CN102507921B - Method for detecting microcystin - Google Patents
Method for detecting microcystin Download PDFInfo
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- CN102507921B CN102507921B CN201110312171.4A CN201110312171A CN102507921B CN 102507921 B CN102507921 B CN 102507921B CN 201110312171 A CN201110312171 A CN 201110312171A CN 102507921 B CN102507921 B CN 102507921B
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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 Tesing and Solution 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.When blue-green algae death, after cell rupture, Microcystin will dissolve in water, and water body periphery people and animals' life security is worked the mischief.World Health Organization's regulation, the content of microcystin in drinking water-LR must not exceed 1 μ g/L.All the time, the report of the environmental problem causing about Microcystin is both at home and abroad of common occurrence, and blue algae bloom almost all will occur every year in the freshwater lakes such as Taihu Lake, Chaohu, the serious environment event of the contaminated grade of resident living water.Therefore, the trace detection of Microcystin is for ensureing that people's life has great importance safely.
The method of the detection Microcystin of widely applying 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, as enzyme connection analytic approach, phosphoprotein phosphatase inhibition analysis method etc.Wherein, the method of liquid chromatography and combined gas chromatography mass spectrometry is owing to having higher separation efficiency and detection sensitivity widespread use in actual Microcystin detects preferably, but its sample pretreatment process complexity, need large-scale instrument, cost is high, and testing process mostly needs the large-scale instruments such as chromatogram, mass spectrum, in processing procedure, need to introduce some toxic solvents (acetonitrile etc.), be unfavorable for the detection of actual water sample; Electrochemical method is applied various material modified electrodes and is realized the indirect detection to Microcystin, but majority need to, with enzyme labeling Microcystin molecule (can not guarantee productive rate), by competition indirect detection, reduce the scope of practical application; Enzyme connection analytic approach is the method for current conventional detection Microcystin, high commercial kit utilizes competing method, possessing under microcystin monoclonal antibody, the pure toxin of standard and the prerequisite about reagent, carrying out chromogenic assay according to the chromogenic substrate adding, is that one is very easy, efficient, method fast, but the antibody type needing is many, and consumption is large, if multiple homolog is identified and needed wide spectrum antibody.Compared with enzyme connection analytic approach, phosphoprotein phosphatase inhibition analysis method has higher sensitivity, can detect all Microcystin homolog total amounts in sample, but conventionally need radioisotopic mark, make subsequent treatment more difficult, cannot develop into conventional method of environmental monitoring.Recently, Raman spectrum is also applied in the detection of Microcystin, concentration by Microcystin in concentrating sample in hydrophobic substrate realizes fast detecting, but need in sample pretreatment process, use the steps such as Solid-Phase Extraction, pretreatment process complexity, length consuming time, needs professional to operate, and brings certain inconvenience to practical application.
FRET (fluorescence resonance energy transfer) is a kind of high-sensitive analytical technology, it refers to and is subject to excite transitions when the out orbit when donor electronics, can with the electron interaction of acceptor, realize energy transmission, cause acceptor electronics to be excited, and donor electronics itself is got back to the process of ground state.This process is subject to the impact of distance between donor and acceptor, generally within the scope of 1～10nm, occurs, and the further increase of distance can cause FRET (fluorescence resonance energy transfer) to weaken until disappear.Therefore the selection of donor and acceptor is very important.In recent years Graphene and derivant thereof because of its superior performance as: electric conductivity is high, and surface area is large, is easy to biomolecule modification etc., is widely used in the preparation of various sensors.In the application of FRET (fluorescence resonance energy transfer), graphene oxide is all being brought into play very large advantage aspect donor and acceptor two.As acceptor, graphene oxide can be accepted from quantum dot, and the excitation electron of various fluorescence molecules, as the quencher of fluorescence.Typical application is the mispairing that detects DNA molecular and DNA base.As donor, Graphene can provide the electronics of excited state under the excitation wavelength of 400nm, and energy is transferred on 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 FRET (fluorescence resonance energy transfer) principle to detect the method for Microcystin, there is practical significance.
