CN108181283A - Double emission ratios type fluorescent optical sensors and its preparation method and application - Google Patents
Double emission ratios type fluorescent optical sensors and its preparation method and application Download PDFInfo
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- CN108181283A CN108181283A CN201810022982.2A CN201810022982A CN108181283A CN 108181283 A CN108181283 A CN 108181283A CN 201810022982 A CN201810022982 A CN 201810022982A CN 108181283 A CN108181283 A CN 108181283A
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of double emission ratios type fluorescent optical sensors.Double emission ratios type fluorescent optical sensors, are obtained using glutathione modified nano gold particle preparation, and stronger fluorescence emission peak is respectively provided at 600nm and at 800nm.Double emission ratios type fluorescent optical sensors are applied in acetylcholinesterase and Pesticides Testing, based on Au S Covalent bonding togethers, substance with SH is generated by acetylcholinesterase hydrolyzable acetylthiocholine or inhibits the activity of enzyme, the property of the double fluorescence emission peaks of nanogold particle itself modified with reference to glutathione, have many advantages, such as that easy to operate, sample treatment is simple, efficient, highly sensitive detection, application prospect is good.Based on double emission ratios type fluorescent optical sensors, the preparation method the present invention provides a kind of double emission ratios type fluorescent optical sensors and its application in detection acetylcholinesterase and pesticide residue.
Description
Technical field
The present invention relates to nanometer material science and biochemistry field of sensing technologies, and in particular to a kind of double emission ratios
Type fluorescent optical sensor and its preparation method and application.
Background technology
Pesticide, including Insecticides (tech) ＆ Herbicides (tech) and bacteria remover, due to having higher toxicity, so as to extensively should
Among modern agriculture, although pest disaster can be prevented, also it is permeated with simultaneously among mankind's publilc health health.According to the world
The data analysis of health organization, about 1,500,000,000 person-times of children's generations include poisoning by food caused by pesticide residue every year；Wherein
Typical Representative be exactly carbamates and organophosphorus insecticide, mechanism key is carbamate and organic phosphates agriculture
Medicine can inhibit a kind of enzyme with catalytic activity --- the activity of acetylcholinesterase in nervous system, so as to inhibit neural biography
It passs, leads to organ failure until dead.Therefore, it protects the mankind from the remaining injury of pesticide in pollution by pesticides and food, seeks
The simple and convenient efficient detection remaining method of Environmental Pesticide is looked for be of great significance.
Traditional Pesticides Testing method includes mass spectrum, gas-chromatography, high performance liquid chromatography and enzyme linked immunoassay etc.,
However these methods not only took time and effort, but also needed expensive reagent, complicated sample treatment, accurate instrument and skilled behaviour
Make personnel.
In consideration of it, the defects of overcoming present in the above-mentioned prior art, provides a kind of simple, convenient, quick, highly sensitive
Detection method is this field urgent problem to be solved.
Invention content
The technical problem to be solved in the present invention is to provide a kind of double emission ratios type fluorescent optical sensors, can be quick, convenient, high
Sensitivity acetylcholinesterase and pesticide residue are detected.
To solve the above-mentioned problems, the present invention provides a kind of double emission ratios type fluorescent optical sensors.The technical solution
It is as follows：
A kind of double emission ratios type fluorescent optical sensors, are obtained using glutathione modified nano gold particle preparation,
At 600nm and it is respectively provided with stronger fluorescence emission peak at 800nm.
Further, grain diameter is 2.5 ± 0.5nm in double emission ratios type fluorescent optical sensors.
The present invention also provides a kind of preparation methods of double emission ratios type fluorescent optical sensors, include the following steps：
Glutathione solution under 90-95 DEG C of oil bath environment is heated, while being vigorously stirred, adds in chlorauric acid solution,
The nanogold particle solution of glutathione modification is prepared；Preferably, glutathione solution is distinguished with chlorauric acid solution dosage
For：The chlorauric acid solution of the glutathione solution of a concentration of 3mM of 50mL and 1500 a concentration of 100mM of μ L；
Cooling, centrifugation, ultrafiltration purification obtain double emission ratios type fluorescent optical sensors.
Further, centrifugation, ultrafiltration purification, dispersion include the following steps：
Centrifugation：The nanogold particle solution that glutathione is modified is placed in a centrifuge 21000g centrifugations, removes bulky grain
Deposit；
Ultrafiltration purification：In the case where rotating speed is 10000rpm, it is extra in solution that supernatant progress hyperfiltration treatment is removed three times
Glutathione；
Dispersion：Nanogold particle obtained by ultrafiltration HEPES buffer solution is pressed 1:2 volume ratios are disperseed again, obtain double hairs
Penetrate Ratio-type fluorescent optical sensor；The wherein a concentration of 2M, pH=8.0 of HEPES buffer solution.
