CN105572090B - A kind of terramycin fluorescence detection method based on graphene-based composite hydrogel - Google Patents
A kind of terramycin fluorescence detection method based on graphene-based composite hydrogel Download PDFInfo
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- CN105572090B CN105572090B CN201610037352.3A CN201610037352A CN105572090B CN 105572090 B CN105572090 B CN 105572090B CN 201610037352 A CN201610037352 A CN 201610037352A CN 105572090 B CN105572090 B CN 105572090B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6491—Measuring fluorescence and transmission; Correcting inner filter effect
Abstract
The invention belongs to environmental monitoring technology field, it is related to a kind of fluorescence sense detection method of antibiotic in the water body of graphene-based composite hydrogel.Graphene dispersing solution links together the graphene oxide layer of script dispersion in the solution collectively as crosslinking agent as monomer, adenosine and aptamers, forms three-dimensional macro structure.Simple soaking process is carried out by the way that the terramycin aqueous solution of various concentration to be added in aquogel system, supernatant is taken to carry out fluorescent strength determining, you can realizes the fluorogenic quantitative detection of terramycin concentration.In aquogel system by the different proportion of preparation, controllable adjusting is realized to the detection range of terramycin, is advantageously applied to different types of practical water body and is detected.Compared with Conventional nano material sensing detection method, this method nano material prepare quickly, easy, preparation condition is mild, the environmental stability of hydrogel material and highly practical.
Description
Technical field
The invention belongs to environmental monitoring technology fields, are related to a kind of terramycin fluorescence based on graphene-based composite hydrogel
Sensing detection method.
Background technology
Tetracycline is a kind of a kind of broad-spectrum antibiotic generated by actinomyces, including naturally occurring tetracycline
(tetracycline, TET), terramycin (oxytetracycline, OTC), fortimicin (Doxycycline), metacycline
(methacycline) etc., structure basic framework containing aphthacene, is widely used in various bacteria and rickettsia, clothing are former
Body, mycoplasma infection.But antibiotic includes the abuse of Tetracyclines, and antibiotic can be made to be accumulated in surrounding medium
(Environmental Science＆Technology, 2015,49 (11), 6772-6782.), then the effects that pass through food chain
It is enriched with (Environmental Science＆Technology, 2015,49 (8), 5070-5079.) in human body.In addition, anti-
Raw element abuse can also induce generation drug resistant gene, seriously threaten living environment (the Proceedings of the of the mankind
National Academy of Sciences of the United States of America,2013,110(9),
3435-3440.).Terramycin is grasped its environment distribution situation and is controlled the pollution of antibiotic as a kind of widely applied tetracycline
Reason has great directive significance.Therefore, urgent need is sent out sensitive, is quick, is efficient, real-time detection method is to terramycin in environment
Real-time concentration is monitored, and data supporting is provided for subsequent processing or disposing task.
Traditional antibiotic detection method has instrument detection method, bioanalysis/biochemical process etc., since its intrinsic defect is difficult
To cope with current environmental monitoring demand.For example, high performance liquid chromatography is time-consuming, laborious, of high cost；Enzyme linked immunosorbent assay (ELISA)
Although having higher sensitivity and selectivity, operating process is complicated, and system hinders this to the bad adaptability of extreme environment
Method is promoted the use of.In recent years, the electrochemistry based on nano material or nanomaterial assembly body or optical sensor, due to having
The properties such as the intrinsic electricity of nano material, optics are conducive to the conduction of signal, have been widely used in sensing analysis detection.Mesh
Before, people also using nano material be successfully established some for antibiotic detection sensing platform (RSC Advances,
2015,5(72),58895-58901；MicrochimicaActa,2013,180(9-10),829-835；Biosensors and
Bioelectronics,2010,26(4),1644-1649；Analyst, 2013,138 (6), 1886-1890), including electrification
, colorimetric, fluorescence sense platform etc., but these methods have a respective no defect, such as cumbersome modified electrode program, easily by
Complex environment influence, excessively high background signal etc., affect conventional efficient and testing result.
