CN102507921A - Method for detecting microcystin - Google Patents
Method for detecting microcystin Download PDFInfo
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- CN102507921A CN102507921A CN2011103121714A CN201110312171A CN102507921A CN 102507921 A CN102507921 A CN 102507921A CN 2011103121714 A CN2011103121714 A CN 2011103121714A CN 201110312171 A CN201110312171 A CN 201110312171A CN 102507921 A CN102507921 A CN 102507921A
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- microcystin
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- stranded dna
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Abstract
The invention belongs to the field of water pollutant detection and treatment and particularly relates to a method for detecting the content of microcystin by the graphene oxide fluorescence quenching caused by fluorescence resonance energy transfer between graphene oxide and gold nanoparticles. The method has high sensitivity and specificity and is suitable for the microcystin trace detection. The limit of detection of the method is lower than the standard of the world health organization.
Description
Technical field
The present invention relates to water pollutant and detect and the improvement field particularly a kind of method that detects Microcystin.
Background technology
Microcystin is a kind of common water pollutant, is produced by prolific blue-green algae in eutrophication water.Dead when blue-green algae, behind the cell rupture, Microcystin will dissolve in the water, and water body periphery people and animals' life security is worked the mischief.World Health Organization's regulation, the content of microcapsule algae toxin must not surpass 1 μ g/L in the potable water.Serious environmental incidents such as all the time, the report of the environmental problem that causes about Microcystin both at home and abroad is of common occurrence, and freshwater lakes such as Taihu Lake, Chaohu almost all blue algae bloom will take place every year, and resident living water is contaminated.Therefore, the trace detection of Microcystin is for ensureing that people's life safety has great importance.
The method of the detection Microcystin of widely using at present mainly contains Capillary Electrophoresis, high performance liquid chromatography; Liquid chromatograph mass spectrography, gas chromatography-mass spectrography, raman detection; Electrochemical method and some biological methods join analytic approach, phosphoprotein phosphatase inhibition analysis method etc. like enzyme.Wherein, the method for liquid chromatography and combined gas chromatography mass spectrometry is owing to have higher separation efficiency and quite good detecting sensitivity widespread use in the Microcystin of reality detects, but its sample pretreatment process complicacy; Need large-scale instrument; Cost is high, and testing process mostly needs large-scale instruments such as chromatogram, mass spectrum; Need in the processing procedure to introduce some toxic solvents (acetonitrile etc.), be unfavorable for the detection of actual water sample; Electrochemical method is used various material modified electrodes and is realized the indirect detection to Microcystin, but majority need through the competition indirect detection, reduce the scope of practical application with enzyme labeling Microcystin molecule (can not guarantee productive rate); Enzyme joins the method that analytic approach is the present detection Microcystin of using always, and high commercial kit utilizes competing method, under the prerequisite that possesses microcystin monoclonal antibody, the pure toxin of standard and relevant reagent; Chromogenic substrate according to adding carries out chromogenic assay, is a kind of very easy, and is efficient; Method fast; But the antibody type that needs is many, and consumption is big, then needs wide spectrum antibody as if multiple homolog is discerned.Joining analytic approach with enzyme compares; Phosphoprotein phosphatase inhibition analysis method has higher sensitivity, can detect all Microcystin homolog total amounts in the sample, but need radioisotopic mark usually; Make relatively difficulty of subsequent treatment, can't develop into conventional method of environmental monitoring.Recently; Raman spectrum also is applied in the detection of Microcystin, and the concentration through Microcystin in the concentrating sample on hydrophobic substrate realizes fast detecting, but need in the sample pretreatment process, use step such as SPE; The pre-treatment process is complicated; Length consuming time needs the professional to operate, and brings certain inconvenience to practical application.
FRET is a kind of high-sensitive analytical technology; It is meant when the donor electronics and receives excite transitions when the out orbit, can with the electron interaction of acceptor, realize energy delivery; Cause the acceptor electronics to be excited, and the donor electronics itself is got back to the process of ground state.This process receives the influence of distance between donor and the acceptor, generally in 1～10nm scope, takes place, and the further increase of distance can cause FRET to weaken until disappearance.Therefore the selection of donor and acceptor is very important.Graphene and derivant thereof are high like: electric conductivity because of its superior performance in recent years, and surface area is big, is easy to biomolecule modification etc., is widely used in the preparation of various sensors.In the application of FRET, graphene oxide is all being brought into play very big advantage aspect donor and the acceptor two.As acceptor, graphene oxide can be accepted from quantum dot, and the excitation electron of various fluorescence molecules is as the quencher of fluorescence.Typical application is to detect the mispairing of dna molecular and DNA base.As donor, Graphene can provide the electronics of excited state under the excitation wavelength of 400nm, energy is transferred on the acceptor, makes the fluorescent quenching of itself.This character is applied to pathogen, the detection of bacterium.
Therefore, provide a kind of based on graphene oxide, utilize the FRET principle to detect the method for Microcystin, have practical significance.
Summary of the invention
In view of this; The present invention provides a kind of method that detects Microcystin; The graphene oxide fluorescent quenching that this method causes through generation FRET between graphene oxide and the golden nanometer particle detects the content of Microcystin; Have higher sensitivity, selectivity, detectability is lower than WHO standard, is applicable to the trace detection of Microcystin.