Summary of the invention
In view of this, the invention provides a kind of method that detects Microcystin, the method detects the content of Microcystin by the graphene oxide fluorescent quenching that FRET (fluorescence resonance energy transfer) occurs between graphene oxide and golden nanometer particle causes, there is higher sensitivity, selectivity, detectability, lower than WHO standard, is applicable to the trace detection of Microcystin.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of method that detects Microcystin, comprise the following steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Described graphene oxide is connected by electrostatic force with described substrate;
Get Microcystin antibody and described graphene oxide is covalently bound, obtain donor substrate;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with described golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Described single stranded DNA has 12～20 bases;
Getting described single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations, the minor groove binding of described Microcystin and described single stranded DNA, obtain standard items receptor complex, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA again, react by antigen and antibody specific, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound, described graphene oxide and described golden nanometer particle generation FRET (fluorescence resonance energy transfer), obtain the first fluorescent quenching intensity by fluoroscopic examination; The complementary strand of described single stranded DNA and described single stranded DNA specific binding, eliminate the non-specific binding of described single stranded DNA and described graphene oxide;
After getting described single stranded DNA-golden nano-complexes and mixing with testing sample, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA successively again, obtain the second fluorescent quenching intensity by fluoroscopic examination, with the concentration of described standard items and described the first fluorescent quenching strength ratio, obtain the content of Microcystin in described testing sample.Microcystin (microcystin, MC), its main architectural feature is N-methyl dehydroalanine and two L-amino acid residue X and Z, according to Microcystin (Microcystins or MCYST) the nomenclature regulation of formulating for 1988.The various combination of X, Z bis-residues is distinguished by the letter suffix of represented amino acid.Common are LR, RR, tri-kinds of toxin of YR, L, R, Y represent respectively leucine, arginine, tyrosine.The general structure of Microcystin is ring (D-alanine-L-X-red-Beta-methyl-D-different aspartic acid-L-Z-Adda-D-isoglutamic acid-N-methyl dehydroalanine), wherein Adda (3 amino 9-methylamino-2,6,8-trimethyl 10-phenyl-4,6-dienoic acid) be that Microcystin biologically active is expressed necessary.Therefore, in the present invention, select the antibody of Adda group of Microcystin as Microcystin antibody.
Microcapsule algae toxin is the algae toxin of being illustrated the earliest chemical constitution. in to the research of algae toxin also mainly with it as research object.It is 7 peptide molecules of a ring-type, and molecular weight is about 1000 dalton, and the event being caused by algae toxin that many countries occur is mostly relevant with microcapsule algae toxin.Therefore, the present invention studies mainly for microcapsule algae toxin and Microcystin-RR.
The drinking water standard that the World Health Organization (WHO) (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 Microcystins that the existing promulgation of China is carried out is 0.001mg/L, water environment quality standard (GB3838-2002) regulation, 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 by detection, with the concentration of described standard items and described the first fluorescent quenching strength ratio, obtain the content of Microcystin in described testing sample, this content is as long as lower than World Health Organization's specified standard 1 μ g/L, and it is qualified to be.In above-mentioned testing process, testing sample can be simulated the lake water that contains Microcystin and detects by added the Microcystin of concentration known in lake water, obtains the second fluorescent quenching intensity.Again with the concentration of Microcystin standard items and its corresponding the first fluorescent quenching strength ratio, if difference is not remarkable, prove accuracy and the feasibility of detection method provided by the invention.Also can be in blue algae bloom season, the detection method that water sampling generally uses according to detection method provided by the invention, at present detects respectively relatively, if difference is not remarkable, also can show that detection method provided by the invention has feasibility and accuracy, but it is larger that the method for sampling is affected by environment, season, water pollution degree, generally take the lake water that simulation contains Microcystin to detect as testing sample.
Preparing in 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 under be adsorbed onto donor substrate surface, based on single doubly-linked DNA in graphene oxide surface compatibility difference, and the interaction between single stranded DNA and graphene oxide is disturbed in the combination meeting of complementary strand, the single stranded DNA that causes non-specific absorption comes off from graphene oxide surface, single stranded DNA in elimination single stranded DNA-golden nanometer particle and the non-specific adsorption of Graphene, make to form dependence between the concentration of Microcystin and fluorescent quenching intensity.Therefore,, in detection method provided by the invention, utilize the complementary strand of single stranded DNA and standard items receptor complex, donor substrate jointly to hatch.
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 APTES ((3-aminopropyl) triethoxysilane; APTE S) modify substrate, make base strap positive electricity, be combined by electrostatic force with electronegative graphene oxide.
Microcystin can with the minor groove binding of DNA, as preferably, DNA is strand adenine and strand thymine.
As preferably, in 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 method detects the content of Microcystin by the graphene oxide fluorescent quenching that FRET (fluorescence resonance energy transfer) occurs between graphene oxide and golden nanometer particle causes, there is higher sensitivity, selectivity, detectability, lower than WHO standard, is applicable to the trace detection of Microcystin.
