CN105572090A - Oxytetracycline fluorescence detection method based on graphene-based compound hydrogel - Google Patents
Oxytetracycline fluorescence detection method based on graphene-based compound hydrogel Download PDFInfo
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Abstract
The invention belongs to the technical field of environmental monitoring, and relates to an oxytetracycline fluorescence detection method for antibiotics in water based on graphene-based compound hydrogel. Graphene dispersed liquid is adopted as a monomer, adenosine and an aptamer are adopted as a cross-linking agent together, graphene oxide lamellas originally dispersed in a solution are connected together, and a three-dimensional macrostructure is formed. Oxytetracycline aqueous solutions with different concentrations are added to a hydrogel system for a simple soaking process, supernate is taken for fluorescence intensity testing, and quantitative fluorescence detection of the concentration of oxytetracycline can be achieved. In the prepared hydrogel system with different proportions, the detection range of oxytetracycline can be controllably adjusted, and the method can be applied to different types of actual water for detection. Compared with a traditional nano material sensing detecting method, the method is fast in nano material preparation, simple, convenient to use, mild in preparation condition and high in environmental stability and practicality of the hydrogel material.
Description
Technical field
The invention belongs to environmental monitoring technology field, relate to a kind of terramycin fluorescence sense detection method based on graphene-based composite aquogel.
Background technology
Tetracycline is the class broad-spectrum antibiotic that a class is produced by actinomyces, comprise naturally occurring tetracycline (tetracycline, TET), terramycin (oxytetracycline, OTC), fortimicin (Doxycycline), metacycline (methacycline) etc., its structure, all containing aphthacene basic framework, is widely used in various bacteria and rickettsia, Chlamydia, mycoplasma infection.But, microbiotic comprises the abuse of Tetracyclines, microbiotic can be made at surrounding medium accumulation (EnvironmentalScience & Technology, 2015,49 (11), 6772-6782.), (the EnvironmentalScience & Technology of enrichment in human body is acted on again by food chain etc., 2015,49 (8), 5070-5079.).In addition, abuse of antibiotics also can induce generation drug resistant gene, living environment (ProceedingsoftheNationalAcademyofSciencesoftheUnitedStat esofAmerica, 2013 of the serious threat mankind, 110 (9), 3435-3440.).Terramycin, as a kind of tetracycline of widespread use, is grasped its environment distribution situation and is had great directive significance to antibiotic pollution control.Therefore, be badly in need of sending out sensitive, quick, efficient, real-time detection method monitors the real-time concentration of terramycin in environment, for subsequent treatment or disposing task provide data supporting.
Traditional microbiotic detection method has instrument detection method, bioanalysis/biochemical process etc., because its intrinsic defect has been difficult to tackle current environmental monitoring demand.Such as, high performance liquid chromatography is consuming time, effort, cost are high; Although enzyme linked immunosorbent assay (ELISA) has higher sensitivity and selectivity, operating process is complicated, and system, to the bad adaptability of extreme environment, hinders promoting the use of of this method.In recent years, based on galvanochemistry or the optical sensor of nano material or nanomaterial assembly body, be conducive to the conduction of signal owing to having the character such as the intrinsic electricity of nano material, optics, be widely used in sensing assays detects.At present, people also utilized nano material successfully establish some for microbiotic detect sensing platform (RSCAdvances, 2015,5 (72), 58895-58901; MicrochimicaActa, 2013,180 (9-10), 829-835; BiosensorsandBioelectronics, 2010,26 (4), 1644-1649; Analyst, 2013,138 (6), 1886-1890), comprise galvanochemistry, colorimetric, fluorescence sense platform etc., but these methods have respective no defect, as loaded down with trivial details modified electrode program, be subject to complex environment impact, too high background signal etc., have impact on conventional efficient and testing result.