In order to realize the foregoing invention purpose, the present invention provides following technical scheme:
The invention provides a kind of method that detects Microcystin, may further comprise the steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Said graphene oxide is connected through electrostatic force with said substrate;
Get Microcystin antibody and said graphene oxide is covalently bound, obtain at the bottom of the donor-based;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with said golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Said single stranded DNA has 12～20 bases;
Getting said single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations; Said Microcystin combines with the ditch of said single stranded DNA; Obtain the standard items receptor complex; Again with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly, through the antigen and antibody specific reaction, obtains graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound; Said graphene oxide and said golden nanometer particle generation FRET obtain the first fluorescent quenching intensity through fluoroscopic examination; The complementary strand of said single stranded DNA combines with said single stranded DNA specificity, eliminates the non-specific binding of said single stranded DNA and said graphene oxide;
After getting said single stranded DNA-golden nano-complexes and testing sample mixing; Again successively with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly; Obtain the second fluorescent quenching intensity through fluoroscopic examination; With the concentration of said standard items and the said first fluorescent quenching strength ratio, obtain the content of Microcystin in the said testing sample.(microcystin, MC), its main architectural feature is N-methyl dehydroalanine and two L-amino acid residue X and Z to Microcystin, according to Microcystin (Microcystins or MCYST) the nomenclature regulation of formulating in 1988.The various combination of X, Z two residues is distinguished by the letter suffix of represented amino acid.Common have a LR, RR, and three kinds of toxin of YR, L, R, Y represent leucine, arginine, tyrosine respectively.The general structure of Microcystin is ring (D-alanine-L-X-red-the different aspartic acid of Beta-methyl-D--L-Z-Adda-D-isoglutamic acid-N-methyl dehydroalanine); Adda (3 amino 9-methylaminos-2 wherein; 6; 8-trimethyl 10-phenyl-4, the 6-dienoic acid) be that the Microcystin biologically active is expressed necessary.Therefore, the antibody of Adda group of selecting Microcystin among the present invention is as Microcystin antibody.
Microcapsule algae toxin is the algae toxin of being illustrated chemical constitution the earliest. in to the research of algae toxin also many with it as research object.It is 7 peptide molecules of a ring-type, and molecular weight is about 1000 dalton, and the incident by the initiation of algae toxin that many countries occur is mostly relevant with microcapsule algae toxin.Therefore, the present invention is primarily aimed at microcapsule algae toxin and Microcystin-RR studies.
The drinking water standard that The World Health Organization (WHO) is recommended instructs in (second edition) and has stipulated that the examination criteria of Microcystin is: microcapsule algae toxin, 1 μ g/L; Drinking Water hygienic quality standard (2001) the regulation Microcystin content that the existing promulgation of China is carried out is 0.001mg/L, water environment quality standard (GB3838-2002) regulation, and the content of microcapsule algae toxin is 0.001mg/L.Therefore, detection method provided by the invention, the concentration of standard items is chosen as 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1μ g/L, 1 μ g/L, 2.5 μ g/L.Obtain the second fluorescent quenching intensity through detecting; With the concentration of said standard items and the said first fluorescent quenching strength ratio; Obtain the content of Microcystin in the said testing sample, as long as this content is lower than World Health Organization's specified standard 1 μ g/L, it is qualified to be.In the above-mentioned testing process, the lake water that testing sample can contain Microcystin through the Microcystin simulation of in lake water, adding concentration known detects, and obtains the second fluorescent quenching intensity.Again with the concentration of Microcystin standard items and its corresponding first fluorescent quenching strength ratio, if difference is not remarkable, promptly prove the accuracy and the feasibility of detection method provided by the invention.Also can be in blue algae bloom season; Water sampling detects comparison respectively according to detection method provided by the invention, the present detection method of generally using; If difference is not remarkable; Can show that also detection method provided by the invention has feasibility and accuracy, but the method for sampling receives environment, season, water pollution degree affect bigger, generally takes to simulate the lake water that contains Microcystin and detects as testing sample.
In preparation graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound process; Single stranded DNA in single stranded DNA-golden nanometer particle compound can not have Microcystin in the presence of be adsorbed onto the donor-based basal surface; Compatibility is different on the graphene oxide surface based on single doubly-linked DNA; With complementary strand combine can disturb the interaction between single stranded DNA and graphene oxide; The single stranded DNA that causes non-special absorption comes off from the graphene oxide surface, eliminates single stranded DNA and the non-specific adsorption of Graphene in single stranded DNA-golden nanometer particle, makes between concentration and the fluorescent quenching intensity of Microcystin and forms dependence.Therefore, in the detection method provided by the invention, hatch jointly at the bottom of utilizing complementary strand and standard items receptor complex, the donor-based of single stranded DNA.
As preferably, in the detection method of Microcystin provided by the invention, substrate is selected from glass sheet, mica sheet or silicon chip.
As preferably, in the detection method of Microcystin provided by the invention, get 3-aminopropyltriethoxywerene werene ((3-aminopropyl) triethoxysilane; APTE S) modifies substrate, make base strap positive electricity, combine through electrostatic force with electronegative graphene oxide.
Microcystin can combine with the ditch of DNA, and as preferably, DNA is strand adenine and strand thymine.
As preferably, in the detection method provided by the invention, Microcystin standard items concentration is respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1μ g/L, 1 μ g/L, 2.5 μ g/L.
As preferably, the condition of hatching in moist chamber 37 ℃ cultivate 3h.
The invention provides a kind of method that detects Microcystin; The graphene oxide fluorescent quenching that this method causes through generation FRET between graphene oxide and the golden nanometer particle detects the content of Microcystin; Have higher sensitivity, selectivity; Detectability is lower than WHO standard, is applicable to the trace detection of Microcystin.