Accompanying drawing explanation
Fig. 1 shows the interaction of Microcystin and DNA; Wherein, Fig. 1 (a) shows Red Shift Phenomena, 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 240nm) of ultraviolet absorption value and the DNA of Microcystin, shows the ultraviolet absorption value (under 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 in the situation that does not have DNA, Microcystin does not have cancellation effect to the fluorescence of EB; Fig. 2 (b) is shown under DNA existence, and Microcystin and EB-DNA compound interact, to the cancellation effect of EB fluorescence; Wherein, curve a to f successively corresponding Microcystins Concentration be 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, ordinate is the ultraviolet absorption peak of Microcystin, 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 of curve shown in from 0 to 10 (increasing progressively with 2 μ mol/L);
Fig. 4 shows that the Microcystin of variable concentrations is on the impact of DNA molecular; Wherein, the concentration of the corresponding Microcystin of Fig. 4 (a) is 0ng/ μ L, the concentration of the corresponding Microcystin of Fig. 4 (b) is 1ng/ μ L, 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 few chain DNA A15 effect;
Fig. 6 shows golden nanometer particle and few chain DNA A
15the variation of absorbance after effect;
Fig. 7 shows the non-specific adsorption effect of single stranded DNA and graphene oxide, and utilizes the complementary strand of this single stranded DNA to eliminate the effect of this non-specific adsorption; Wherein, Fig. 7 (a) shows the atomic force microscopy picture of graphene oxide-Microcystin antibody; Fig. 7 (b) shows the atomic force microscopy 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, through the complementary strand atomic force microscopy picture after treatment of this single stranded DNA; Fig. 7 (d) shows the atomic force microscopy picture after graphene oxide-Microcystin antibody is combined with Microcystin-single stranded DNA-golden nanometer particle; After Fig. 7 (e) shows that graphene oxide-Microcystin antibody is combined with Microcystin-single stranded DNA-golden nanometer particle, through the complementary strand atomic force microscopy picture after treatment of this single stranded DNA;
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 of donor substrate; Fig. 8 (b) shows the atomic force phenogram of eliminating the non-specific 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 specific binding rear surface pattern 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; Cylindricality corresponding graphene oxide, 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 successively from left to right, then through the complementary strand processing of this single stranded DNA; Fig. 9 (b) shows the variation of the fluorescence intensity in the microcapsule algae toxin standard items of variable concentrations; Corresponding graphene oxide, blank group, concentration are respectively 10 to cylindricality successively from left to right
-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 Microcystin-RR standard items of variable concentrations; Corresponding graphene oxide, blank group, concentration are respectively 10 to cylindricality successively from left to right
-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 of the fluorescence intensity that contains microcapsule algae toxin in simulation testing sample; Corresponding graphene oxide, blank group, concentration are respectively 10 to cylindricality successively from left to right
-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 of the fluorescence intensity that contains Microcystin-RR in simulation testing sample; Corresponding graphene oxide, blank group, concentration are respectively 10 to cylindricality successively from left to right
-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, after graphene oxide-Microcystin antibody incubation, the respectively variation of fluorescence intensity, corresponding graphene oxide, 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 to cylindricality successively from left to right.
Embodiment
The invention discloses a kind of method that detects Microcystin.Those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of method that detects Microcystin, comprise the following steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Described graphene oxide is connected by electrostatic force with described substrate;
Get Microcystin antibody and described graphene oxide is covalently bound, obtain donor substrate;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with described golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Described single stranded DNA has 12～20 bases;
Getting described single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations, the minor groove binding of described Microcystin and described single stranded DNA, obtain standard items receptor complex, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA again, react by antigen and antibody specific, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound, described graphene oxide and described golden nanometer particle generation FRET (fluorescence resonance energy transfer), obtain the first fluorescent quenching intensity by fluoroscopic examination; The complementary strand of described single stranded DNA and described single stranded DNA specific binding, eliminate the non-specific binding of described single stranded DNA and described graphene oxide;
After getting described single stranded DNA-golden nano-complexes and mixing with testing sample, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA successively again, obtain the second fluorescent quenching intensity by fluoroscopic examination, according to the concentration of described standard items and described the first fluorescent quenching intensity, obtain the content of Microcystin in described testing sample.
