CN104677863B - A kind of organic phosphorus detection method based on the resonance in vitro such as local surface - Google Patents
A kind of organic phosphorus detection method based on the resonance in vitro such as local surface Download PDFInfo
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- CN104677863B CN104677863B CN201510100740.7A CN201510100740A CN104677863B CN 104677863 B CN104677863 B CN 104677863B CN 201510100740 A CN201510100740 A CN 201510100740A CN 104677863 B CN104677863 B CN 104677863B
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Abstract
The present invention relates to field of biochemistry detection, more particularly to a kind of organic phosphorus detection method based on the resonance in vitro such as local surface.In the present invention, by by organophosphor sample, the sensing chip of acetylcholinesterase is fully contacted with immobilization, and forming organophosphor in censorchip surface suppresses enzyme membrane；The mixed liquor of the above-mentioned sensing chip immersion acetylthiocholine and gold chloride for suppressing enzyme membrane containing organophosphor is fully reacted, gold nano grain is formed in censorchip surface；The above-mentioned sensing chip containing gold nano grain is irradiated with visible or infrared light, the extinction spectra of transmitted light is measured, LSPR collection of illustrative plates is obtained；LSPR collection of illustrative plates is compared with benchmark LSPR collection of illustrative plates, whether is offset according to absworption peak, determine whether contain organophosphor in organophosphor sample；According to the displacement of absworption peak, the concentration of organophosphor is determined.The present invention need not make metal nanoparticle before fixed acetylcholine ester enzyme membrane, simplify detection process, realize low cost.
Description
Technical field
The present invention relates to field of biochemistry detection, more particularly to a kind of organophosphor based on the resonance in vitro such as local surface
Detection method.
Background technology
Organic phosphorus compound refers generally in chemical molecular formula the organic gas containing C-P bond or containing organo-functional group
Phosphate compounds, generally comprise the nerve gas commonly used on various organophosphorus pesticides and biochemical battlefield.Organic phosphorus compound
To the acetylcholinesterase in organism（Acetyl-cholinesterase, AChE）With strong inhibitory action, can cause
Acetylcholine（Acetylcholine, ACh）Accumulation, causes central nervous system function disorderly, organism is lost function until
Death, is a kind of efficient biological poison.With extensive use of the organic phosphorus compound in today's society, it is to environment, life
The negative effect of thing circle or even human life also receives the close attention of countries in the world researcher simultaneously.Therefore, it is organic
The identification of phosphorus compound has turned into the emphasis of various countries' research with detection technique, has important meaning to environmental monitoring and national defense safety
Justice.
The detection of current organic phosphorus compound is mainly carried out with identification by indoor large analysis and detecting instrument, such as color
Spectrometer, infrared spectrometer, color-matter combination analysis instrument etc..Such testing equipment volume is big, analysis process is complicated, portability
Difference, is unsuitable for the outdoor portable on-line checking to organic phosphorus compound.
In recent years, with going deep into that plasma optics is studied, the local surface plasma based on metal Nano structure
Resonance (LSPR) organophosphor method for sensing the advantages of it is easily achieved portable, quick response, multifunctional unit because being answered
With.As shown in figure 1, when the size of metal Nano structure 101 is much smaller than the wavelength of incident light, the electric field component of incident light is caused
Electric charge on metal Nano structure 101 polarizes, and the negative ions for producing that polarize collect with the change of incident electric fields
Body vibrates, and electron cloud 102 is formed, so as to produce polarized electric field 103.When this frequency of oscillation is consistent with incident light frequency,
Just there is LSPR so that the incident light of stimulating frequency is absorbed, obtain LSPR spectrums.The LSPR collection of illustrative plates environment folding residing nearby with it
Penetrate that rate is relevant, therefore the aspect such as detect with important application in biomolecule detection, organic gas.