Description of drawings
Fig. 1 shows the interaction of Microcystin and DNA; Wherein, Fig. 1 (a) shows the red shift phenomenon, from the bottom to top curve successively the concentration of corresponding DNA be 0 to 18ng/ μ L (increasing progressively) with 2ng/ μ L, the concentration of Microcystin is 2 μ mol/L; Fig. 1 (b) shows hypochromic effect, is the ultraviolet absorption value summation (under the 240nm) of the ultraviolet absorption value and the DNA of Microcystin, shows the ultraviolet absorption value (under the 240nm) of the compound that Microcystin and DNA form;
Fig. 2 shows the quenching effect of Microcystin to EB-DNA compound fluorescence; Wherein, Fig. 2 (a) shows that Microcystin under the situation that does not have DNA does not have the cancellation effect to the fluorescence of EB; Fig. 2 (b) is shown in DNA and exists down, and Microcystin and EB-DNA compound interact, to the cancellation effect of EB fluorescence; Wherein, the corresponding successively Microcystin concentration of curve a to f is 0,2,4,6,8,10 μ mol/L;
Fig. 3 shows the interaction between Microcystin and DNA base; Wherein, Fig. 3 (a) shows the interaction between adenine and Microcystin; Fig. 3 (b) shows the interaction between thymine and Microcystin; Fig. 3 (c) shows the interaction between guanine and Microcystin; Fig. 3 (d) shows the interaction between cytimidine and Microcystin; Fig. 3 (a) is to Fig. 3 (d); Horizontal ordinate is for detecting wavelength, and ordinate is the ultraviolet absorption peak of Microcystin, and the concentration of Microcystin is 2 μ mol/L; The concentration of A, T, C, G is 0.3 μ mol/L, respectively corresponding 0 to the 20 μ mol/L (increasing progressively with 2 μ mol/L) of curve shown in from 0 to 10;
Fig. 4 shows the influence of the Microcystin of variable concentrations to dna molecular; Wherein, the concentration of the corresponding Microcystin of Fig. 4 (a) is 0ng/ μ L, and the concentration of the corresponding Microcystin of Fig. 4 (b) is 1ng/ μ L, and the concentration of the corresponding Microcystin of Fig. 4 (c) is 2.5ng/ μ L, and the concentration of the corresponding Microcystin of Fig. 4 (d) is 4ng/ μ L;
Fig. 5 shows the ultraviolet absorption curve after microcapsule algae toxin (MC-LR) and Microcystin-RR (MC-RR) and the few chain DNA A15 effect;
Fig. 6 shows golden nanometer particle and few chain DNA A
15The variation of effect back absorbance;
Fig. 7 shows the non-specific adsorption effect of single stranded DNA and graphene oxide, and the effect that utilizes this non-specific adsorption of complementary strand elimination of this single stranded DNA; Wherein, Fig. 7 (a) shows the atomic force displaing micro picture of graphene oxide-Microcystin antibody; Fig. 7 (b) shows the atomic force displaing micro picture after graphene oxide-Microcystin antibody and single stranded DNA-golden nanometer particle are hatched; After Fig. 7 (c) shows that graphene oxide-Microcystin antibody and single stranded DNA-golden nanometer particle are hatched, the atomic force displaing micro picture after the complementary strand of this single stranded DNA is handled; Atomic force displaing micro picture after Fig. 7 (d) shows graphene oxide-Microcystin antibody and Microcystin-single stranded DNA-golden nanometer particle combines; After Fig. 7 (e) shows graphene oxide-Microcystin antibody and Microcystin-single stranded DNA-golden nanometer particle combines, the atomic force displaing micro picture after the complementary strand of this single stranded DNA is handled;
Fig. 8 shows the atomic force phenogram of graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle synthesis step; Wherein, Fig. 8 (a) shows the atomic force phenogram at the bottom of the donor-based; Fig. 8 (b) shows the atomic force phenogram of eliminating the non-special absorption between Microcystin and Graphene; Fig. 8 (c) shows the atomic force phenogram on the Graphene surface of Microcystin antibody modification; Fig. 8 (d) shows the atomic force phenogram that Microcystin and Microcystin antibody specificity combine the back surface topography to change; Fig. 8 (e) shows that golden nanometer particle is attached to the atomic force phenogram of Graphene surface topography;
Fig. 9 shows that the Microcystin of variable concentrations causes the reduction of Graphene fluorescence intensity; Wherein, Fig. 9 (a) shows the variation of the fluorescence intensity in graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle building-up process; The corresponding successively from left to right graphene oxide of cylindricality, graphene oxide-Microcystin antibody, graphene oxide-Microcystin antibody-Microcystin, graphene oxide-Microcystin antibody-single stranded DNA-golden nanometer particle, graphene oxide-Microcystin antibody-single stranded DNA-golden nanometer particle are handled through the complementary strand of this single stranded DNA again; Fig. 9 (b) shows the variation of the fluorescence intensity in the microcapsule algae toxin standard items of variable concentrations; The corresponding successively from left to right graphene oxide of cylindricality, blank group, concentration are respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The microcapsule algae toxin of μ g/L, 1 μ g/L, 2.5 μ g/L; Fig. 9 (c) shows the variation of the fluorescence intensity in the Microcystin-RR standard items of variable concentrations; The corresponding successively from left to right graphene oxide of cylindricality, blank group, concentration are respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin-RR of μ g/L, 1 μ g/L, 2.5 μ g/L; Fig. 9 (d) shows the variation that contains the fluorescence intensity of microcapsule algae toxin in the simulation testing sample; The corresponding successively from left to right graphene oxide of cylindricality, blank group, concentration are respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The microcapsule algae toxin of μ g/L, 1 μ g/L, 2.5 μ g/L; Fig. 9 (e) shows the variation that contains the fluorescence intensity of Microcystin-RR in the simulation testing sample; The corresponding successively from left to right graphene oxide of cylindricality, blank group, concentration are respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin-RR of μ g/L, 1 μ g/L, 2.5 μ g/L; After Fig. 9 (f) shows that Microcystin and saxitoxin (NEO and STX) are hatched with single stranded DNA-golden nanometer particle respectively; Behind graphene oxide-Microcystin antibody incubation; The respectively variation of fluorescence intensity, the corresponding successively from left to right graphene oxide of cylindricality, blank group, concentration are that the microcapsule algae toxin of 1 μ g/L, Microcystin-RR that concentration is 1 μ g/L, saxitoxin-NEO, the concentration that concentration is 1 μ g/L are saxitoxin-STX of 1 μ g/L.