Ultraviolet is passed through in the interaction of microcapsule algae toxin, Microcystin-RR and DNA, fluorescence, and the methods such as atomic force microscope prove.As can be seen from Figure 1, along with adding of DNA, the uv absorption of Microcystin has obvious increase, and Red Shift Phenomena is as shown in Fig. 1 (a), and hypochromic effect is as shown in Fig. 1 (b), and this shows to exist and interact between the two.Fig. 2 has embodied microcapsule algae toxin, the cancellation of Microcystin-RR to EB-DNA compound fluorescence.Because the fluorescence of DNA itself is very weak, can not direct-detection, the ethidium bromide (EB) that we utilize can be inserted between DNA base, and the fluorescence intensity of the two compound is strengthened, and reaches the intensity that can detect.Fig. 2 (a) has shown that in the situation that does not have DNA, microcapsule algae toxin does not have cancellation effect to the fluorescence of EB, Fig. 2 (b) has shown that microcapsule algae toxin in the situation that has DNA, Microcystin-RR have and have cancellation effect the fluorescence of EB, therefore, the fluorescent quenching phenomenon in Fig. 2 causes because microcapsule algae toxin, Microcystin-RR and EB-DNA compound interact.In Fig. 3, embody the interaction between microcapsule algae toxin, Microcystin-RR and DNA base.Along with the increase of base concentration, microcapsule algae toxin, the ultraviolet absorption peak of Microcystin-RR obviously increases and red shift, but the signal of adenine and thymine is obviously better than guanine and cytimidine, and with DNA molecular and microcapsule algae toxin in Fig. 1 (a), signal similar after Microcystin-RR effect, microcapsule algae toxin, Microcystin-RR easily combines with adenine and thymine, and the compact district of adenine and thymine forms ditch in DNA molecular, therefore judge microcapsule algae toxin, the combination of Microcystin-RR and DNA is minor groove binding.In addition by calculating the binding constant between microcapsule algae toxin, Microcystin-RR and DNA, in conjunction with the minor groove binding mode than having confirmed between the two.Microcapsule algae toxin, the impact of Microcystin-RR on DNA molecular of variable concentrations in Fig. 4, are shown, along with the increase of microcapsule algae toxin, Microcystin-RR concentration, DNA molecular is progressively wound around, and finally forms bar-shaped aggegation, has further proved the interaction of the two.In Fig. 5, show the ultraviolet absorption curve after microcapsule algae toxin, Microcystin-RR and few chain DNA A15 effect, Fig. 6 has shown the variation of golden nanometer particle and the rear absorbance of few chain DNA A15 effect, has all proved that Au-DNA-Microcystin compound can form.
Preparing in 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 under be adsorbed onto the donor substrate surface as shown in Fig. 7 (a), see Fig. 7 (b).Based on single doubly-linked DNA in graphene oxide surface compatibility difference, and the interaction between single stranded DNA and graphene oxide is disturbed in the combination meeting of complementary strand, the single stranded DNA that causes non-specific absorption comes off from graphene oxide surface, single stranded DNA in elimination single stranded DNA-golden nanometer particle and the non-specific adsorption of Graphene, as point-like thing in Fig. 7 (c) is significantly less than point-like thing in Fig. 7 (b), point-like thing is significantly less than point-like thing in Fig. 7 (d) in Fig. 7 (e), makes to form dependence between the concentration of Microcystin and fluorescent quenching intensity.Therefore,, in detection method provided by the invention, utilize the complementary strand of single stranded DNA and standard items receptor complex, donor substrate jointly to hatch.
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 APTES ((3-aminopropyl) triethoxysilane; APTES) modify substrate, make base strap positive electricity, be combined by electrostatic force with electronegative graphene oxide.
Microcystin can with the minor groove binding of DNA, as preferably, DNA is strand adenine and strand thymine.
As preferably, in 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 method detects the content of Microcystin by the graphene oxide fluorescent quenching that FRET (fluorescence resonance energy transfer) occurs between graphene oxide and golden nanometer particle causes, there is higher sensitivity, selectivity, detectability, lower than WHO standard, is applicable to the trace detection of Microcystin.
In detection method provided by the invention, agents useful for same all can be buied by market.Wherein, Microcystin (microcapsule algae toxin, Microcystin-RR), saxitoxin (STX, NEO) and the antibody for Microcystin Adda group all Jim Press Science and Technology Ltd. (Beijing) are buied from her; Few chain DNA (SH-A
15, T
15, SH-G
12, C
12, SH-A
20, T
20, SH-T
18, A
18) synthetic purchased from Sheng Gong biotechnology company, directly do not use through purifying; Ethyl dimethyl amine propyl carbodiimide diimine (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; EDC), NHS (N-hydroxy sulfosuccinimide; Sulfo-NHS), APTES ((3-aminopropyl) triethoxysilane; And N APTES), N-diisopropyl ethyl amine borane complex (N, N-diisopropylethylamine), agents useful for same be analyze pure; Antibody damping fluid is phosphate buffer (pH=7), and all solution is 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 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, 1g potassium persulfate, in 2.5g phosphorus pentoxide mixed solution, cool to room temperature after constant temperature 4.5h, deionized water dilution, filters, dry.The dag of handling well is added to 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir and keep temperature lower than 20 ℃, be finally heated to 35 ℃ and stir 2h.Add 280mL water and add 8mL30% hydrogen peroxide, now solution becomes glassy yellow and has bubble formation at once, filters, and cleans respectively with hydrochloric acid and water, and dry after dialysis, ultrasonic 30min forms 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, pass into argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splashes in two small containers in exsiccator bottom and continues logical argon gas 3min.Put into after clean glass sheet, more then logical argon gas 3min shift out, make glass sheet be exposed to 2h in APTES steam.Then, shift out APTES, sealing vacuum dryer, ready-made glass sheet is kept in exsiccator until use always.