Currently used LSPR sensing technologies are as shown in Fig. 2 first, metal is made in clean substrate of glass 201
Nanostructured 202；Secondly, one layer of sensitive material 203 corresponding with object to be measured is combined on the surface of metal Nano structure 202, such as
Chemical-sensitive material, biology enzyme, biological antibody etc.；Then, sensitive material 203 will adsorb the testing molecule 204 dissociated in environment,
Change the refractive index around metal Nano structure 202；LSPR collection of illustrative plates is detected using the device shown in Fig. 3 finally.Light source
The 301 visible or infrared lights for sending, are irradiated on sensing chip 304 by optical fiber 302 and condenser lens 303, and transmitted light is through poly-
Focus lens 303 and optical fiber 302 enter Infrared-Visible spectrometer 305, and the spectral information for obtaining is detected by data by spectrometer 305
Line 306 enters computer 307, obtains LSPR collection of illustrative plates 308.The LSPR collection of illustrative plates before and after detection absorption testing molecule 204, can obtain respectively
Obtain presence and the concentration information of object to be measured.
It can be seen that, currently used LSPR sensing technologies needs were fabricated separately efficiently controllable before sensitive material is combined
Metal Nano structure.Although metal Nano structure preparation method such as chemical synthesis, self-assembly method and the electronics commonly used at present
Beam/ion beam direct write method etc. has been widely studied, but the step of making metal Nano structure is still extremely complex, and price is high
It is expensive, do not meet the developing direction of current integrated inexpensive sensor.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of organophosphor inspection based on local surface plasma resonance
Survey method, without making layer of metal nanostructured thereon before substrate combination sensitive material, directly using detection process
In chemical reaction generation metal nanoparticle, and presence using organophosphor and concentration regulate and control generated metal nanoparticle
Amount, simplify detection process, repeatability stabilization, accuracy rate is high, and Portable belt is used, reduces cost.
A kind of organic phosphorus detection method based on local surface plasma resonance in the present invention of problem above is solved,
It is characterized in that：Comprise the following steps：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, by acetylcholinesterase（AChE）It is fixed on the glass substrate into
Enzyme sensing chip；
（2）Organophosphor sample drop is coated onto on enzyme sensing chip, the organophosphor suppression that AChE activity is partly suppressed is formed
Enzyme membrane；
（3）By step（2）Sensing chip after middle treatment is immersed in acetylthiocholine (ATCh) and gold chloride at room temperature
(HAuCl4) react in mixed liquor, the reaction time is 0.5 ~ 2.5h, and a certain amount of gold nano grain is obtained on sensing chip.
（4）With visible or infrared light irradiating step（3）Sensing chip after middle treatment, then detect saturating with spectral investigator
Light is penetrated, obtains measuring LSPR collection of illustrative plates；
（5）LSPR collection of illustrative plates is compared with benchmark LSPR collection of illustrative plates, whether is offset according to absworption peak, determined described organic
Whether contain organophosphor in phosphorus sample, and according to the displacement of absworption peak, determine the concentration of organophosphor, when absworption peak has to the right partially
Move and determine to contain organophosphor in sample, side-play amount is bigger, and organic phosphorus concentration is higher.
The step（1）In substrate of glass be K9 glass.
The step（2）Acetylthiocholine 0.5 containing molar concentration in acetylthiocholine and gold chloride mixed liquor ~
20mM, the gold chloride 0.01 ~ 0.5% of mass percent, remaining is phosphate buffer（PBS）.
Optimization molar concentration acetylthiocholine is 1 ~ 10mM, and mass percent gold chloride is 0.05 ~ 0.2%.
The step（1）In fixing means be absorption method, investment, covalently bonded be legal or cross-linking method.
The absorption method is comprised the following steps：
By acetylcholinesterase（AChE）PBS solution drip in substrate of glass, stand 1h at 4 DEG C；It is slow with phosphate again
Fliud flushing is rinsed 2 ~ 4 times, and room temperature is dried or dried up.