Graphene-based hydrogel is the class nanometer three-dimensional structure assembly obtaining extensive concern in recent years.There is because of it advantages such as larger specific surface area, stimuli responsive characteristic, more conjugation region and oxygen-containing functional group, make it in insoluble drug release, dyestuff or heavy metals removal, have huge advantage.In addition the graphene-based hydrogel reduced also has stronger mechanical property, electric conductivity, thermal stability, environmental stability, chemistry or electrochemical stability, make it in energy storage, there is larger application prospect in the fields such as such as ultracapacitor, lithium battery, fuel cell and DSSC.Nowadays, graphene-based hydrogel is also applied to sensing detection field (SensorsandActuatorsB:Chemical, 2016,223,76-82; AnalyticalChemistry, 2015,87 (19), 9567-9571).Graphene-based hydrogel is applied to antibiotic detection in water body by the present invention, sets up a kind of fluorescence analysis method of terramycin, and solve loaded down with trivial details consuming time, the shortcoming such as cost is high, poor practicability of classic method, application prospect widely.
Summary of the invention
The invention solves that the preparation of existing microbiotic detection method nano material is complicated, energy consumption is high, instability is easily reunited, the deficiency such as consuming time, poor practicability, provide the method for terramycin in a kind of easy, quick, economic, practical detection water body.
Graphene-based composite aquogel in the present invention by simply, rapidly graphene dispersing solution, crosslinking chemical (adenosine and aptamers) being obtained through physical mixed, can avoid complicated modification program and the consumption of High Temperature High Pressure.Wherein, graphene sheet layer is as molecule of the skeleton, and GO lamella is joined together to form three-dimensional macro structure as crosslinking chemical by adenosine and aptamers jointly.And the quantitative detection of terramycin can be realized by a convenient-to-running immersion process.In immersion process, terramycin can form object-adaptor complex with fluorescently-labeled aptamers specific binding as object.Due to the change of aptamers configuration, weaken the acting force between aptamers and GO lamella, object-adaptor complex is come off from GO lamella and enters into supernatant.In supernatant fluorescence intensity within the specific limits with the concentration positive correlation of terramycin, thus provide foundation for the quantitative test of terramycin.And the specific recognition capability that aptamers is intrinsic, can ensure that graphene-based composite aquogel is applied to the specificity of detection.
The invention provides a kind of method of detection terramycin of fluorescence sense platform of graphene-based composite aquogel, concrete steps are as follows:
(1) graphene oxide is prepared: graphene oxide adopts the Hummers chemical method preparation improved.Step is as follows: get a certain amount of concentrated sulphuric acid (98%) and slowly join in dag (concentrated sulphuric acid volume: dag quality=23:1), after fully stirring, in 0 DEG C of ice-water bath, slowly adds KMnO
4(KMnO
4be 3:1 with dag mass ratio), fully stir simultaneously.Then by after potpourri continuous ultrasound 5 ~ 7h, deep brown solution is obtained.In deep brown solution, slowly add high purity water, after 5 ~ 15min is boiled in heating, add the hydrogen peroxide cessation reaction of high purity water and 30% successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, remove impurity 2 ~ 3 times with watery hydrochloric acid centrifuge washing under the condition of 8000 ~ 10000r/min, then remove impurity 5 ~ 7 times with high purity water centrifuge washing under the condition of 8000 ~ 10000r/min.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse 5 ~ 7 days to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) the graphene-based compound water congealing being applied to terramycin detection composes: choose specific recognition with the aptamers in conjunction with terramycin as recognition component, the 5' end of the single stranded DNA of this aptamers is carried out to the fluorescence labeling of FAM group; Graphene uniform dispersion step (1) prepared in aqueous, be that 2 ~ 15 μMs of aptamers solution hatch 4 ~ 12 hours at 0 ~ 25 DEG C by the graphene dispersing solution of 10mg/mL and concentration, mix with the adenosine solution of 10mg/mL again, potpourri acutely rocks, and 5 ~ 10min is heated at 85 ~ 95 DEG C, form uniform graphite oxide thiazolinyl composite aquogel, wherein the volume ratio of graphene dispersing solution, aptamers solution and adenosine solution three is 10:1:2.
(3) the quantitative detection of terramycin: in graphite oxide thiazolinyl composite aquogel prepared by step (2), slowly add the PBS solution of the terramycin of 0 ~ 2000 μ g/L, through the immersion of 0.5 ~ 2 hour, get supernatant and carry out fluorescent strength determining.