The preparation of donor substrate:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise on the glass sheet that APTES modifies and forms array drying by electrostatic force in moist chamber.Through washing with after nitrogen dries up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) drips and cultivates at 37 ℃, and 3h carrys out the carboxyl on active oxidation Graphene surface, then washes and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to graphene oxide surface in same point.
The preparation of golden nanometer particle:
Sodium citrate solution (0.1mol/L, 1.94mL) is added drop-wise to the HAuCl of boiling fast
4aqueous solution (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 a clean bottle, with 20 μ L, 10 μ mol/L HS-A
15(15mer:5 '-SH AAA AAA AAA AAA AAA-3 ') jointly hatch.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution obtaining turns per minute with 13000,15min at every turn.Au-DNA (about 10nmol/L) is kept at (5mmol/L PB, 0.1mol/L NaCl, pH=7) in phosphoric acid buffer (PB) solution.
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 and contains 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1microcystin standard items-single stranded DNA-golden nanometer particle 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 aqueous solution of μ g/L, 1 μ g/L, 2.5 μ g/L, simulation actual water sample, as testing sample, mixes 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:
Get the donor substrate of preparation, 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 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 (fluorescence resonance energy transfer) occurs, and the fluorescence intensity of Graphene weakens, by detecting, with Leica TCS SP2 Laser Scanning Confocal Microscope excitation wavelength 534nm obtain scanning fluoroscopic image, obtain the first fluorescent quenching intensity.
According to the method described above, get the donor substrate of testing sample-single stranded DNA-golden nanometer particle compound, preparation, the complementary strand of single stranded DNA---few chain DNA T
15(15mer:5 '-TTT TTT TTT TTTTTT-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, then according to the concentration of Microcystin standard items and the first fluorescent quenching intensity, obtain the content of Microcystin.
The preparation of water soluble oxidized Graphene:
Take 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, 1g potassium persulfate, in 2.5g phosphorus pentoxide mixed solution, cool to room temperature after constant temperature 4.5h, deionized water dilution, filters, dry.The dag of handling well is added to 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir and keep temperature lower than 20 ℃, be finally heated to 35 ℃ and stir 2h.Add 280mL water and add 8mL30% hydrogen peroxide, now solution becomes glassy yellow and has bubble formation at once, filters, and cleans respectively with hydrochloric acid and water, and dry after dialysis, ultrasonic 30min forms 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, pass into argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splashes in two small containers in exsiccator bottom and continues logical argon gas 3min.Put into after clean glass sheet, more then logical argon gas 3min shift out, make glass sheet be exposed to 2h in APTES steam.Then, shift out APTES, sealing vacuum dryer, ready-made glass sheet is kept in exsiccator until use always.
The preparation of donor substrate:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise on the glass sheet that APTES modifies and forms array drying by electrostatic force in moist chamber.Through washing with after nitrogen dries up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) drips and cultivates at 37 ℃, and 3h carrys out the carboxyl on active oxidation Graphene surface, then washes and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to graphene oxide surface in same point.
The preparation of golden nanometer particle:
Sodium citrate solution (0.1mol/L, 1.94mL) is added drop-wise to the HAuCl of boiling fast
4aqueous solution (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 a clean bottle, with 20 μ L, 10 μ mol/L HS-T
12(12mer:5 '-SH TTT TTT TTT TTT-3 ') jointly cultivate.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution obtaining turns per minute with 13000,15min at every turn.Au-DNA (about 10nmol/L) is kept at (5mmol/L PB, 0.1mol/L NaCl, pH=7) in phosphoric acid buffer (PB) solution.
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 and contains 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1microcystin standard items-single stranded DNA-golden nanometer particle 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 aqueous solution of μ g/L, 1 μ g/L, 2.5 μ g/L, simulation actual water sample, as testing sample, mixes 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:
Get the donor substrate of preparation, 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 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 (fluorescence resonance energy transfer) occurs, and the fluorescence intensity of Graphene weakens, by detecting, with Leica TCSSP2 Laser Scanning Confocal Microscope excitation wavelength 534nm obtain scanning fluoroscopic image, obtain the first fluorescent quenching intensity.