PBS solution refers to phosphate buffer, is the most frequently used a kind of buffer solution in Biochemistry Experiment, general concentration
It is 0.01 ~ 0.1M, pH value is 7 ~ 8.
Rate of addition speed is added dropwise that consumption is how many to be determined according to glass substrate area, and such as glass substrate area more greatly, can
To drip, it is also possible to quicker in speed more.
Flushing can be that sensing chip is immersed in PBS solution, then immerse in PBS solution again after taking out, and operate repeatedly
Make, purpose rinses out the enzyme do not fixed.It is to dry sensing chip that room temperature is dried or dried up, and is made thereon without moisture
Untill.
The cross-linking method is Euplotes woodruffi or sulfo-LC-SPDP cross-linking methods.（Sulfo-LC-SPDP is ProductName
Claim, there is no Chinese translation, sulfo-LC-SPDP is produced by pierce companies of the U.S..）
The Euplotes woodruffi is comprised the following steps：
By the mixed solution of acetylcholinesterase, the glutaraldehyde 0.1 ~ 10% of mass percent and bovine serum albumin 0.1 ~ 10%
Drip in substrate of glass, 8 ~ 12h is stood at 4 DEG C；Again 2 ~ 4 are rinsed with the phosphate buffer of the glycine 1% of mass percent
Secondary, room temperature is dried or is dried up.
The glutaraldehyde of mass percent is 1 ~ 10% and bovine serum albumin is 1 ~ 10% in prioritization scheme.
It is added drop-wise to after chip surface by some times, the enzyme of a part is fixed on chip, then uses glycine solution
By unnecessary material, such as loose enzyme, do not participate in glutaraldehyde and bovine serum albumin of reaction etc. and wash off.
The sulfo-LC-SPDP cross-linking methods are comprised the following steps：
By the sodium acetate of the phosphate buffer of acetylcholinesterase, the sulfo-LC-SPDP aqueous solution and dithiothreitol (DTT)
Solution by volume 1：0.5~1.5：0.5 ~ 1.5 is well mixed, and drips in substrate of glass, 1h is stood at 4 DEG C, then use phosphate
Wash buffer 2 ~ 4 times, room temperature is dried or is dried up, and wherein the pH value of the sodium acetate buffer of dithiothreitol (DTT) is 4.5, therein
The molar concentration of dithiothreitol (DTT) is 1 ~ 50mM, and the molar concentration of sodium acetate is 0.1M, the sulfo-LC-SPDP aqueous solution mole
Concentration is 5 ~ 50mM.
The sodium acetate of the phosphate buffer, the sulfo-LC-SPDP aqueous solution and dithiothreitol (DTT) of acetylcholinesterase is molten
Liquid optimization volume ratio is 1：1：1.
The acetylcholine ester enzyme concentration is 50 ~ 500u/ml, and u is active unit.
The step（2）Middle organophosphor sample is volatility organo-phosphorus gas such as nerve gas sarin, tabun, soman, dimension
Ai Kesi etc., or be organophosphor liquid, such as parathion, malathion, Rogor, metrifonate, DDVP etc..
The step step（5）In benchmark LSPR collection of illustrative plates by following steps obtain：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, acetylcholinesterase is fixed and is sensed into enzyme on the glass substrate
Chip；
（2）Reacted during enzyme sensing chip is immersed in into acetylthiocholine and gold chloride mixed liquor at room temperature, the reaction time
It is 0.5 ~ 2.5h, gold nano grain is obtained on sensing chip.
（3）With visible or infrared light irradiating step（3）Sensing chip after middle treatment, then detect saturating with spectral investigator
Light is penetrated, benchmark LSPR collection of illustrative plates is obtained.