Embodiment
The invention discloses a kind of method that detects Microcystin.Those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order to realize the foregoing invention purpose, the present invention provides following technical scheme:
The invention provides a kind of method that detects Microcystin, may further comprise the steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Said graphene oxide is connected through electrostatic force with said substrate;
Get Microcystin antibody and said graphene oxide is covalently bound, obtain at the bottom of the donor-based;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with said golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Said single stranded DNA has 12～20 bases;
Getting said single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations; Said Microcystin combines with the ditch of said single stranded DNA; Obtain the standard items receptor complex; Again with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly, through the antigen and antibody specific reaction, obtains graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound; Said graphene oxide and said golden nanometer particle generation FRET obtain the first fluorescent quenching intensity through fluoroscopic examination; The complementary strand of said single stranded DNA combines with said single stranded DNA specificity, eliminates the non-specific binding of said single stranded DNA and said graphene oxide;
After getting said single stranded DNA-golden nano-complexes and testing sample mixing; Again successively with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly; Obtain the second fluorescent quenching intensity through fluoroscopic examination; According to the concentration and the said first fluorescent quenching intensity of said standard items, obtain the content of Microcystin in the said testing sample.
Ultraviolet is passed through in the interaction of microcapsule algae toxin, Microcystin-RR and DNA, fluorescence, and methods such as atomic force microscope prove.As can be seen from Figure 1, along with the adding of DNA, the uv absorption of Microcystin has tangible increase, and the red shift phenomenon is shown in Fig. 1 (a), and hypochromic effect is shown in Fig. 1 (b), and this shows that existence interacts between the two.Fig. 2 has embodied the cancellation to EB-DNA compound fluorescence of microcapsule algae toxin, Microcystin-RR.Since the fluorescence of DNA itself very a little less than, can not directly detect, the ethidium bromide (EB) that we utilize can be inserted between the DNA base, makes the fluorescence intensity enhancing of the two compound, reaches the intensity that can detect.Fig. 2 (a) has shown that microcapsule algae toxin under the situation that does not have DNA does not have the cancellation effect to the fluorescence of EB; Fig. 2 (b) has shown that microcapsule algae toxin under the situation that has DNA, Microcystin-RR have the fluorescence of EB the cancellation effect is arranged; Therefore, the fluorescent quenching phenomenon among Fig. 2 causes owing to microcapsule algae toxin, Microcystin-RR and EB-DNA compound interact.Embodied the interaction between microcapsule algae toxin, Microcystin-RR and DNA base among Fig. 3.Increase along with base concentration; The ultraviolet absorption peak of microcapsule algae toxin, Microcystin-RR obviously increases and red shift; But the signal of adenine and thymine obviously is better than guanine and cytimidine; And with the signal similar after dna molecular and microcapsule algae toxin, the Microcystin-RR effect among Fig. 1 (a); Microcapsule algae toxin, Microcystin-RR are prone to combine with adenine and thymine, and the compact district of adenine and thymine forms ditch in dna molecular, therefore judge microcapsule algae toxin, Microcystin-RR and DNA to combine be the ditch combination.In addition through calculating the binding constant between microcapsule algae toxin, Microcystin-RR and the DNA, in conjunction with than the ditch combination that has confirmed between the two.Shown the influence of the microcapsule algae toxin, Microcystin-RR of variable concentrations among Fig. 4 to dna molecular; Increase along with microcapsule algae toxin, Microcystin-RR concentration; Dna molecular progressively twines, and finally forms bar-shaped aggegation, has further proved the interaction of the two.Shown the ultraviolet absorption curve after microcapsule algae toxin, Microcystin-RR and the few chain DNA A15 effect among Fig. 5; Fig. 6 has shown the variation of golden nanometer particle and few chain DNA A15 effect back absorbance, has proved that all Au-DNA-Microcystin compound can form.
In preparation graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound process; Single stranded DNA in single stranded DNA-golden nanometer particle compound can not have Microcystin in the presence of be adsorbed onto the donor-based basal surface shown in Fig. 7 (a), see Fig. 7 (b).Compatibility is different on the graphene oxide surface based on single doubly-linked DNA; With complementary strand combine can disturb the interaction between single stranded DNA and graphene oxide; The single stranded DNA that causes non-special absorption comes off from the graphene oxide surface; The single stranded DNA in elimination single stranded DNA-golden nanometer particle and the non-specific adsorption of Graphene; Significantly be less than Fig. 7 (b) dotted thing like Fig. 7 (c) dotted thing, Fig. 7 (e) dotted thing significantly is less than Fig. 7 (d) dotted thing, makes between concentration and the fluorescent quenching intensity of Microcystin and forms dependence.Therefore, in the detection method provided by the invention, hatch jointly at the bottom of utilizing complementary strand and standard items receptor complex, the donor-based of single stranded DNA.
As preferably, in the detection method of Microcystin provided by the invention, substrate is selected from glass sheet, mica sheet or silicon chip.
As preferably, in the detection method of Microcystin provided by the invention, get 3-aminopropyltriethoxywerene werene ((3-aminopropyl) triethoxysilane; APTES) modify substrate, make base strap positive electricity, combine through electrostatic force with electronegative graphene oxide.
Microcystin can combine with the ditch of DNA, and as preferably, DNA is strand adenine and strand thymine.
As preferably, in the detection method provided by the invention, Microcystin standard items concentration is respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1μ g/L, 1 μ g/L, 2.5 μ g/L.
The invention provides a kind of method that detects Microcystin; The graphene oxide fluorescent quenching that this method causes through generation FRET between graphene oxide and the golden nanometer particle detects the content of Microcystin; Have higher sensitivity, selectivity; Detectability is lower than WHO standard, is applicable to the trace detection of Microcystin.