According to the method described above, get the donor substrate of testing sample-single stranded DNA-golden nanometer particle compound, 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, detect and obtain the second fluorescent quenching intensity, then according to the concentration of Microcystin standard items and the first fluorescent quenching intensity, obtain the content of Microcystin.
The preparation of water soluble oxidized Graphene:
Take 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, 1g potassium persulfate, in 2.5g phosphorus pentoxide mixed solution, cool to room temperature after constant temperature 4.5h, deionized water dilution, filters, dry.The dag of handling well is added to 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir and keep temperature lower than 20 ℃, be finally heated to 35 ℃ and stir 2h.Add 280mL water and add 8mL30% hydrogen peroxide, now solution becomes glassy yellow and has bubble formation at once, filters, and cleans respectively with hydrochloric acid and water, and dry after dialysis, ultrasonic 30min forms 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, pass into argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splashes in two small containers in exsiccator bottom and continues logical argon gas 3min.Put into after clean glass sheet, more then logical argon gas 3min shift out, make glass sheet be exposed to 2h in APTES steam.Then, shift out APTES, sealing vacuum dryer, ready-made glass sheet is kept in exsiccator until use always.
The preparation of donor substrate:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise on the glass sheet that APTES modifies and forms array drying by electrostatic force in moist chamber.Through washing with after nitrogen dries up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) drips and cultivates at 37 ℃, and 3h carrys out the carboxyl on active oxidation Graphene surface, then washes and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to graphene oxide surface in same point.
The preparation of golden nanometer particle:
Sodium citrate solution (0.1mol/L, 1.94mL) is added drop-wise to the HAuCl of boiling fast
4aqueous solution (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 a clean bottle, with 20 μ L, 10 μ mol/L HS-A
20(20mer:5 '-SH AAAAAAAAAAAAAAAAAAAA-3 ') jointly cultivate.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution obtaining turns per minute with 13000,15min at every turn.Au-DNA (about 10nmol/L) is kept at (5mmol/L PB, 0.1mol/L NaCl, pH=7) in phosphoric acid buffer (PB) solution.
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 and contains 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1microcystin standard items-single stranded DNA-golden nanometer particle 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 aqueous solution of μ g/L, 1 μ g/L, 2.5 μ g/L, simulation actual water sample, as testing sample, mixes 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:
Get the donor substrate of preparation, 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 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 (fluorescence resonance energy transfer) occurs, and the fluorescence intensity of Graphene weakens, by detecting, with Leica TCS SP2 Laser Scanning Confocal Microscope excitation wavelength 534nm obtain scanning fluoroscopic image, obtain the first fluorescent quenching intensity.
According to the method described above, get the donor substrate of testing sample-single stranded DNA-golden nanometer particle compound, 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, detect and obtain the second fluorescent quenching intensity, according to the concentration of Microcystin standard items and the first fluorescent quenching intensity, obtain the content of Microcystin again.
The preparation of water soluble oxidized Graphene:
Take 1.2g dag and join 80 ℃ of 4.8mL concentrated sulphuric acids, 1g potassium persulfate, in 2.5g phosphorus pentoxide mixed solution, cool to room temperature after constant temperature 4.5h, deionized water dilution, filters, dry.The dag of handling well is added to 0 ℃, in the 48mL concentrated sulphuric acid, slowly add 6g potassium permanganate, stir and keep temperature lower than 20 ℃, be finally heated to 35 ℃ and stir 2h.Add 280mL water and add 8mL30% hydrogen peroxide, now solution becomes glassy yellow and has bubble formation at once, filters, and cleans respectively with hydrochloric acid and water, and dry after dialysis, ultrasonic 30min forms 0.1mg/mL dispersion liquid.
The preparation of the positively charged glass sheet that APTES modifies:
In vacuum dryer, pass into argon gas 2min, 30 μ L APTES and 15 μ L N, N-diisopropylethylamine splashes in two small containers in exsiccator bottom and continues logical argon gas 3min.Put into after clean glass sheet, more then logical argon gas 3min shift out, make glass sheet be exposed to 2h in APTES steam.Then, shift out APTES, sealing vacuum dryer, ready-made glass sheet is kept in exsiccator until use always.