Graphene-based hydrogel is a kind of nanometer three-dimensional structure assembly for obtaining extensive concern in recent years.Because it has
The advantages that larger specific surface area, stimuli responsive characteristic, more conjugation region and oxygen-containing functional group, make its drug release,
There is huge advantage in terms of dyestuff or heavy metals removal.In addition the graphene-based hydrogel restored also has stronger mechanicalness
Energy, electric conductivity, thermal stability, environmental stability, chemistry or electrochemical stability make it in energy storage, such as ultracapacitor, lithium
There is larger application prospect in the fields such as battery, fuel cell and dye-sensitized solar cells.Nowadays, graphene-based hydrogel
It is applied to sensing detection field (Sensors and Actuators B:Chemical,2016,223,76-82；
Analytical Chemistry,2015,87(19),9567-9571).Graphene-based hydrogel is applied to water body by the present invention
A kind of fluorescence analysis method of terramycin is established in the detection of middle antibiotic, and it is cumbersome time-consuming, of high cost, real to solve conventional method
The shortcomings of poor with property, application prospect is very extensive.
Invention content
The present invention solves existing antibiotic detection method nano material and prepares complexity, high energy consumption, unstable easy reunion, consumption
When, poor practicability the deficiencies of, the method that provides terramycin in a kind of easy, quick, economic, practical detection water body.
Graphene-based composite hydrogel in the present invention can be by simply and rapidly by graphene dispersing solution, crosslinking agent
(adenosine and aptamers) are obtained through physical mixed, avoid the consumption of complicated modification program and high temperature and pressure.Wherein,
GO lamellas are joined together to form as molecule of the skeleton, adenosine and aptamers collectively as crosslinking agent by graphene sheet layer
Three-dimensional macro structure.And the quantitative detection of terramycin can be realized by a convenient-to-running soaking process.In soaking process
In, terramycin can be specifically bound to form object-adaptor complex as object with the aptamers of fluorescent marker.By
In the variation of aptamers configuration, weakens the active force between aptamers and GO lamellas, make object-adaptor complex from GO pieces
It is shedded into supernatant on layer.In supernatant fluorescence intensity in a certain range with the concentration positive correlation of terramycin, to
Foundation is provided for the quantitative analysis of terramycin.And the specific recognition capability that aptamers are intrinsic, it is ensured that graphene-based compound
Hydrogel is applied to the specificity of detection.
The present invention provides a kind of method of the detection terramycin of fluorescence sense platform of graphene-based composite hydrogel, tools
Steps are as follows for body：
(1) graphene oxide is prepared：Graphene oxide is prepared using improved Hummers chemical methods.Steps are as follows：It takes
A certain amount of concentrated sulfuric acid (98%) is slowly added into graphite powder (concentrated sulfuric acid volume:Graphite powder quality=23:1) it in, is sufficiently stirred
Afterwards, it is slowly added to KMnO in 0 DEG C of ice-water bath4(KMnO4It is 3 with graphite powder mass ratio:1) it, is sufficiently stirred simultaneously.Then it will mix
After closing 5~7h of object continuous ultrasound, deep brown solution is obtained.It is slowly added to high purity water into deep brown solution, heating boils 5~
After 15min, high purity water and 30% hydrogen peroxide termination reaction are sequentially added, the graphene oxide for obtaining glassy yellow is water-soluble
Liquid.After centrifugation, with dilute hydrochloric acid under conditions of 8000~10000r/min the removal of centrifuge washing 2~3 times impurity, then use
High purity water 5~7 removal impurity of centrifuge washing under conditions of 8000~10000r/min.The graphite oxygen of purifying is taken out after washing
Compound is packed into bag filter (MW=14000) and dialyses 5~7 days further to remove impurity, and finally freeze-drying obtains solid oxidation
Graphene.
(2) the graphene-based compound water congealing for being applied to terramycin detection composes：It chooses specific recognition and combines soil mould
The aptamers of element carry out the ends 5' of the single stranded DNA of the aptamers fluorescent marker of FAM groups as recognition component；By step
(1) in aqueous solution, the graphene dispersing solution of 10mg/mL is adapted to a concentration of 2~15 μM for the graphene uniform dispersion prepared
Liquid solution is hatched 4~12 hours at 0~25 DEG C, then is mixed with the adenosine solution of 10mg/mL, and mixture acutely shakes
It is dynamic, and 5~10min is heated at 85~95 DEG C, form uniform graphite oxide alkenyl composite hydrogel, wherein graphene dispersion
The volume ratio of liquid, adaptation liquid solution and adenosine solution three is 10:1:2.
