible spectrophotometer.Result of study shows, the recovery measuring MC-LR content in lake water sample with the aptamer colorimetric sensor constructed is between 97%, and relative standard deviation (n=3) is 9.2%.From the result of test, the aptamer colorimetric sensor of preparation shows higher sensitivity and accuracy when measuring MC-LR.The analysis being prepared as MC-LR in water body of this aptamer colorimetric sensor detects and provides a kind of simple, fast analytical approach.
Above-mentioned is can understand and apply the invention for the ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to embodiment here, those skilled in the art are according to announcement of the present invention, and the improvement made for the present invention and amendment all should within protection scope of the present invention.
Claims (4)
1. a preparation method for the aptamer colorimetric sensor detected fast for MC-LR, is characterized in that concrete steps are as follows:
(1) in the there-necked flask of cleaning, the gold chloride (HAuCl that 40 ~ 60 mL concentration are 0.03 ~ 0.06 mol/L is added
4) solution, continuous heating is to boiling; Under magnetic agitation, be the HAuCl that 0.03 ~ 0.04 mol/L sodium citrate solution joins boiling by 4 ~ 6 mL concentration
4in solution, continue to stop heating after 20 ~ 40 minutes; Treat that solution is cooled to room temperature, obtain golden nanometer particle colloidal sol with the membrane filtration of 0.22 μm;
(2) the golden nanometer particle colloidal sol that the step (1) adding 400 ~ 450 μ L to sample hose obtains, and add the aptamer solution of the MC-LR of 30 ~ 40 μ L and the NaCl solution of 20 ~ 30 μ L respectively, react after 10 ~ 20 minutes, obtain aptamer colorimetric sensor; Wherein, the base sequence of the aptamer of the MC-LR of use is:
5’-GGCGC-CAAAC-AGGAC-CACCA-TGACA-ATTAC
-CCATA-CCACC-TCATT-ATGCC-CCATC-TCCGC-3’。
2. the aptamer colorimetric sensor that preparation method obtains as claimed in claim 1 is applied to a detection of MC-LR, it is characterized in that concrete steps are as follows:
Joined respectively by the MC-LR standard solution of variable concentrations in aptamer colorimetric sensor solution, incubated at room temperature 10 ~ 20 minutes, measures the uv-visible absorption spectra of different sample, respectively according to absorbance A with ultraviolet-visible spectrophotometer
646/ A
521ratio and the logarithm of MC-LR concentration between linear relationship set up working curve.
3. the aptamer colorimetric sensor that preparation method obtains as claimed in claim 1 is applied to a selective enumeration method of MC-LR, it is characterized in that concrete steps are as follows:
In the solution containing aptamer colorimetric sensor, add the chaff interference solution of MC-LR solution and 50 times of MC-LR concentration respectively, adopt ultraviolet-visible spectrophotometer to measure the uv-visible absorption spectra of mixed liquor.
4. according to the golden nanometer particle absorbance ratio A that the MC-LR added and chaff interference cause
646/ A
521change realize selectivity analysis to MC-LR; Wherein: described chaff interference is any one in Acetamiprid, glyphosate, metrifonate, clofentezine or Atrazine.
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| CN105866044A (en) * | 2016-06-23 | 2016-08-17 | 湖北出入境检验检疫局检验检疫技术中心 | Quick detector for microcystic toxins |
| CN107153041A (en) * | 2017-05-11 | 2017-09-12 | 同济大学 | The preparation and application for the aptamer colorimetric sensor that Polychlorinated biphenyls 77 is detected |
| CN107505298A (en) * | 2017-08-17 | 2017-12-22 | 上海应用技术大学 | A kind of method that cyromazine in milk is detected based on G tetrad aptamers fluorescence probe |
| CN108344863A (en) * | 2018-02-08 | 2018-07-31 | 中国科学院重庆绿色智能技术研究院 | A kind of new method of the nano-pore detection Microcystin based on aptamers-nanogold sensing |
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Non-Patent Citations (8)
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866044A (en) * | 2016-06-23 | 2016-08-17 | 湖北出入境检验检疫局检验检疫技术中心 | Quick detector for microcystic toxins |
| CN108732167A (en) * | 2017-04-17 | 2018-11-02 | 天津大学 | The method that rapid field detection is carried out to Polychlorinated biphenyls total amount with papery detection device |
| CN107153041A (en) * | 2017-05-11 | 2017-09-12 | 同济大学 | The preparation and application for the aptamer colorimetric sensor that Polychlorinated biphenyls 77 is detected |
| CN107505298A (en) * | 2017-08-17 | 2017-12-22 | 上海应用技术大学 | A kind of method that cyromazine in milk is detected based on G tetrad aptamers fluorescence probe |
| CN107505298B (en) * | 2017-08-17 | 2020-05-12 | 上海应用技术大学 | Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe |
| CN108344863A (en) * | 2018-02-08 | 2018-07-31 | 中国科学院重庆绿色智能技术研究院 | A kind of new method of the nano-pore detection Microcystin based on aptamers-nanogold sensing |
| CN109490262A (en) * | 2018-10-23 | 2019-03-19 | 中国科学院重庆绿色智能技术研究院 | Microcystin sensor and preparation method, application based on zinc oxide nanowire |
| CN110261340A (en) * | 2019-05-24 | 2019-09-20 | 同济大学 | A kind of quick visualization analyzes the method and sensor of a variety of phthalate total amount of pollutant |
| CN112680451A (en) * | 2021-03-15 | 2021-04-20 | 中南大学 | CRISPR/Cas fluorescence sensor based on metal organic framework material and preparation method and application thereof |
| CN112680451B (en) * | 2021-03-15 | 2021-09-21 | 中南大学 | CRISPR/Cas fluorescence sensor based on metal organic framework material and preparation method and application thereof |
| CN113959968A (en) * | 2021-11-18 | 2022-01-21 | 南京林业大学 | Method for detecting microcystin MC-LR by ratio type colorimetric aptamer sensor based on AuNPs |
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