Agents useful for same all can be buied by market in the detection method provided by the invention.Wherein, Microcystin (microcapsule algae toxin, Microcystin-RR), saxitoxin (STX, NEO) and all Jim Press Science and Technology Ltd. (Beijing) buy from her to the antibody of Microcystin Adda group; Few chain DNA (SH-A
15, T
15, SH-G
12, C
12, SH-A
20, T
20, SH-T
18, A
18) worker's biotechnology company is synthetic available from giving birth to, directly do not use through purifying; Ethyl dimethyl amine propyl carbodiimide diimine (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; EDC), N-hydroxysulphosuccinimide (N-hydroxy sulfosuccinimide; Sulfo-NHS), 3-aminopropyltriethoxywerene werene ((3-aminopropyl) triethoxysilane; APTES) and N, N-diisopropyl ethyl amine borane complex (N, N-diisopropylethylamine), agents useful for same be analyze pure; The antibody damping fluid is phosphate buffer (pH=7), and all solution are all used ultrapure water (18.2M Ω cm) preparation.
Below in conjunction with embodiment, further set forth the present invention:
Embodiment 1
The preparation of water soluble oxidized Graphene:
Take by weighing the 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, the 1g potassium persulfate, in the 2.5g phosphorus pentoxide mixed solution, cool to room temperature behind the constant temperature 4.5h, drying is filtered in the deionized water dilution.The dag of handling well is added 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir the maintenance temperature and be lower than 20 ℃, be heated to 35 ℃ at last and stir 2h.Add 280mL water and add the 8mL30% hydrogen peroxide at once, solution became glassy yellow and had bubble to generate this moment, filtered, and cleaned with hydrochloric acid and water respectively, and the dialysis back is dry, and ultrasonic 30min forms the 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, feed argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splash in two small containers in exsiccator bottom and continue logical argon gas 3min.After putting into clean glass sheet, logical again argon gas 3min shifts out then, makes glass sheet be exposed to 2h in the APTES steam.Then, shift out APTES, the sealing vacuum dryer, ready-made glass sheet is kept in the exsiccator up to use always.
Preparation at the bottom of the donor-based:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise to and leans on electrostatic force to form array on the glass sheet that APTES modifies and in moist chamber, dry.After process washing and nitrogen dried up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) dripped and also cultivate at 37 ℃, and 3h comes the carboxyl on active oxidation Graphene surface, washes then and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to the graphene oxide surface on same point.
The preparation of golden nanometer particle:
(0.1mol/L 1.94mL) is added drop-wise to the HAuCl of boiling to sodium citrate solution fast
4The WS (50mL H
2O, 0.167mL 10%HAuCl
4) in, and continue to stir.Potpourri continues boiling 40min, forms the aurosol of claret, and in stirring cool to room temperature.
The preparation of single stranded DNA-golden nanometer particle:
Get the synthetic golden nanometer particle of 3mL and transfer in the clean bottle, with 20 μ L, 10 μ mol/L HS-A
15(15mer:5 '-SH AAA AAA AAA AAA AAA-3 ') hatch jointly.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution that obtains changes per minutes with 13000, at every turn 15min.Au-DNA (about 10nmol/L) be kept in phosphoric acid buffer (PB) solution (5mmol/L PB, 0.1mol/L NaCl, pH=7).
The preparation of single stranded DNA-golden nanometer particle-Microcystin standard items:
Get concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items pure water solution of μ g/L, 1 μ g/L, 2.5 μ g/L mixes with golden nanometer particle-single stranded DNA respectively, obtains to contain 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin standard items-single stranded DNA-golden nanometer particle the compound of the Microcystin standard items of μ g/L, 1 μ g/L, 2.5 μ g/L.
The preparation of single stranded DNA-golden nanometer particle-testing sample:
Gather South Lake, Changchun water sample, prepare concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items South Lake WS of μ g/L, 1 μ g/L, 2.5 μ g/L, the simulation actual water sample as testing sample, is mixed with single stranded DNA-golden nanometer particle respectively, obtains testing sample-single stranded DNA-golden nanometer particle compound.
The preparation of graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound:
At the bottom of getting the donor-based of preparation, the Microcystin standard items-single stranded DNA-golden nanometer particle compound of preparation, the complementary strand of single stranded DNA---few chain DNA T
15(15mer:5 '-TTT TTT TTT TTT TTT-3 ') 37 ℃ of cultivation 3h in moist chamber; Utilize the antigen and antibody specific recognition reaction between Microcystin and the Microcystin antibody, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound.Because the space length of graphene oxide and golden nanometer particle shortens; FRET takes place, and the fluorescence intensity of Graphene weakens, through detecting; Obtain scanning fluoroscopic image in excitation wavelength at 534nm with Leica TCS SP2 Laser Scanning Confocal Microscope, obtain the first fluorescent quenching intensity.
According to the method described above, get at the bottom of the donor-based of testing sample-single stranded DNA-golden nanometer particle compound, preparation, complementary strand---the few chain DNA T of single stranded DNA
15(15mer:5 '-TTT TTT TTT TTTTTT-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, again according to the concentration and the first fluorescent quenching intensity of Microcystin standard items, obtain the content of Microcystin.
The preparation of water soluble oxidized Graphene:
Take by weighing the 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, the 1g potassium persulfate, in the 2.5g phosphorus pentoxide mixed solution, cool to room temperature behind the constant temperature 4.5h, drying is filtered in the deionized water dilution.The dag of handling well is added 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir the maintenance temperature and be lower than 20 ℃, be heated to 35 ℃ at last and stir 2h.Add 280mL water and add the 8mL30% hydrogen peroxide at once, solution became glassy yellow and had bubble to generate this moment, filtered, and cleaned with hydrochloric acid and water respectively, and the dialysis back is dry, and ultrasonic 30min forms the 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, feed argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splash in two small containers in exsiccator bottom and continue logical argon gas 3min.After putting into clean glass sheet, logical again argon gas 3min shifts out then, makes glass sheet be exposed to 2h in the APTES steam.Then, shift out APTES, the sealing vacuum dryer, ready-made glass sheet is kept in the exsiccator up to use always.
Preparation at the bottom of the donor-based:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise to and leans on electrostatic force to form array on the glass sheet that APTES modifies and in moist chamber, dry.After process washing and nitrogen dried up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) dripped and also cultivate at 37 ℃, and 3h comes the carboxyl on active oxidation Graphene surface, washes then and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to the graphene oxide surface on same point.