Based on double emission ratios type fluorescent optical sensors, the present invention also provides a kind of double emission ratios type fluorescent optical sensors
Application in acetylcholinesterase is detected.
A kind of method of double emission ratios type fluorescent optical sensor detection acetylcholinesterases, includes the following steps：
Acetylcholinesterase, substrate acetylthiocholine and double emission ratios type fluorescent optical sensors are mixed in proportion
It closes, reaction system is made, and 25min is incubated at 37 DEG C；
Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm and 800nm
The fluorescence intensity change at place realizes the detection to acetylcholinesterase；
Preferably, Fluorescence Spectrometer measures the fluorescence spectrum of 500-900nm, excitation wavelength 400nm.
Further, reaction system preparation method includes：
Add in the substrate acetylthiocholine, the bis- emission ratios type fluorescent optical sensors of 3 μ L, second to be measured of 10 a concentration of 1mM of μ L
100 μ L reaction systems are made in acetylcholinesterase and suitable ultra-pure water.
Based on double emission ratios type fluorescent optical sensors, the present invention also provides a kind of double emission ratios type fluorescent optical sensors
Application in pesticide residue is detected.
A kind of method of double emission ratios type fluorescent optical sensor detection pesticide residues, includes the following steps：
By acetylcholinesterase and pestsides synthesis to be measured, 20min is incubated at 37 DEG C；
Substrate acetylthiocholine and double emission ratios type fluorescent optical sensors are proportionally added into, reaction system is made, 37
5min is incubated at DEG C；
Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm and 800nm
The fluorescence intensity change at place realizes the detection to pesticide residue；
Preferably, Fluorescence Spectrometer measures the fluorescence spectrum of 500-900nm, excitation wavelength 400nm.
Further, reaction system preparation includes：
Add in a concentration of 1U/mL acetylcholinesterases of 8 μ L, a concentration of 1mM acetylthiocholines of 10 μ L, the 3 bis- transmitting ratios of μ L
100 μ L reaction systems are made in rate type fluorescent optical sensor, pesticide to be measured and suitable ultra-pure water.
Compared with prior art, double emission ratios type fluorescent optical sensors provided by the invention, advantageous effect are：It is applied to
In acetylcholinesterase and Detecting Pesticide, based on Au-S Covalent bonding togethers, pass through acetylcholinesterase hydrolyzable sulphur
The substance of band-SH is generated for acetylcholine or pesticide inhibits the activity of enzyme, the nanogold particle itself modified with reference to glutathione
The double emission peak properties of fluorescence have many advantages, such as that easy to operate, sample treatment is simple, efficient, highly sensitive detection.
Description of the drawings
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments of the present invention, for
For those of ordinary skill in the art, without creative efforts, other are can also be obtained according to these attached drawings
Attached drawing.
Fig. 1 is the material characterization figure of double emission ratios type fluorescent optical sensors provided by the invention；
Fig. 2 is the testing principle schematic diagram of double emission ratios type fluorescent optical sensors provided by the invention；
Fig. 3 is reaction mechanism fluorescence spectra of the present invention；
Fig. 4 is the fluorescence spectra of various concentration acetylcholinesterase；
Fig. 5 is ratio figure of the fluorescence intensity of various concentration acetylcholinesterase at 800nm and 600nm；
Fig. 6 is the fluorescence spectra of pesticide acetylcholine esterase inhibition reaction of the various concentration by taking Aldicarb as an example；
Fig. 7 is the logarithmic relationship figure of fluorescence intensity ratio at 800nm and 600nm of various concentration Aldicarb.