(3) the quantitative detection of terramycin：In the graphite oxide alkenyl composite hydrogel prepared to step (2), it is slowly added to 0
The PBS solution of the terramycin of~2000 μ g/L took supernatant to carry out fluorescent strength determining by immersion in 0.5~2 hour.
The aptamers sequence is
5'-FAM-CGTACGGAATTCGCTAGCCGAGGCACAGTCGCTGGTGCCTACCTGGTTGCCGTTGTGTGGAT
CCGAGCTCCACGTG-3'。
Beneficial effects of the present invention：
(1) aptamers content is 10 μM, and when 50 μ L, the Monitoring lower-cut of the graphene-based composite hydrogel system is up to 25 μ
G/L, detection range is in 25~1000 μ g/L.
(2) formation of graphene-based composite hydrogel is the mixing plastic process by a step quickly, easy, does not need height
The synthesis condition of the complexity such as pressure, high temperature.
(3) detection process of terramycin is convenient, it is only necessary to pass through simple soaking process, supernatant is taken to carry out fluorescence intensity
Measurement.
(4) the controllable of aquogel system can be realized by the content of aptamers in the graphene-based composite hydrogel of change
Detection range.
(5) the graphene-based composite hydrogel can realize the detection of other objects by the type of replacement aptamers,
Has the function of versatility detection.
Description of the drawings
Fig. 1 is the preparation process and detection mechanism schematic diagram of graphene-based composite hydrogel of the present invention.
Fig. 2A is that the graphene-based aquogel system that the method for the present invention obtains is applied to soil when aptamers content is 2 μM
The standard working curve of mycin detection.
Fig. 2 B are that the graphene-based aquogel system that the method for the present invention obtains is applied to soil when aptamers content is 2 μM
The range of linearity and linear equation of mycin detection.
Fig. 3 A are that the graphene-based composite hydrogel system that the method for the present invention obtains is applied when aptamers content is 5 μM
In the standard working curve of terramycin detection.
Fig. 3 B are that the graphene-based composite hydrogel system that the method for the present invention obtains is applied when aptamers content is 5 μM
In the range of linearity and linear equation of terramycin detection.
Fig. 4 A are that the graphene-based composite hydrogel system that the method for the present invention obtains is answered when aptamers content is 10 μM
Standard working curve for terramycin detection.
Fig. 4 B are that the graphene-based composite hydrogel system that the method for the present invention obtains is answered when aptamers content is 10 μM
The range of linearity and linear equation for terramycin detection.
Fig. 5 A are that the graphene-based composite hydrogel system that the method for the present invention obtains is answered when aptamers content is 15 μM
Standard working curve for terramycin detection.
Fig. 5 B are that the graphene-based composite hydrogel system that the method for the present invention obtains is answered when aptamers content is 15 μM
The range of linearity and linear equation for terramycin detection.
Specific implementation mode
The specific implementation mode of the present invention is illustrated below in conjunction with technical solution.
Embodiment 1
Configure the measurement of terramycin content in water sample：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution needs of 500 μ L (10mg/mL) are adapted to liquid solution with the fluorescent marker of 50 μ L (2 μM) in advance and exist
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes, and
5min is heated at 90 DEG C, forms uniform graphite oxide alkenyl composite hydrogel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with antibiotic concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.92 (Fig. 2A
With Fig. 2 B).
(5) measurement of terramycin content in water sample is configured：
With the water sample of a concentration of 500 μ g/L of PBS buffer solutions configuration terramycin.Sample is carried out for step (3) method
Detection, the standard working curve that testing result is obtained with step (4) compare, and calculate the concentration of terramycin.Experimental result is measured
520 μ g/L of terramycin content, the rate of recovery 104%.Relative standard deviation RSD is 2.20% (n=5).