The preparation of donor substrate:
1 μ L graphene oxide (0.05mg/mL) is added drop-wise on the glass sheet that APTES modifies and forms array drying by electrostatic force in moist chamber.Through washing with after nitrogen dries up, 1 μ L EDC (15mmol/L), NHS (30mmol/L) drips and cultivates at 37 ℃, and 3h carrys out the carboxyl on active oxidation Graphene surface, then washes and removes remainder.Microcystin antibody-solutions (1 μ L, 1ng/ μ L) is cultivated 3h and is guaranteed that antibody is covalently bound to graphene oxide surface in same point.
The preparation of golden nanometer particle:
Sodium citrate solution (0.1mol/L, 1.94mL) is added drop-wise to the HAuCl of boiling fast
4aqueous solution (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 a clean bottle, with 20 μ L, 10 μ mol/L HS-T
18(18mer:5 '-SH TTT TTT TTT TTT TTT TTT-3 ') jointly cultivate.Through the reaction of 24h with leave standstill, it is at room temperature centrifugal twice that the Au-DNA solution obtaining turns per minute with 13000,15min at every turn.Au-DNA (about 10nmol/L) is kept at (5mmol/L PB, 0.1mol/L NaCl, pH=7) in phosphoric acid buffer (PB) solution.
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 and contains 10
-4μ g/L, 10
-3μ g/L, 10
-2μ g/L, 10
-1microcystin standard items-single stranded DNA-golden nanometer particle 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 aqueous solution of μ g/L, 1 μ g/L, 2.5 μ g/L, simulation actual water sample, as testing sample, mixes 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:
Get the donor substrate of preparation, 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 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 (fluorescence resonance energy transfer) occurs, and the fluorescence intensity of Graphene weakens, by detecting, with LeicaTCS SP2 Laser Scanning Confocal Microscope excitation wavelength 534nm obtain scanning fluoroscopic image, obtain the first fluorescent quenching intensity.
According to the method described above, get the donor substrate of testing sample-single stranded DNA-golden nanometer particle compound, preparation, the complementary strand of single stranded DNA---few chain DNA A
18(18mer:5 '-AAAAAAAAAAAAAAAAAA-3 ') 37 ℃ of cultivation 3h in moist chamber, detect and obtain the second fluorescent quenching intensity, then according to the concentration of Microcystin standard items and the first fluorescent quenching intensity, obtain the content of Microcystin.
The testing result of embodiment 5 embodiment 1 to embodiment 4
The pattern profile of each step in synthesizing with graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound that AFM analysis embodiment 1 to embodiment 4 obtains.In 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 nearly cover whole surface, stoped the non-specific absorption of Microcystin on the glass sheet of positively charged.Take microcapsule algae toxin as example, synthesis step characterizes with AFM.Fig. 8 (b) has shown does not have microcapsule algae toxin to be adsorbed on graphene oxide surface, has eliminated the impact of non-specific absorption.Then, under the effect of EDC and NHS, what antibody was a large amount of is adsorbed onto graphene oxide surface, causes that the height of donor substrate is increased to 2nm, sees Fig. 8 (c).Significantly pattern variation occurs in microcapsule algae toxin is added drop-wise to donor substrate surface, is accompanied by and is highly increased to 4nm, shows that the specific adsorption between Microcystin antibody and microcapsule algae toxin can occur, and sees Fig. 8 (d).After Microcystin-single stranded DNA-golden nanometer particle compound and antibody effect, the pattern of resulting composite is embodied in Fig. 8 (e).A large amount of golden nanometer particles and resulting composite overall height are 17nm, prove that golden nanometer particle and graphene oxide enough approach and can carry out FRET detection, generally detect in the time that distance is less than 10nm.
Be presented at the every single step reaction in graphene oxide surface by fluorescent scanning image and relative intensity of fluorescence.From the change in fluorescence in building-up process shown in Fig. 9 (a), the average fluorescent strength of original graphene oxide is considered as to 1, and the absorption of Microcystin antibody (Adda group) and Microcystin does not almost affect the fluorescence of graphene oxide.In the time that single stranded DNA-golden nanometer particle compound is added drop-wise to donor substrate surface, fluorescence intensity obviously reduces (being about 58 ± 4.8%).After the complementary strand of above-mentioned single stranded DNA is processed, fluorescence intensity returns to 92 ± 0.9%, shows again to process with the complementary strand of above-mentioned single stranded DNA the importance of donor substrate.