Step（3）Described in gold nano grain refer to HAuCl4Obtained gold nano is reduced by thiocholine (TCh)
Particle, with generation chlorinated thio choline (TCl), specific reaction equation is：
Compared with prior art, the advantage of the invention is that：
(1) without metal Nano structure was fabricated separately before sensitive material is combined, step is simple, and step is simple, can grasp
The property made is strong, can realize low cost and miniaturization；
(2) regulate and control the amount of generated gold nano grain using the concentration of organic phosphorus compound, be a kind of brand-new detection
Method；
(3) have in terms of organo-phosphorus gas small molecule is detected to the mechanism of AChE activity suppressions using organic phosphorus compound
There is highly sensitive advantage.
Brief description of the drawings
Fig. 1 is LSPR mechanism schematic diagrames in the present invention；
Fig. 2 is based on LSPR organophosphor Cleaning Principle schematic diagrames in the present invention according to prior art；
Fig. 3 is the LSPR collection of illustrative plates detection devices of prior art in the present invention；
Fig. 4 is the LSPR organic phosphorus detection method block diagrams according to the present invention-better embodiment in the present invention；
Fig. 5 is to detect the extinction spectra that the metrifonate of various concentrations is obtained in the present invention in the embodiment of the present invention 1；
Fig. 6 is the simulant methyl acid phosphate two of the Schain poison gas for detecting various concentrations in the present invention in the embodiment of the present invention 2
The extinction spectra that methyl esters (DMMP) is obtained.
Specific embodiment
A kind of organic phosphorus detection method based on local surface plasma resonance in the present invention, it is characterised in that：Bag
Include following steps：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, by acetylcholinesterase（AChE）It is fixed on the glass substrate into
Enzyme sensing chip；
（2）Organophosphor sample drop is coated onto on enzyme sensing chip, the organophosphor suppression that AChE activity is partly suppressed is formed
Enzyme membrane；
（3）By step（2）Sensing chip after middle treatment is immersed in acetylthiocholine (ATCh) and gold chloride at room temperature
(HAuCl4) react in mixed liquor, the reaction time is 0.5 ~ 2.5h, and a certain amount of gold nano grain is obtained on sensing chip.
（4）With visible or infrared light irradiating step（3）Sensing chip after middle treatment, then detect saturating with spectral investigator
Light is penetrated, obtains measuring LSPR collection of illustrative plates；
（5）LSPR collection of illustrative plates is compared with benchmark LSPR collection of illustrative plates, whether is offset according to absworption peak, determined described organic
Whether contain organophosphor in phosphorus sample, and according to the displacement of absworption peak, determine the concentration of organophosphor, when absworption peak has to the right partially
Move and determine to contain organophosphor in sample, side-play amount is bigger, and organic phosphorus concentration is higher.
The step（1）In substrate of glass be K9 glass.
The step（2）Acetylthiocholine 0.5 containing molar concentration in acetylthiocholine and gold chloride mixed liquor ~
20mM, the gold chloride 0.01 ~ 0.5% of mass percent, remaining is phosphate buffer（PBS）.
Optimization molar concentration acetylthiocholine is 1 ~ 10mM, and mass percent gold chloride is 0.05 ~ 0.2%.
The step（1）In fixing means be absorption method, investment, covalently bonded be legal or cross-linking method.
The absorption method is comprised the following steps：
By acetylcholinesterase（AChE）PBS drip in substrate of glass, stand 1h at 4 DEG C；Phosphate buffer is used again
Rinse 2 ~ 4 times, room temperature is dried or dried up.
PBS refers to phosphate buffer, is the most frequently used a kind of buffer solution in Biochemistry Experiment, and general concentration is
0.01 ~ 0.1M, pH value is 7 ~ 8.
Rate of addition speed is added dropwise that consumption is how many to be determined according to glass substrate area, and such as glass substrate area more greatly, can
To drip, it is also possible to quicker in speed more.
Flushing can be that sensing chip is immersed in PBS solution, then immerse in PBS solution again after taking out, and operate repeatedly
Make, purpose rinses out the enzyme do not fixed.It is to dry sensing chip that room temperature is dried or dried up, and is made thereon without moisture
Untill.