Specific embodiment
In order to which those skilled in the art is made to more fully understand the technical solution in the embodiment of the present invention, and make the present invention's
Above-mentioned purpose, feature and advantage can be more obvious understandable, and the specific embodiment of the present invention is made into one below in conjunction with the accompanying drawings
The explanation of step.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
Embodiment 1：The preparation of double emission ratios type fluorescent optical sensors
The preparation method of double emission ratios type fluorescent optical sensors, includes the following steps：
Step S1：Prepare the nanogold particle solution of glutathione modification；
It specifically includes：
Step S11：By concentrated nitric acid and concentrated hydrochloric acid according to 1:3 ratio uniform mixing, is made chloroazotic acid, stands 30min, make it
Oxidability reaches most strong, then continues in flask, gently shakes, and after 30min, shifts chloroazotic acid, with super water clean flask and its
He reacts glassware, and is baked to spare；
Step S12：In the reaction glassware cleaned in step S11, the glutathione for adding in a concentration of 3mM of 50mL is molten
Liquid while being vigorously stirred, adds in the chlorauric acid solution of 1500 a concentration of 100mM of μ L, and whole device is placed in 95 DEG C of oil bath pan
Middle reaction 35min, is prepared the nanogold particle solution of finely dispersed glutathione modification, and solution is clarified to be golden yellow
Liquid；
Step S2：Cooling, centrifugation, ultrafiltration purification, dispersion obtain double emission ratios type fluorescent optical sensors；
It specifically includes：
Step S21：The nanogold particle solution that the glutathione of gained is modified after step S1 is reacted is cooled to room temperature, and is put
21000g is centrifuged in ultracentrifuge, removes bulky grain deposit；
Step S22：Under rotating speed 10000rpm, hyperfiltration treatment is carried out to supernatant three times to remove paddy extra in solution
The sweet peptide of Guang；
Step S23：Nanogold particle obtained by ultrafiltration is resuspended in a concentration of 2M, the HEPES buffer solutions of pH=8.0
In, it obtains bio-sensing and detects required double emission ratios type fluorescent optical sensors；Nanogold particle wherein obtained by ultrafiltration with
The volume ratio of HEPES buffer solution is 1:2.
Referring to Fig. 1, the material characterization figure for double emission ratios type fluorescent optical sensors provided by the invention.Wherein, curve a
Represent to prepare the ultraviolet-ray visible absorbing of raw material glutathione that nanogold particle uses and gold chloride mixed solution in the present invention；
Curve b represents the ultraviolet-ray visible absorbing of chlorauric acid solution used in the present invention；Curve c represents double transmittings provided by the invention
The ultraviolet-ray visible absorbing of Ratio-type fluorescent optical sensor；Curve d represents double emission ratios type fluorescent optical sensors provided by the invention
Fluorescence emission spectrum.As seen from Figure 1, double emission ratios type fluorescent optical sensors provided by the invention are at 600nm and 800nm
Place is respectively provided with stronger fluorescence emission peak.
By detection, nanometer in the double emission ratios type fluorescent optical sensors being prepared using the preparation method of embodiment 1
Gold particle grain size is 2.5 ± 0.5nm.
It should be noted that in the preparation method of 1 pair of emission ratios type fluorescent optical sensor of embodiment, oil bath temperature can be
Other temperature in 90-95 DEG C, such as 90 DEG C, 91 DEG C, 92 DEG C, 93 DEG C, 94 DEG C.
Double emission ratios type fluorescent optical sensors provided by the invention are described in detail in detection second below by way of specific embodiment
Application in acetylcholinesterase and pesticide residue.Before this, first to double emission ratios type fluorescence senses provided by the invention
The testing principle of device is illustrated.
Fig. 2 is please referred to, is the testing principle schematic diagram of double emission ratios type fluorescent optical sensors provided by the invention.Second
Acetylcholinesterase under certain condition can hydrolyze acetylthiocholine, form thiocholine and acetic acid, and thiocholine is
A kind of positively charged thiol compound can be covalently bound together by forming Au-S keys with nanogold particle, and increase is received
The quantity of rice gold particle surface ligand, and this process finally causes gold nano grain glimmering since electronics transfer effect has occurred
The variation at light emitting center so that fluorescent emission enhances at 600nm, and fluorescent emission synchronizes decrease at 800nm；Unhydrolysed bottom
Due to the obstruction of acetyl group ,-SH is not exposed in object acetylthiocholine, it is impossible to react to form Au-S with nanogold particle
Covalent bond increases the amount of ligand on nanogold particle surface, so as to cause the variations of the double transmitting peak intensities of gold nano grain.
Fig. 3 is please referred to, is reaction mechanism fluorescence spectra of the present invention.Wherein curve a represents provided by the invention double
The fluorescence spectra of emission ratios type fluorescent optical sensor nanogold particle；Curve b represents nanogold particle and acetylthiocholine
Fluorescence spectra；Curve c represents nanogold particle and acetylcholine fluorescence spectra；Curve d represents nanogold particle and acetyl
The fluorescence spectra of cholinesterase；Curve e represents the fluorescence light of nanogold particle, acetylcholine and acetylcholine ester enzymatic mixture
Spectrogram；Curve f represents the fluorescence spectra of nanogold particle, acetylthiocholine and acetylcholine ester enzymatic mixture.It can by Fig. 3
To find out, before and after acetylcholine ester enzyme hydrolysis, fluorescence intensity difference is apparent, is the selectivity of acetylcholinesterase and its inhibitor
Identification provides feasibility.