Embodiment 2
Configure the measurement of terramycin content in water sample：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution needs of 500 μ L (10mg/mL) are adapted to liquid solution with the fluorescent marker of 50 μ L (5 μM) in advance and exist
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes, and
5min is heated at 90 DEG C, forms uniform graphite oxide alkenyl composite hydrogel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with terramycin concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.99 (Fig. 3 A
With Fig. 3 B).
(5) measurement of terramycin content in water sample is configured：
With the water sample of a concentration of 800 μ g/L of PBS buffer solutions configuration terramycin.Sample is carried out for step (3) method
Detection, the standard working curve that testing result is obtained with step (4) compare, and calculate the concentration of terramycin.Experimental result is measured
782 μ g/L of terramycin content, the rate of recovery 97.8%.Relative standard deviation RSD is 1.86% (n=5).
Embodiment 3
Configure the measurement of terramycin content in water sample：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution of 500 μ L (10mg/mL) needs to be adapted to liquid solution with the fluorescent marker of 50 μ L (10 μM) in advance
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes,
And 5min is heated at 90 DEG C, form uniform graphite oxide alkenyl composite hydrogel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with terramycin concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.99 (Fig. 4 A
With Fig. 4 B).
(5) measurement of terramycin content in water sample is configured：
With the water sample of a concentration of 250 μ g/L of PBS buffer solutions configuration terramycin.Sample is carried out for step (3) method
Detection, the standard working curve that testing result is obtained with step (4) compare, and calculate the concentration of terramycin.Experimental result is measured
245 μ g/L of terramycin content, the rate of recovery 98.0%.Relative standard deviation RSD is 3.30% (n=5).
Embodiment 4
Configure the measurement of terramycin content in water sample：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution of 500 μ L (10mg/mL) needs to be adapted to liquid solution with the fluorescent marker of 50 μ L (15 μM) in advance
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes,
And 5min is heated at 90 DEG C, form uniform graphite oxide alkenyl composite hydrogel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with terramycin concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.90 (Fig. 5 A
With Fig. 5 B).
(5) measurement of terramycin content in water sample is configured：
With the water sample of a concentration of 100 μ g/L of PBS buffer solutions configuration terramycin.Sample is carried out for step (3) method
Detection, the standard working curve that testing result is obtained with step (4) compare, and calculate the concentration of terramycin.Experimental result is measured
119 μ g/L of terramycin content, the rate of recovery 119%.Relative standard deviation RSD is 2.70% (n=5).
Embodiment 5
Originally in water sample terramycin content measurement：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution of 500 μ L (10mg/mL) needs to be adapted to liquid solution with the fluorescent marker of 50 μ L (10 μM) in advance
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes,
And 5min is heated at 90 DEG C, form uniform graphene oxide based aquagel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with terramycin concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.99 (Fig. 4 A
With Fig. 4 B).
(5) measurement of terramycin content in water sample is configured：
Since terramycin being not detected in originally water sample, therefore use recovery testu.It is mould with tap water sample preparation soil
Plain solution, a concentration of 2500 μ g/L.The originally water sample of 200 μ L mark-ons is taken to be mixed with the PBS buffer solutions of 1800 μ L, mixed liquor
Middle a concentration of 250 μ g/L of terramycin.Sample is detected for step (3) method, the mark that testing result is obtained with step (4)
Quasi- working curve comparison, calculates the concentration of terramycin.Experimental result measures 240 μ g/L of terramycin content, the rate of recovery 96%.
Relative standard deviation RSD is 1.75% (n=5).