The cross-linking method is Euplotes woodruffi or sulfo-LC-SPDP cross-linking methods.（Sulfo-LC-SPDP is ProductName
Claim, there is no Chinese translation, sulfo-LC-SPDP is produced by pierce companies of the U.S..）
The Euplotes woodruffi is comprised the following steps：
By the mixed solution of acetylcholinesterase, the glutaraldehyde 0.1 ~ 10% of mass percent and bovine serum albumin 0.1 ~ 10%
Drip in substrate of glass, 8 ~ 12h is stood at 4 DEG C；Again 2 ~ 4 are rinsed with the phosphate buffer of the glycine 1% of mass percent
Secondary, room temperature is dried or is dried up.
The glutaraldehyde of mass percent is 1 ~ 10% and bovine serum albumin is 1 ~ 10% in prioritization scheme.
It is added drop-wise to after chip surface by some times, the enzyme of a part is fixed on chip, then uses glycine solution
By unnecessary material, such as loose enzyme, do not participate in glutaraldehyde and bovine serum albumin of reaction etc. and wash off.
The sulfo-LC-SPDP cross-linking methods are comprised the following steps：
By the sodium acetate of the phosphate buffer of acetylcholinesterase, the sulfo-LC-SPDP aqueous solution and dithiothreitol (DTT)
Solution by volume 1：0.5~1.5：0.5 ~ 1.5 is well mixed, and drips in substrate of glass, 1h is stood at 4 DEG C, then use phosphate
Wash buffer 2 ~ 4 times, room temperature is dried or is dried up, and wherein the pH value of the sodium acetate buffer of dithiothreitol (DTT) is 4.5, therein
The molar concentration of dithiothreitol (DTT) is 1 ~ 50mM, and the molar concentration of sodium acetate is 0.1M, the sulfo-LC-SPDP aqueous solution mole
Concentration is 5 ~ 50mM.
The sodium acetate of the phosphate buffer, the sulfo-LC-SPDP aqueous solution and dithiothreitol (DTT) of acetylcholinesterase is molten
Liquid optimization volume ratio is 1：1：1.
The acetylcholine ester enzyme concentration is 50 ~ 500u/ml, and u is active unit.
The step（2）Middle organophosphor sample is volatility organo-phosphorus gas such as nerve gas sarin, tabun, soman, dimension
Ai Kesi etc., or be organophosphor liquid, such as parathion, malathion, Rogor, metrifonate, DDVP etc..
The step step（5）In benchmark LSPR collection of illustrative plates by following steps obtain：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, acetylcholinesterase is fixed and is sensed into enzyme on the glass substrate
Chip；
（2）Reacted during enzyme sensing chip is immersed in into acetylthiocholine and gold chloride mixed liquor at room temperature, the reaction time
It is 0.5 ~ 2.5h, gold nano grain is obtained on sensing chip.
（3）With visible or infrared light irradiating step（3）Sensing chip after middle treatment, then detect saturating with spectral investigator
Light is penetrated, benchmark LSPR collection of illustrative plates is obtained.
To make the object, technical solutions and advantages of the present invention more protrude, below in conjunction with the accompanying drawings and specific embodiment is detailed
It is thin to introduce the present invention.But protection scope of the present invention is not limited in following instance, should be comprising all interior in claims
Hold.
Embodiment 1
Detecting step is as shown in Figure 4 to be detected to organophosphorus pesticide metrifonate using detection method of the invention.
(1) step 4-I, prepares enzyme sensing chip：Selection size is the K9 glass 401 of 10mm × 20mm × 2mm as core
Piece substrate, is cleaned to substrate, is dried.AChE is fixed using absorption method, after glass basic surface is fixed
AChE 402.