Embodiment 2：Application of double emission ratios type fluorescent optical sensors in acetylcholinesterase is detected
The method of double emission ratios type fluorescent optical sensor detection acetylcholinesterases, includes the following steps：
Step S1：Reaction system is prepared, and 25min is incubated at 37 DEG C；
Specifically, reaction system preparation method includes：
Add in the substrate acetylthiocholine of 10 a concentration of 1mM of μ L, double emission ratios types that 3 μ L embodiments 1 are prepared
100 μ L reaction systems are made in fluorescent optical sensor, acetylcholinesterase to be measured and suitable ultra-pure water；
Step S2：Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm
With the fluorescence intensity change at 800nm, the detection to acetylcholinesterase is realized；
Specifically, it is detected using fluorescence detector；According to double emission ratios type fluorescent optical sensors provided by the invention
The characteristics of, testing conditions are：Excitation wavelength is 400nm, and launch wavelength is 600nm and 800nm, fluoroscopic examination ranging from 500-
900nm, excitation and transmite slit are 5nm.
Fig. 4 and Fig. 5 are please referred to, wherein Fig. 4 is the fluorescence spectra of various concentration acetylcholinesterase；Fig. 5 is not
With ratio figure of the fluorescence intensity at 800nm and 600nm of concentration acetylcholinesterase.The second it can be seen from Fig. 4, Fig. 5
Minimum detection limit of the acetylcholinesterase in the detection method is about 0.0025mU/mL.
Embodiment 3：Application of double emission ratios type fluorescent optical sensors in pesticide residue is detected
The method of double emission ratios type fluorescent optical sensor detection pesticide residues, includes the following steps：
Step S1：By acetylcholinesterase and pestsides synthesis to be measured, 20min is incubated at 37 DEG C；
Specifically, adding in a concentration of 1U/mL acetylcholinesterases of 8 μ L and pesticide to be measured, incubation temperature is 37 DEG C, during reaction
Between be 20min；
Step S2：It is proportionally added into substrate acetylthiocholine and double emission ratios type fluorescence that embodiment 1 is prepared
Reaction system is made in sensor, and 5min is incubated at 37 DEG C；
Specifically, add in a concentration of 1mM acetylthiocholines of 10 μ L, double emission ratios types that 3 μ L embodiments 1 are prepared
100 μ L reaction systems are made in fluorescent optical sensor and suitable water, and incubation temperature is 37 DEG C, time 5min；
Step S3：Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm
With the fluorescence intensity change at 800nm, the detection to pesticide residue is realized；
Specifically, it is detected using fluorescence detector；According to double emission ratios type fluorescent optical sensors provided by the invention
The characteristics of, testing conditions are：Excitation wavelength is 400nm, and launch wavelength is 600nm and 800nm, fluoroscopic examination ranging from 500-
900nm, excitation and transmite slit are 5nm.
Fig. 6 and Fig. 7 are please referred to, wherein Fig. 6 is that pesticide of the various concentration by taking Aldicarb as an example inhibits acetylcholine ester
The fluorescence spectra of enzyme reaction；Fig. 7 is that the logarithm of fluorescence intensity ratio at 800nm and 600nm of various concentration Aldicarb closes
System's figure.By Fig. 6, Fig. 7 analysis it is found that when double emission ratios type fluorescent optical sensors provided by the invention detect pesticide Aldicarb,
503nhibiting concentration (concentration of inhibitor when being suppressed half) and minimum detection limit are respectively 5.37 × 10-9M and 8.76 × 10- 11M。
The present invention is strong and weak by the activity of catalyzing hydrolysis, generate thiol product number, gold nano grain surface is caused to be matched
The variation of body quantity leads to the variation of the double emission peak fluorescence intensities of gold nano grain, to acetylcholinesterase and its inhibitor into
Row detection.Bio-sensing system provided by the invention based on acetylcholinesterase and nanogold particle passes through nanogold
The variation of the double emission peak fluorescence intensities of particle itself carries out Ratio-type detection, accurate without complicated operation without considering background
Instrument, expensive reagent realizes well to the simple, convenient, quick, special of acetylcholinesterase and its pesticide inhibitor
Opposite sex detection, also, compared with conventional method, this method further improves the spirit of acetylcholinesterase and Pesticides Testing
Quick property and selectivity, while this method sample treatment is simple to operate, settles at one go, detection time is short, simple using instrument,
It is pollution-free, to realize that disease and the residual detection of environment agriculture provide more simple and rapid powerful measure and platform.