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 Microcystins Concentration, more golden nanometer particle is connected to donor substrate surface, FRET (fluorescence resonance energy transfer) phenomenon is more obvious, shows as green fluorescence intensity and reduces gradually.The relative cancellation efficiency of microcapsule algae toxin is respectively 24 ± 2.5%, 29 ± 1.7%, 41 ± 1.2% as calculated, 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%, as 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 aqueous solution of μ g/L, 1 μ g/L, 2.5 μ g/L, simulation actual water sample, as testing sample, mix with single stranded DNA-golden nanometer particle, donor substrate respectively, detect fluorescent quenching intensity, along with the increase of Microcystins Concentration, more golden nanometer particle is connected to donor substrate surface, FRET (fluorescence resonance energy transfer) phenomenon is more obvious, shows as green fluorescence intensity and reduces gradually.The relative cancellation efficiency of microcapsule algae toxin is respectively 29 ± 2.0% as calculated, 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%, as shown in Fig. 9 (d), Fig. 9 (e), compare with the relative cancellation efficiency 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.Get respectively the microcapsule algae toxin that concentration is 1 μ g/L, Microcystin-RR, saxitoxin-NEO, saxitoxin-STX standard items pure water solution is hatched with golden nanometer particle-single stranded DNA respectively, detect fluorescent quenching intensity, microcapsule algae toxin, Microcystin-RR, saxitoxin-NEO, the relative cancellation efficiency of saxitoxin-STX is respectively 55 ± 1.0% as calculated, 54 ± 1.9%, 10 ± 0.8%, 10 ± 0.5%, as 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 is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. a method that detects Microcystin, is characterized in that, comprises the following steps:
Obtain graphene oxide;
Obtain the substrate of positively charged; Described graphene oxide is connected by electrostatic force with described substrate;
Get Microcystin antibody and described graphene oxide is covalently bound, obtain donor substrate;
Obtain golden nanometer particle;
The single stranded DNA of getting sulfydryl modification mixes with described golden nanometer particle, obtains single stranded DNA-golden nano-complexes; Described single stranded DNA has 12～20 bases;
Getting described single stranded DNA-golden nano-complexes mixes with the Microcystin standard items of variable concentrations, obtain standard items receptor complex, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA again, react by antigen and antibody specific, obtain graphene oxide-Microcystin antibody-Microcystin-single stranded DNA-golden nanometer particle compound, described graphene oxide and described golden nanometer particle generation FRET (fluorescence resonance energy transfer), obtain the first fluorescent quenching intensity by fluoroscopic examination;
After getting described single stranded DNA-golden nano-complexes and mixing with testing sample, jointly hatch with the complementary strand of described donor substrate, described single stranded DNA successively again, obtain the second fluorescent quenching intensity by fluoroscopic examination, with the concentration of described standard items and described the first fluorescent quenching strength ratio, obtain the content of Microcystin in described testing sample.
2. method according to claim 1, is characterized in that, described substrate is selected from glass sheet, mica sheet or silicon chip.
3. method according to claim 1, is characterized in that, described single stranded DNA is strand adenylic acid or strand thymine.
4. method according to claim 1, is characterized in that, described 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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| CN102866139B (en) * | 2012-09-21 | 2014-08-06 | 南开大学 | Establishment method based on surface plasma reinforcing energy transferring biosensor |
| CN103233078B (en) * | 2013-05-14 | 2014-11-26 | 华南师范大学 | Multi-gene detection method of Listeria monocytogenes based on quantum dot/graphene oxide nanometer platform |
| CN103411933B (en) * | 2013-08-04 | 2015-09-16 | 吉林大学 | Based on the preparation method of the surface plasmon resonance DNA sensor of graphene oxide |
| CN104181136B (en) * | 2014-08-25 | 2017-03-29 | 广西师范大学 | A kind of Resonance Rayleigh Scattering energy transfer spectrographic method for determining formaldehyde |
| CN105067582A (en) * | 2015-07-31 | 2015-11-18 | 无锡市疾病预防控制中心 | Quantum dot fluorescence detection method for fast determination of MC-RR in water |
| CN106568748A (en) * | 2016-10-09 | 2017-04-19 | 江南大学 | Method for detecting microcystin LR based on fluorescence resonance energy transfer of shell-core type up-conversion material and molybdenum disulfide |
| CN106525796B (en) * | 2016-11-08 | 2020-04-28 | 北京化工大学 | Recyclable fluorescence sensor for detecting microcystin and application method thereof |
| CN108956743B (en) * | 2018-07-24 | 2021-01-08 | 哈尔滨工程大学 | Detection method of field effect transistor biosensor enhanced by AuNPs |
| CN109696430B (en) * | 2019-03-01 | 2021-07-27 | 长江师范学院 | Method for measuring concentration of microcystin |
| CN111273022B (en) * | 2020-02-06 | 2023-11-28 | 上海市胸科医院 | Troponin concentration detection method based on nanogold-graphene quantum dots |
| 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 |
| CN116465872B (en) * | 2023-05-09 | 2023-11-28 | 临沂大学 | Method for rapidly detecting microcystin |
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