The preparation of golden nanometer particle:
(0.1mol/L 1.94mL) is added drop-wise to the HAuCl of boiling to sodium citrate solution fast
4The WS (50mL H
2O, 0.167mL 10%HAuCl
4) in, and continue to stir.Potpourri continues boiling 40min, forms the aurosol of claret, and in stirring cool to room temperature.
The preparation of single stranded DNA-golden nanometer particle:
Get the synthetic golden nanometer particle of 3mL and transfer in the clean bottle, with 20 μ L, 10 μ mol/L HS-T
12(12mer:5 '-SH TTT TTT TTT TTT-3 ') the common cultivation.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution that obtains changes per minutes with 13000, at every turn 15min.Au-DNA (about 10nmol/L) be kept in phosphoric acid buffer (PB) solution (5mmol/L PB, 0.1mol/L NaCl, pH=7).
The preparation of single stranded DNA-golden nanometer particle-Microcystin standard items:
Get concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items pure water solution of μ g/L, 1 μ g/L, 2.5 μ g/L mixes with golden nanometer particle-single stranded DNA respectively, obtains to contain 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin standard items-single stranded DNA-golden nanometer particle the compound of the Microcystin standard items of μ g/L, 1 μ g/L, 2.5 μ g/L.
The preparation of single stranded DNA-golden nanometer particle-testing sample:
Gather South Lake, Changchun water sample, prepare concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items South Lake WS of μ g/L, 1 μ g/L, 2.5 μ g/L, the simulation actual water sample as testing sample, is mixed with single stranded DNA-golden nanometer particle respectively, obtains testing sample-single stranded DNA-golden nanometer particle compound.
The preparation of graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound:
At the bottom of getting the donor-based of preparation, the Microcystin standard items-single stranded DNA-golden nanometer particle compound of preparation, the complementary strand of single stranded DNA---few chain DNA A
12(12mer:5 '-AAA AAA AAAAAA-3 ') 37 ℃ of cultivation 3h in moist chamber; Utilize the antigen and antibody specific recognition reaction between Microcystin and the Microcystin antibody, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound.Because the space length of graphene oxide and golden nanometer particle shortens; FRET takes place, and the fluorescence intensity of Graphene weakens, through detecting; Obtain scanning fluoroscopic image in excitation wavelength at 534nm with Leica TCSSP2 Laser Scanning Confocal Microscope, obtain the first fluorescent quenching intensity.
According to the method described above, get at the bottom of the donor-based of testing sample-single stranded DNA-golden nanometer particle compound, preparation, complementary strand---the few chain DNA A of single stranded DNA
12(12mer:5 '-AAA AAA AAAAAA-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, again according to the concentration and the first fluorescent quenching intensity of Microcystin standard items, obtain the content of Microcystin.
The preparation of water soluble oxidized Graphene:
Take by weighing the 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, the 1g potassium persulfate, in the 2.5g phosphorus pentoxide mixed solution, cool to room temperature behind the constant temperature 4.5h, drying is filtered in the deionized water dilution.The dag of handling well is added 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir the maintenance temperature and be lower than 20 ℃, be heated to 35 ℃ at last and stir 2h.Add 280mL water and add the 8mL30% hydrogen peroxide at once, solution became glassy yellow and had bubble to generate this moment, filtered, and cleaned with hydrochloric acid and water respectively, and the dialysis back is dry, and ultrasonic 30min forms the 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, feed argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splash in two small containers in exsiccator bottom and continue logical argon gas 3min.After putting into clean glass sheet, logical again argon gas 3min shifts out then, makes glass sheet be exposed to 2h in the APTES steam.Then, shift out APTES, the sealing vacuum dryer, ready-made glass sheet is kept in the exsiccator up to use always.
Preparation at the bottom of the donor-based:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise to and leans on electrostatic force to form array on the glass sheet that APTES modifies and in moist chamber, dry.After process washing and nitrogen dried up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) dripped and also cultivate at 37 ℃, and 3h comes the carboxyl on active oxidation Graphene surface, washes then and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to the graphene oxide surface on same point.
The preparation of golden nanometer particle:
(0.1mol/L 1.94mL) is added drop-wise to the HAuCl of boiling to sodium citrate solution fast
4The WS (50mL H
2O, 0.167mL 10%HAuCl
4) in, and continue to stir.Potpourri continues boiling 40min, forms the aurosol of claret, and in stirring cool to room temperature.
The preparation of single stranded DNA-golden nanometer particle:
Get the synthetic golden nanometer particle of 3mL and transfer in the clean bottle, with 20 μ L, 10 μ mol/L HS-A
20(20mer:5 '-SH AAAAAAAAAAAAAAAAAAAA-3 ') the common cultivation.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution that obtains changes per minutes with 13000, at every turn 15min.Au-DNA (about 10nmol/L) be kept in phosphoric acid buffer (PB) solution (5mmol/L PB, 0.1mol/L NaCl, pH=7).
The preparation of single stranded DNA-golden nanometer particle-Microcystin standard items:
Get concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items pure water solution of μ g/L, 1 μ g/L, 2.5 μ g/L mixes with golden nanometer particle-single stranded DNA respectively, obtains to contain 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin standard items-single stranded DNA-golden nanometer particle the compound of the Microcystin standard items of μ g/L, 1 μ g/L, 2.5 μ g/L.
The preparation of single stranded DNA-golden nanometer particle-testing sample:
Gather South Lake, Changchun water sample, prepare concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items South Lake WS of μ g/L, 1 μ g/L, 2.5 μ g/L, the simulation actual water sample as testing sample, is mixed with single stranded DNA-golden nanometer particle respectively, obtains testing sample-single stranded DNA-golden nanometer particle compound.