Embodiment 6
The measurement of terramycin content in river sample：
(1) preparation of graphene oxide：
Graphene oxide is prepared using improved Hummers chemical methods.It is as follows：It is slow to weigh 1.0g graphite powders
Slowly it is added in the 23mL concentrated sulfuric acids, after being sufficiently stirred, 3g KMnO is slowly added in 0 DEG C of ice-water bath4, it is slowly added to fill simultaneously
Divide stirring, then after continuous ultrasound 12h, obtains deep brown solution, be then slowly added into 46mL deionized waters, heating is boiled
After 15min, sequentially adds 140mL high purity waters and 10mL hydrogen peroxide terminates reaction, obtain the graphene oxide water of glassy yellow
Solution.After centrifugation, with 5% 2 removal impurity of dilute hydrochloric acid 10000r/min centrifuge washings, high purity water is then used
5 removal impurity of 10000r/min centrifuge washings.The graphite oxide of purifying is taken out after washing, is packed into bag filter (MW=
14000) one week is dialysed further to remove impurity, and finally freeze-drying obtains solid oxidation graphene.
(2) preparation of graphene-based composite hydrogel：
The graphene dispersing solution of 500 μ L (10mg/mL) needs to be adapted to liquid solution with the fluorescent marker of 50 μ L (15 μM) in advance
To hatch a few houres at 4 DEG C, adenosine solution (100 μ L, 10mg/mL) is added later, mixed liquor is vigorously mixed, shakes,
And 5min is heated at 90 DEG C, form uniform graphene oxide based aquagel.
(3) detection method：
In the aquogel system prepared to step (2), being slowly added to the terramycin aqueous solution of 0~2000 μ g/L, (solvent is
The PBS solution of 20mM, including 100mM NaCl, 3mM KCl, 10mM MgCl2, pH=7.5).It is reacted by immersions in 2 hours,
Supernatant is taken to carry out fluorescent strength determining, excitation wavelength 486nm, launch wavelength 518nm, at ambient temperature, in measurement
Clear liquid fluorescence intensity is with terramycin concentration situation of change.
(4) drafting of standard working curve
As the increase of terramycin concentration in sample, the fluorescence intensity of supernatant are continuously increased in step (3), 25~
Within the scope of 1000 μ g/L, fluorescence intensity has good linear relationship, linearly dependent coefficient R with terramycin concentration2=0.99 (Fig. 5 A
With Fig. 5 B).
(5) in river sample terramycin content measurement：
Since terramycin being not detected in river sample, therefore use recovery testu.River sample first passes through 0.22 μm
Membrane filtration is filtered to remove suspended solid.The water sample of river sample is used to prepare terramycin solution, a concentration of 2500 μ g/L later.It takes
The river sample of 200 μ L mark-ons is mixed with the PBS buffer solutions of 1800 μ L, a concentration of 250 μ g/L of terramycin in mixed liquor.By sample
Product are detected for step (3) method, and the standard working curve that testing result is obtained with step (4) compares, and are calculated unearthed mould
The concentration of element.Experimental result measures 273 μ g/L of terramycin content, the rate of recovery 109%.Relative standard deviation RSD is 4.15%
(n=5).
Claims (1)
1. a kind of terramycin fluorescence detection method based on graphene-based composite hydrogel, which is characterized in that steps are as follows：
(1) graphene oxide is prepared
(2) the graphene-based compound water congealing for being applied to terramycin detection composes：It chooses specific recognition and combines terramycin
Aptamers carry out the ends 5' of the single stranded DNA of the aptamers fluorescent marker of FAM groups as recognition component；Step (1) is made
The graphene dispersing solution of 10mg/mL in aqueous solution, is adapted to liquid solution by standby graphene uniform dispersion with a concentration of 2~15 μM
To hatch 4~12 hours at 0~25 DEG C, then is mixed with the adenosine solution of 10mg/mL, mixture acutely shakes, and
5~10min is heated at 85~95 DEG C, forms uniform graphite oxide alkenyl composite hydrogel, wherein graphene dispersing solution, adaptation
The volume ratio of liquid solution and adenosine solution three are 10:1:2；
(3) the quantitative detection of terramycin：In the graphite oxide alkenyl composite hydrogel prepared to step (2), it is slowly added to 0~
The PBS solution of the terramycin of 2000 μ g/L took supernatant to carry out fluorescent strength determining by immersion in 0.5~2 hour；
The aptamers sequence is
5'-FAM-CGTACGGAATTCGCTAGCCGAGGCACAGTCGCTGGTGCCTACCTGGTTGCCGTTGTGTGGATCCGA
GCTCCACGTG-3'。
Priority Applications (1)
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