Described aptamers sequence is
5'-FAM-CGTACGGAATTCGCTAGCCGAGGCACAGTCGCTGGTGCCTACCTGGTTGCCGTTGTGTGGATCCGAGCTCCACGTG-3'。
Beneficial effect of the present invention:
(1) aptamers content is 10 μMs, and during 50 μ L, the Monitoring lower-cut of this graphene-based composite aquogel system can reach 25 μ g/L, and sensing range is at 25 ~ 1000 μ g/L.
(2) formation of graphene-based composite aquogel is by quick, the easy mixing plastic process of a step, does not need the synthesis condition that high pressure, high temperature etc. are complicated.
(3) testing process of terramycin is convenient, only needs through simple immersion process, gets supernatant and carry out fluorescent strength determining.
(4) by changing the content of aptamers in graphene-based composite aquogel, the controlled sensing range of aquogel system can be realized.
(5) this graphene-based composite aquogel by changing the kind of aptamers, can realize the detection of other objects, namely has the function that versatility detects.
Accompanying drawing explanation
Fig. 1 is the preparation process of graphene-based composite aquogel of the present invention and detection mechanism schematic diagram.
Fig. 2 A be method of the present invention obtain graphene-based aquogel system be applied to when aptamers content is 2 μMs terramycin detection standard working curve.
Fig. 2 B be method of the present invention obtain graphene-based aquogel system be applied to when aptamers content is 2 μMs terramycin detection the range of linearity and linear equation.
Fig. 3 A be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 5 μMs terramycin detection standard working curve.
Fig. 3 B be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 5 μMs terramycin detection the range of linearity and linear equation.
Fig. 4 A be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 10 μMs terramycin detection standard working curve.
Fig. 4 B be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 10 μMs terramycin detection the range of linearity and linear equation.
Fig. 5 A be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 15 μMs terramycin detection standard working curve.
Fig. 5 B be method of the present invention obtain graphene-based composite aquogel system be applied to when aptamers content is 15 μMs terramycin detection the range of linearity and linear equation.
Embodiment
The specific embodiment of the present invention is illustrated below in conjunction with technical scheme.
Embodiment 1
The mensuration of terramycin content in configuration water sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (2 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphite oxide thiazolinyl composite aquogel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with antibiotic concentration situation of change.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.92 (Fig. 2 A and Fig. 2 B).
(5) mensuration of terramycin content in water sample is configured:
With the water sample that PBS buffer solution configuration terramycin concentration is 500 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 520 μ g/L, and the recovery is 104%.Relative standard deviation RSD is 2.20% (n=5).
Embodiment 2
The mensuration of terramycin content in configuration water sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (5 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphite oxide thiazolinyl composite aquogel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with terramycin concentration change situation.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.99 (Fig. 3 A and Fig. 3 B).
(5) mensuration of terramycin content in water sample is configured:
With the water sample that PBS buffer solution configuration terramycin concentration is 800 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 782 μ g/L, and the recovery is 97.8%.Relative standard deviation RSD is 1.86% (n=5).
Embodiment 3
The mensuration of terramycin content in configuration water sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (10 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphite oxide thiazolinyl composite aquogel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with terramycin concentration change situation.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.99 (Fig. 4 A and Fig. 4 B).
(5) mensuration of terramycin content in water sample is configured:
With the water sample that PBS buffer solution configuration terramycin concentration is 250 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 245 μ g/L, and the recovery is 98.0%.Relative standard deviation RSD is 3.30% (n=5).
Embodiment 4
The mensuration of terramycin content in configuration water sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (15 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphite oxide thiazolinyl composite aquogel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with terramycin concentration change situation.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.90 (Fig. 5 A and Fig. 5 B).
(5) mensuration of terramycin content in water sample is configured:
With the water sample that PBS buffer solution configuration terramycin concentration is 100 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 119 μ g/L, and the recovery is 119%.Relative standard deviation RSD is 2.70% (n=5).