The cleaning process of substrate of glass is carried out in accordance with the following steps：
K9 substrate of glass is distinguished in the sodium hydroxide solution of 1.2 moles every liter (M) and the hydrochloric acid solution of 1.2M first
5 minutes (min) of immersion, is then washed with deionized water net；Then, in immersion concentrated hydrochloric acid solution, 1min is soaked, is washed with deionized water
Only；Finally, 5min is soaked respectively in ethanol, acetone and deionized water successively；Normal temperature is dried up or dried.
AChE is fixed in substrate to be carried out in accordance with the following steps：
First, it is that (concentration is 0.1M to 100 every milliliter of PBS of the AChE of (u/mL) of activity, and pH value is 8.0) molten by concentration
10 microlitres of liquid (μ L) is dripped in the clean substrate of glass by pre-processing, and 1 hour (h) is placed at 4 DEG C；Then rinsed with PBS,
Room temperature is dried or is dried up again, and enzyme sample film is formed on the glass substrate.
(2) step 4-II, organophosphor sample is attached on enzyme sensing chip, and it is organic that formation activity is partly suppressed
Phosphorus suppresses enzyme membrane.Specific method is as follows：Sensing chip containing enzyme sample film is immersed in concentration for 0 nanogram is every for totally 4 respectively
Milliliter (ng/mL), in the metrifonate solution of 100ng/mL, 200ng/mL and 300ng/mL, in reacting 25 min at 37 DEG C,
Dried up after being cleaned using deionized water, obtain the AChE 403 of reduced activity.
(3) step 4-III, by by step 4-I and 4-II treatment after substrate be immersed in concentration be 3mM ATCh and
Concentration is 0.05% HAuCl4Mixed liquor in, make its reaction 1.5h.Can observe that 4 substrates have not under high-power microscope
Produced with the gold nano grain 404 of sparse degree, metrifonate solution concentration is bigger, the gold nano grain on corresponding substrate is diluter
Dredge.
(4) step 4-IV, measures LSPR collection of illustrative plates.The test device be given using Fig. 3 is passed to 4 kinds of different organophosphors above
Sense chip carries out spectrum test.The light sent using visible light source halogen tungsten lamp 301 is transmitted by optical fiber 302, saturating by focusing on
By the organophosphor sensing chip 304 containing different gold nano grains, transmitted light is coupled into the irradiation of mirror 303 by condenser lens 303
Enter optical fiber 302, and detected into spectrometer 305.By the treatment of computer 307,1 benchmark LSPR collection of illustrative plates and 3 are obtained
Individual measurement LSPR collection of illustrative plates.
(5) Fig. 5 is the corresponding LSPR collection of illustrative plates of sensing chip under different metrifonate concentration.It can be seen that, benchmark LSPR collection of illustrative plates
The peak value of 501 (correspondence metrifonate solution concentration is 0ng/mL) is at 619nm, and peak value is in 642nm, 667nm, 718nm
It is 100ng/mL, 200ng/mL and 300ng/mL that the measurement LSPR collection of illustrative plates 502,503,504 at place corresponds to metrifonate concentration respectively.Can
See, it is known that chip Spectral Extinction wavelength information just can learn metrifonate concentration information.
Embodiment 2, using detection method of the invention to simulant methyl-phosphoric acid dimethyl ester (DMMP) gas of Schain poison gas
Body is detected that detecting step is as shown in Figure 4.
(1) step 4-I, prepares enzyme sensing chip：Selection size is the K9 glass 401 of 10mm × 20mm × 2mm as core
Piece substrate, is cleaned to substrate, is dried.AChE is fixed using cross-linking method, after glass basic surface is fixed
AChE 402.
The cleaning process of substrate of glass is carried out in accordance with the following steps：
K9 substrate of glass is soaked into 5min respectively in the sodium hydroxide solution of 1.2M and the hydrochloric acid solution of 1.2M first, so
After be washed with deionized water it is net；Then, in immersion concentrated hydrochloric acid solution, 1min is soaked, is washed with deionized water net；Finally, successively in second
5min is soaked respectively in alcohol, acetone and deionized water；Normal temperature is dried up or dried.