The preparation of graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound:
At the bottom of getting the donor-based of preparation, the Microcystin standard items-single stranded DNA-golden nanometer particle compound of preparation, the complementary strand of single stranded DNA---few chain DNA T
20(20mer:5 '-TTT TTT TTT TTTTTT TTT TT-3 ') 37 ℃ of cultivation 3h in moist chamber; Utilize the antigen and antibody specific recognition reaction between Microcystin and the Microcystin antibody, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound.Because the space length of graphene oxide and golden nanometer particle shortens; FRET takes place, and the fluorescence intensity of Graphene weakens, through detecting; Obtain scanning fluoroscopic image in excitation wavelength at 534nm with Leica TCS SP2 Laser Scanning Confocal Microscope, obtain the first fluorescent quenching intensity.
According to the method described above, get at the bottom of the donor-based of testing sample-single stranded DNA-golden nanometer particle compound, preparation, complementary strand---the few chain DNA T of single stranded DNA
20(20mer:5 '-TTT TTT TTT TTTTTT TTT TT-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, again according to the concentration and the first fluorescent quenching intensity of Microcystin standard items, obtain the content of Microcystin.
The preparation of water soluble oxidized Graphene:
Take by weighing the 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, the 1g potassium persulfate, in the 2.5g phosphorus pentoxide mixed solution, cool to room temperature behind the constant temperature 4.5h, drying is filtered in the deionized water dilution.The dag of handling well is added 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir the maintenance temperature and be lower than 20 ℃, be heated to 35 ℃ at last and stir 2h.Add 280mL water and add the 8mL30% hydrogen peroxide at once, solution became glassy yellow and had bubble to generate this moment, filtered, and cleaned with hydrochloric acid and water respectively, and the dialysis back is dry, and ultrasonic 30min forms the 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, feed argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splash in two small containers in exsiccator bottom and continue logical argon gas 3min.After putting into clean glass sheet, logical again argon gas 3min shifts out then, makes glass sheet be exposed to 2h in the APTES steam.Then, shift out APTES, the sealing vacuum dryer, ready-made glass sheet is kept in the exsiccator up to use always.
Preparation at the bottom of the donor-based:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise to and leans on electrostatic force to form array on the glass sheet that APTES modifies and in moist chamber, dry.After process washing and nitrogen dried up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) dripped and also cultivate at 37 ℃, and 3h comes the carboxyl on active oxidation Graphene surface, washes then and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to the graphene oxide surface on same point.
The preparation of golden nanometer particle:
(0.1mol/L 1.94mL) is added drop-wise to the HAuCl of boiling to sodium citrate solution fast
4The WS (50mL H
2O, 0.167mL 10%HAuCl
4) in, and continue to stir.Potpourri continues boiling 40min, forms the aurosol of claret, and in stirring cool to room temperature.
The preparation of single stranded DNA-golden nanometer particle:
Get the synthetic golden nanometer particle of 3mL and transfer in the clean bottle, with 20 μ L, 10 μ mol/L HS-T
18(18mer:5 '-SH TTT TTT TTT TTT TTT TTT-3 ') the common cultivation.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution that obtains changes per minutes with 13000, at every turn 15min.Au-DNA (about 10nmol/L) be kept in phosphoric acid buffer (PB) solution (5mmol/L PB, 0.1mol/L NaCl, pH=7).
The preparation of single stranded DNA-golden nanometer particle-Microcystin standard items:
Get concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items pure water solution of μ g/L, 1 μ g/L, 2.5 μ g/L mixes with golden nanometer particle-single stranded DNA respectively, obtains to contain 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1Microcystin standard items-single stranded DNA-golden nanometer particle the compound of the Microcystin standard items of μ g/L, 1 μ g/L, 2.5 μ g/L.
The preparation of single stranded DNA-golden nanometer particle-testing sample:
Gather South Lake, Changchun water sample, prepare concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items South Lake WS of μ g/L, 1 μ g/L, 2.5 μ g/L, the simulation actual water sample as testing sample, is mixed with single stranded DNA-golden nanometer particle respectively, obtains testing sample-single stranded DNA-golden nanometer particle compound.
The preparation of graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound:
At the bottom of getting the donor-based of preparation, the Microcystin standard items-single stranded DNA-golden nanometer particle compound of preparation, the complementary strand of single stranded DNA---few chain DNA A
18(18mer:5 '-AAAAAAAAAAAAAAAAAA-3 ') 37 ℃ of cultivation 3h in moist chamber; Utilize the antigen and antibody specific recognition reaction between Microcystin and the Microcystin antibody, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound.Because the space length of graphene oxide and golden nanometer particle shortens; FRET takes place, and the fluorescence intensity of Graphene weakens, through detecting; Obtain scanning fluoroscopic image in excitation wavelength at 534nm with LeicaTCS SP2 Laser Scanning Confocal Microscope, obtain the first fluorescent quenching intensity.
According to the method described above, get at the bottom of the donor-based of testing sample-single stranded DNA-golden nanometer particle compound, preparation, complementary strand---the few chain DNA A of single stranded DNA
18(18mer:5 '-AAAAAAAAAAAAAAAAAA-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, again according to the concentration and the first fluorescent quenching intensity of Microcystin standard items, obtain the content of Microcystin.
The testing result of embodiment 5 embodiment 1 to embodiment 4
With AFM analyze graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound that embodiment 1 to embodiment 4 obtains synthetic in the pattern profile of each step.Among Fig. 8 (a), the individual layer of homogeneous or double-deck graphene oxide are adsorbed on the glass sheet of APTES modification, and the difference in height of interlayer is 1nm.Graphene oxide has almost covered whole surface, has stoped the non-special absorption of Microcystin on the glass sheet of positively charged.With the microcapsule algae toxin is example, and synthesis step characterizes with AFM.Fig. 8 (b) has shown does not have microcapsule algae toxin to be adsorbed on the graphene oxide surface, has eliminated the influence of non-special absorption.Then, under the effect of EDC and NHS, antibody is a large amount of is adsorbed onto the graphene oxide surface, causes that the height at the bottom of the donor-based is increased to 2nm, sees Fig. 8 (c).Significantly morphology change occurs in microcapsule algae toxin is added drop-wise to the donor-based basal surface, is accompanied by and highly is increased to 4nm, shows that the specific adsorption between Microcystin antibody and microcapsule algae toxin can take place, and sees Fig. 8 (d).After Microcystin-single stranded DNA-golden nanometer particle compound and antibody effect, the pattern of resulting composite is embodied among Fig. 8 (e).A large amount of golden nanometer particles and resulting composite overall height are 17nm, prove golden nanometer particle and graphene oxide enough near and can carry out FRET and detect, generally detect during less than 10nm in distance.