Each embodiment in this specification is described by the way of progressive, identical similar portion between each embodiment
Point just to refer each other, and the highlights of each of the examples are the differences with other embodiments.
Embodiments of the present invention are explained in detail above in association with attached drawing, but the present invention is not limited to described reality
Apply mode.To those skilled in the art, without departing from the principles and spirit of the present invention to these embodiments
The a variety of change, modification, replacement and modification carried out are still fallen within protection scope of the present invention.
Claims (10)
1. a kind of double emission ratios type fluorescent optical sensors, which is characterized in that obtained using glutathione modified nano gold particle preparation
It arrives, stronger fluorescence emission peak is respectively provided at 600nm and at 800nm.
2. double emission ratios type fluorescent optical sensors according to claim 1, which is characterized in that double emission ratios types are glimmering
Grain diameter is 2.5 ± 0.5nm in optical sensor.
3. a kind of preparation method of double emission ratios type fluorescent optical sensors described in claim 1, which is characterized in that including as follows
Step：
Glutathione solution under 90-95 DEG C of oil bath environment is heated, while being vigorously stirred, chlorauric acid solution is added in, prepares
Obtain the nanogold particle solution of glutathione modification；Preferably, glutathione solution is respectively with chlorauric acid solution dosage：
The chlorauric acid solution of the glutathione solution of a concentration of 3mM of 50mL and 1500 a concentration of 100mM of μ L；
Cooling, centrifugation, ultrafiltration purification, dispersion obtain double emission ratios type fluorescent optical sensors.
4. the preparation method of double emission ratios type fluorescent optical sensors according to claim 3, which is characterized in that centrifugation surpasses
Filter purifying, dispersion include the following steps：
Centrifugation：The nanogold particle solution that glutathione is modified is placed in a centrifuge 21000g centrifugations, removes bulky grain precipitation
Substance；
Ultrafiltration purification：In the case where rotating speed is 10000rpm, hyperfiltration treatment is carried out to supernatant and removes paddy Guang extra in solution three times
Sweet peptide；
Dispersion：Nanogold particle obtained by ultrafiltration HEPES buffer solution is pressed 1:2 volume ratios are disperseed again, obtain double transmitting ratios
Rate type fluorescent optical sensor；The wherein a concentration of 2M, pH=8.0 of HEPES buffer solution.
5. a kind of application of double emission ratios type fluorescent optical sensors in acetylcholinesterase is detected described in claims 1 or 2.
A kind of 6. method of double emission ratios type fluorescent optical sensor detection acetylcholinesterases, which is characterized in that including walking as follows
Suddenly：
Acetylcholinesterase, substrate acetylthiocholine and double emission ratios type fluorescent optical sensors described in claim 1 are pressed
Ratio mixes, and reaction system is made, and be incubated 25min at 37 DEG C；
Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm and at 800nm
Fluorescence intensity change realizes the detection to acetylcholinesterase.
7. the method for double emission ratios type fluorescent optical sensor detection acetylcholinesterases according to claim 6, feature
It is, reaction system preparation method includes：
Substrate acetylthiocholine, 3 μ the L double emission ratios type fluorescence described in claim 1 for adding in 10 a concentration of 1mM of μ L pass
100 μ L reaction systems are made in sensor, acetylcholinesterase to be measured and suitable ultra-pure water.
8. a kind of application of double emission ratios type fluorescent optical sensors in pesticide residue is detected described in claims 1 or 2.
A kind of 9. method of double emission ratios type fluorescent optical sensor detection pesticide residues, which is characterized in that include the following steps：
By acetylcholinesterase and pestsides synthesis to be measured, 20min is incubated at 37 DEG C；
Substrate acetylthiocholine and double emission ratios type fluorescent optical sensors described in claim 1 are proportionally added into, is made anti-
System is answered, 5min is incubated at 37 DEG C；
Spectral detection is carried out using Fluorescence Spectrometer, by double emission ratios type fluorescent optical sensors at 600nm and at 800nm
Fluorescence intensity change realizes the detection to pesticide residue.
10. the method for double emission ratios type fluorescent optical sensor detection pesticide residues according to claim 9, feature exist
In reaction system preparation method includes：
Add in a concentration of 1U/mL acetylcholinesterases of 8 μ L, a concentration of 1mM acetylthiocholines of 10 μ L, described in 3 μ L claims 1
Double emission ratios type fluorescent optical sensors, pesticide to be measured and suitable ultra-pure water, 100 μ L reaction systems are made.
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|---|---|
| CN108181283B (en) | 2020-10-09 |
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