AChE is fixed in substrate to be carried out in accordance with the following steps：
First, it is the AChE of 150u/mL by concentration, concentration is 5% glutaraldehyde, and the bovine serum albumin that concentration is 1%
PBS (concentration is 0.1M, and pH value is that the μ L of 8 solution 10 are dripped in the clean substrate of glass by pre-processing, and at 4 DEG C overnight, is obtained
Obtain enzyme sensing chip；Then the enzyme sensing chip is rinsed with the PBS solution containing 1% glycine；Last room temperature is dried or is dried up,
Enzyme sample film is formed on enzyme sensing chip.
(2) step 4-II, organophosphor sample is attached on enzyme sensing chip, and it is organic that formation activity is partly suppressed
Phosphorus suppresses enzyme membrane.This example specific method is as follows：Sensing chip containing enzyme sample film is immersed in concentration and is for totally 4 respectively
In the DMMP gases of 0ppm, 10ppm, 20ppm and 30ppm, in 15min is reacted at 25 DEG C, active weakened at different degrees is obtained
AChE.
(3) step 4-III, by by step 4-I and 4-II treatment after substrate be immersed in concentration be 5mM ATCh and
Concentration is 0.1% HAuCl4Mixed liquor in, make its reaction 1.5h.Can observe that 4 substrates have difference under high-power microscope
The gold nano grain of sparse degree is produced.DMMP concentration is bigger, and the gold nano grain on corresponding substrate is more sparse.
(4) step 4-IV, measures LSPR collection of illustrative plates.The test device be given using Fig. 34 kinds of different organophosphors to more than
Sensing chip carries out spectrum test.The light sent using visible light source halogen tungsten lamp 301 is transmitted by optical fiber 302, by focusing on
By the organophosphor sensing chip 304 containing different gold nano grains, transmitted light is coupled the irradiation of lens 303 by condenser lens 303
Into optical fiber 302, and detected into spectrometer 305.By the treatment of computer 307,1 benchmark LSPR collection of illustrative plates is obtained
With 3 measurement LSPR collection of illustrative plates.
(5) Fig. 6 is the corresponding LSPR collection of illustrative plates of sensing chip under different DMMP concentration.It can be seen that, benchmark LSPR collection of illustrative plates
The peak value of 601 (correspondence DMMP concentration is 0ppm) is at 569nm, and peak value is in the measurement at 601nm, 619nm, 678nm
It is 10ppm, 20ppm and 30ppm that LSPR collection of illustrative plates 602,603,604 corresponds to DMMP concentration respectively.It can be seen that, it is known that chip delustring
Spectrum wavelength information just can learn DMMP concentration informations.
The step of description of the invention and example, is divided, and is intended merely to describe clear, and a step can be merged into when realizing
Suddenly or some steps are split, is decomposed into multiple steps, as long as comprising identical logical relation, all in the guarantor of this patent
In the range of shield；To realizing the inessential modification of step addition or introducing inessential design, but do not change of the invention real
The core design of existing step is all in the protection domain of the patent.It will be understood by those skilled in the art that above-mentioned each reality
The mode of applying is to realize specific embodiment of the invention, and in actual applications, can make various to it in the form and details
Change, without departing from the spirit and scope of the present invention.