Be presented at each the step reaction of graphene oxide surface through fluorescent scanning image and relative intensity of fluorescence.Change in fluorescence by in the building-up process shown in Fig. 9 (a) can know, the average fluorescent strength of original graphene oxide is regarded as 1, and the absorption of Microcystin antibody (Adda group) and Microcystin is almost to the not influence of fluorescence of graphene oxide.When single stranded DNA-fluorescence intensity obviously reduced (being about 58 ± 4.8%) when the golden nanometer particle compound was added drop-wise to the donor-based basal surface.After the complementary strand processing through above-mentioned single stranded DNA, fluorescence intensity returns to 92 ± 0.9%, shows again with the importance at the bottom of the complementary strand processing donor-based of above-mentioned single stranded DNA.
Concentration is respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items pure water solution of μ g/L, 1 μ g/L, 2.5 μ g/L mixes with golden nanometer particle-single stranded DNA respectively; Obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound; Detect fluorescent quenching intensity, along with the increase of Microcystin concentration, more golden nanometer particle is connected to the donor-based basal surface; The FRET phenomenon is obvious more, shows as green fluorescence intensity and reduces gradually.The relative cancellation efficient of microcapsule algae toxin is respectively 24 ± 2.5% through calculating, 29 ± 1.7%, 41 ± 1.2%, 47 ± 4.7%; 53 ± 2.1%, 68 ± 2.4%, Microcystin-RR is respectively 26 ± 1.0%; 32 ± 0.6%, 42 ± 4.1%, 47 ± 0.2%; 55 ± 1.9%, 66 ± 1.8%, shown in Fig. 9 (b), Fig. 9 (c).
Get South Lake, Changchun water sample, prepare concentration and be respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1The Microcystin standard items South Lake WS of μ g/L, 1 μ g/L, 2.5 μ g/L, the simulation actual water sample is as testing sample; Mix respectively with at the bottom of single stranded DNA-golden nanometer particle, the donor-based; Detect fluorescent quenching intensity, along with the increase of Microcystin concentration, more golden nanometer particle is connected to the donor-based basal surface; The FRET phenomenon is obvious more, shows as green fluorescence intensity and reduces gradually.The relative cancellation efficient of microcapsule algae toxin is respectively 29 ± 2.0% through calculating, 34 ± 1.5%, 44 ± 1.7%, 50 ± 3.6%; 65 ± 2.7%, 65 ± 0.5%, Microcystin-RR is respectively 29 ± 0.6%, 36 ± 2.3%; 43 ± 1.5%, 50 ± 1.8%, 63 ± 6.2%, 66 ± 2.9%; Shown in Fig. 9 (d), Fig. 9 (e), to compare with the relative cancellation efficient of above-mentioned Microcystin standard items, difference is remarkable (P＞0.05) not.
Detection method selectivity provided by the invention detects:
Saxitoxin is the another kind of toxin of blue-green algae secretion, and normal and Microcystin is also deposited.Getting microcapsule algae toxin, Microcystin-RR, saxitoxin-NEO, the saxitoxin-STX standard items pure water solution that concentration is 1 μ g/L respectively hatches with golden nanometer particle-single stranded DNA respectively; Detect fluorescent quenching intensity, the relative cancellation efficient of microcapsule algae toxin, Microcystin-RR, saxitoxin-NEO, saxitoxin-STX is respectively 55 ± 1.0% through calculating, 54 ± 1.9%; 10 ± 0.8%; 10 ± 0.5%, shown in Fig. 9 (f), uncombined toxin STX and NEO can not cause fluorescent quenching; Demonstrate the fluorescence intensity similar with negative control, high selectivity comes from the specific effect between Microcystin and Microcystin antibody.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (4)
1. a method that detects Microcystin is characterized in that, may further comprise the steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Said graphene oxide is connected through electrostatic force with said substrate;
Get Microcystin antibody and said graphene oxide is covalently bound, obtain at the bottom of the donor-based;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with said golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Said single stranded DNA has 12～20 bases;
Getting said single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations; Obtain the standard items receptor complex; Again with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly; React through antigen and antibody specific; Obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound, said graphene oxide and said golden nanometer particle generation FRET obtain the first fluorescent quenching intensity through fluoroscopic examination;
After getting said single stranded DNA-golden nano-complexes and testing sample mixing; Again successively with at the bottom of the said donor-based, the complementary strand of said single stranded DNA hatches jointly; Obtain the second fluorescent quenching intensity through fluoroscopic examination; With the concentration of said standard items and the said first fluorescent quenching strength ratio, obtain the content of Microcystin in the said testing sample.
2. method according to claim 1 is characterized in that said substrate is selected from glass sheet, mica sheet or silicon chip.
3. method according to claim 1 is characterized in that, said single stranded DNA is strand adenylic acid or strand thymine.
4. method according to claim 1 is characterized in that, said Microcystin standard items concentration is respectively 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1μ g/L, 1 μ g/L, 2.5 μ g/L.
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| CN115057472B (en) * | 2022-06-21 | 2023-10-27 | 中国医学科学院基础医学研究所 | Novel fluorescence sensing system and application thereof in PTP-1B detection |
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| CN116465872B (en) * | 2023-05-09 | 2023-11-28 | 临沂大学 | Method for rapidly detecting microcystin |
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