Embodiment 5
The mensuration of terramycin content in tap water sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (10 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphene oxide based aquagel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with terramycin concentration change situation.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.99 (Fig. 4 A and Fig. 4 B).
(5) mensuration of terramycin content in water sample is configured:
Owing to not detecting terramycin in tap water sample, therefore adopt recovery testu.With tap water sample preparation terramycin solution, concentration is 2500 μ g/L.Get 200 μ L to add target tap water sample and mix with the PBS buffer solution of 1800 μ L, in mixed liquor, terramycin concentration is 250 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 240 μ g/L, and the recovery is 96%.Relative standard deviation RSD is 1.75% (n=5).
Embodiment 6
The mensuration of terramycin content in river sample:
(1) preparation of graphene oxide:
Graphene oxide adopts the Hummers chemical method preparation improved.Concrete steps are as follows: take 1.0g dag and slowly join in the 23mL concentrated sulphuric acid, after fully stirring, in 0 DEG C of ice-water bath, slowly add 3gKMnO
4, slowly add and fully stir, then after continuous ultrasound 12h simultaneously, obtain deep brown solution, then slowly add 46mL deionized water, after 15min is boiled in heating, add 140mL high purity water and 10mL hydrogen peroxide cessation reaction more successively, obtain jonquilleous graphene oxide aqueous solution.After centrifuging, the watery hydrochloric acid 10000r/min centrifuge washing with 5% removes impurity 2 times, then removes impurity 5 times with high purity water 10000r/min centrifuge washing.Take out the graphite oxide of purifying after washing, load bag filter (MW=14000) and dialyse one week to remove impurity further, last freeze drying obtains solid oxidation Graphene.
(2) preparation of graphene-based composite aquogel:
The graphene dispersing solution of 500 μ L (10mg/mL) needs at 4 DEG C, to hatch several hours with the fluorescence labeling aptamers solution of 50 μ L (15 μMs) in advance, add adenosine solution (100 μ L afterwards, 10mg/mL), mixed liquor acutely mixes, rock, and 5min is heated at 90 DEG C, form uniform graphene oxide based aquagel.
(3) detection method:
In aquogel system prepared by step (2), (solvent is the PBS solution of 20mM, comprises 100mMNaCl, 3mMKCl, 10mMMgCl slowly to add the terramycin aqueous solution of 0 ~ 2000 μ g/L
2, pH=7.5).Through the immersion reaction of 2 hours, get supernatant and carry out fluorescent strength determining, excitation wavelength was 486nm, and emission wavelength is 518nm, at ambient temperature, measured supernatant fluorescence intensity with terramycin concentration change situation.
(4) drafting of standard working curve
Along with the increase of terramycin concentration in sample in step (3), the fluorescence intensity of supernatant constantly increases, and within the scope of 25 ~ 1000 μ g/L, fluorescence intensity and terramycin concentration have good linear relationship, linearly dependent coefficient R
2=0.99 (Fig. 5 A and Fig. 5 B).
(5) mensuration of terramycin content in river sample:
Owing to not detecting terramycin in river sample, therefore adopt recovery testu.River sample first filters through the filtering membrane of 0.22 μm to remove suspended solid.Afterwards with the water sample preparation terramycin solution of river sample, concentration is 2500 μ g/L.Get 200 μ L to add target river sample and mix with the PBS buffer solution of 1800 μ L, in mixed liquor, terramycin concentration is 250 μ g/L.Sample is used for step (3) method to detect, the standard working curve that testing result and step (4) obtain contrasts, and calculates the concentration of terramycin.Experimental result measures terramycin content 273 μ g/L, and the recovery is 109%.Relative standard deviation RSD is 4.15% (n=5).