Claims (8)
1. a kind of organic phosphorus detection method based on local surface plasma resonance technology, it is characterised in that including following step
Suddenly：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, acetylcholinesterase is fixed and senses core into enzyme on the glass substrate
Piece；The acetylcholine ester enzyme concentration is 50 ~ 500u/ml, and u is active unit；
（2）Organophosphor sample drop is coated onto on enzyme sensing chip；
（3）By step（2）Sensing chip after middle treatment is immersed in acetylthiocholine and gold chloride mixed liquor anti-at room temperature
Should, the reaction time is 0.5 ~ 2.5h, and gold nano grain is obtained on sensing chip；The acetylthiocholine and gold chloride are mixed
0.5 ~ 20mM of acetylthiocholine containing molar concentration in conjunction liquid, the gold chloride 0.01 ~ 0.5% of mass percent, remaining is phosphorus
Phthalate buffer；
（4）With visible or infrared light irradiating step（3）Sensing chip after middle treatment, then detect transmission with spectral investigator
Light, obtains measuring LSPR collection of illustrative plates；
（5）LSPR collection of illustrative plates is compared with benchmark LSPR collection of illustrative plates, whether is offset according to absworption peak, determine the organophosphor sample
Whether contain organophosphor in product, and according to the displacement of absworption peak, determine the concentration of organophosphor, when absworption peak offsets determining sample
Contain organophosphor in product, side-play amount is bigger, and organic phosphorus concentration is higher.
2. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 1, its feature
It is：The step（1）In substrate of glass be K9 glass.
3. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 1, its feature
It is：The step（1）In fixing means be absorption method, investment, covalently bonded be legal or cross-linking method.
4. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 3, its feature
It is：The absorption method is comprised the following steps：
By the phosphate buffer drop of acetylcholinesterase on the glass substrate, 1h is stood at 4 DEG C；Phosphate buffer is used again
Rinse 2 ~ 4 times, room temperature is dried or dried up.
5. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 3, its feature
It is：The cross-linking method is Euplotes woodruffi or sulfo-LC-SPDP cross-linking methods.
6. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 5, its feature
It is：The Euplotes woodruffi is comprised the following steps：
The mixed solution of acetylcholinesterase, the glutaraldehyde 0.1 ~ 10% of mass percent and bovine serum albumin 0.1 ~ 10% is dripped to
In substrate of glass, 8 ~ 12h is stood at 4 DEG C；Rinsed 2 ~ 4 times with the phosphate buffer of the glycine 1% of mass percent again, room
Temperature is dried or is dried up.
7. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 5, its feature
It is：The sulfo-LC-SPDP cross-linking methods are comprised the following steps：
By the sodium acetate solution of the phosphate buffer of acetylcholinesterase, the sulfo-LC-SPDP aqueous solution and dithiothreitol (DTT)
By volume 1:0.5~1.5:0.5 ~ 1.5 is well mixed, and drips in substrate of glass, 1h is stood at 4 DEG C, then use phosphate-buffered
Liquid is rinsed 2 ~ 4 times, and room temperature is dried or dried up, and wherein the pH value of the sodium acetate buffer of dithiothreitol (DTT) is 4.5, two sulphur therein
The molar concentration of threitol is 1 ~ 50mM, and the molar concentration of sodium acetate is 0.1M, the molar concentration of the sulfo-LC-SPDP aqueous solution
It is 5 ~ 50mM.
8. the organic phosphorus detection method based on local surface plasma resonance technology according to claim 1, its feature
It is：The step（5）In benchmark LSPR collection of illustrative plates by following steps obtain：
（1）Prepare enzyme sensing chip：Cleaning substrate of glass, acetylcholinesterase is fixed and senses core into enzyme on the glass substrate
Piece；
（2）Reacted during enzyme sensing chip is immersed in into acetylthiocholine and gold chloride mixed liquor at room temperature, the reaction time is 0.5
~ 2.5h, obtains gold nano grain on sensing chip；
（3）With visible or infrared light irradiating step（2）Sensing chip after middle treatment, then detect transmission with spectral investigator
Light, obtains benchmark LSPR collection of illustrative plates.
Priority Applications (1)
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| CN103374562A (en) * | 2012-04-11 | 2013-10-30 | 中国人民解放军军事医学科学院毒物药物研究所 | Enzyme immobilization method |
| CN103454253A (en) * | 2013-06-25 | 2013-12-18 | 复旦大学 | Organic phosphorus detection method based on surface plasmon resonance |
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