Claims (1)
1., based on a terramycin fluorescence detection method for graphene-based composite aquogel, it is characterized in that, step is as follows:
(1) graphene oxide is prepared
(2) the graphene-based compound water congealing being applied to terramycin detection composes: choose specific recognition with the aptamers in conjunction with terramycin as recognition component, the 5' end of the single stranded DNA of this aptamers is carried out to the fluorescence labeling of FAM group; Graphene uniform dispersion step (1) prepared in aqueous, be that 2 ~ 15 μMs of aptamers solution hatch 4 ~ 12 hours at 0 ~ 25 DEG C by the graphene dispersing solution of 10mg/mL and concentration, mix with the adenosine solution of 10mg/mL again, potpourri acutely rocks, and 5 ~ 10min is heated at 85 ~ 95 DEG C, form uniform graphite oxide thiazolinyl composite aquogel, wherein the volume ratio of graphene dispersing solution, aptamers solution and adenosine solution three is 10:1:2;
(3) the quantitative detection of terramycin: in graphite oxide thiazolinyl composite aquogel prepared by step (2), slowly add the PBS solution of the terramycin of 0 ~ 2000 μ g/L, through the immersion of 0.5 ~ 2 hour, get supernatant and carry out fluorescent strength determining;
Described aptamers sequence is
5'-FAM-CGTACGGAATTCGCTAGCCGAGGCACAGTCGCTGGTGCCTACCTGGTTGCCGTTGTGTGGATCCGAGCTCCACGTG-3'。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234263A (en) * | 2018-09-27 | 2019-01-18 | 福建海峡石墨烯产业技术研究院有限公司 | A method of the crosslinked action immobilized enzyme based on adenosine monophosphate and graphene |
| CN112362598A (en) * | 2020-10-14 | 2021-02-12 | 厦门大学 | Antibiotic detection method based on hydrogel release mechanism |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7456951B2 (en) * | 2004-11-25 | 2008-11-25 | Hitachi High-Technologies Corporation | Fluorescence detection method, detection apparatus and fluorescence detection program |
| CN103881128A (en) * | 2014-03-10 | 2014-06-25 | 黄鹏 | Preparation method of in-situ glucose detecting membrane based on fluorescence detection method |
| CN104194791A (en) * | 2014-07-24 | 2014-12-10 | 江苏大学 | Preparation method of CdTe quantum dots for fluorescence detection of aspirin |
| CN104387318A (en) * | 2014-11-11 | 2015-03-04 | 中国科学技术大学 | Gelator of supramolecular hydrogel capable of detecting and removing cadmium and preparation method of gelator |
| CN104730008A (en) * | 2015-01-27 | 2015-06-24 | 大连理工大学 | Oxytetracycline sensing detection method based on graphene aptamer hydrogel |
-
2016
- 2016-01-20 CN CN201610037352.3A patent/CN105572090B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7456951B2 (en) * | 2004-11-25 | 2008-11-25 | Hitachi High-Technologies Corporation | Fluorescence detection method, detection apparatus and fluorescence detection program |
| CN103881128A (en) * | 2014-03-10 | 2014-06-25 | 黄鹏 | Preparation method of in-situ glucose detecting membrane based on fluorescence detection method |
| CN104194791A (en) * | 2014-07-24 | 2014-12-10 | 江苏大学 | Preparation method of CdTe quantum dots for fluorescence detection of aspirin |
| CN104387318A (en) * | 2014-11-11 | 2015-03-04 | 中国科学技术大学 | Gelator of supramolecular hydrogel capable of detecting and removing cadmium and preparation method of gelator |
| CN104730008A (en) * | 2015-01-27 | 2015-06-24 | 大连理工大学 | Oxytetracycline sensing detection method based on graphene aptamer hydrogel |
Non-Patent Citations (2)
| Title |
|---|
| BIMALENDU ADHIKARI等: "Graphene Oxide-Based Supramolecular Hydrogels for Making Nanohybrid Systems with Au Nanoparticles", 《LANGMUIR》 * |
| 高升: "基于长链适配子与石墨烯构建的荧光传感方法及其对土霉素的检测研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234263A (en) * | 2018-09-27 | 2019-01-18 | 福建海峡石墨烯产业技术研究院有限公司 | A method of the crosslinked action immobilized enzyme based on adenosine monophosphate and graphene |
| CN112362598A (en) * | 2020-10-14 | 2021-02-12 | 厦门大学 | Antibiotic detection method based on hydrogel release mechanism |
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