CN104345037B - Optical detection sensor for microcystin - Google Patents
Optical detection sensor for microcystin Download PDFInfo
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- CN104345037B CN104345037B CN201410617885.XA CN201410617885A CN104345037B CN 104345037 B CN104345037 B CN 104345037B CN 201410617885 A CN201410617885 A CN 201410617885A CN 104345037 B CN104345037 B CN 104345037B
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Abstract
The invention discloses an optical detection sensor for microcystin. The optical detection sensor comprises a linear polarization light source, a 45-degree/45-degree/90-degree glass prism, a sensing chip and a spectrum analyzer and is characterized in that a silicon circulation tank is arranged on the sensing chip; the sensing chip comprises a glass sheet, a gold layer coated on the surface of one side of the glass sheet as well as a microcystin molecularly imprinted membrane modified on the surface of the gold layer. The optical detection sensor disclosed by the invention has the beneficial effects that firstly, a molecular imprinting technique is adopted for forming a sensitive layer of the sensor, so that specific recognition and adsorption efficiency for the microcystin are enhanced, the specificity of detection means is improved, and the detection time is shortened; secondly, by the use of a surface plasma optical detection technology, the sensitivity is improved, and the performance of the sensor is improved; thirdly, the whole sensor is simple and quick to operate, is low in requirement on operators, and provides effective help for highly sensitive detection of the microcystin.
Description
Technical field
The present invention relates to a kind of Microcystin optical detecting sensor.
Background technology
In Middle And Lower Reaches of Changjiang River is one of area of China's freshwater lake integrated distribution, in the past few decades in, the mankind
Activity creates strong influence to the lake ecosystem of this area, and the most prominent is the increasingly eutrophication of suburb lakes.
Receive the nutrient substance such as excessive nitrogen and phosphorus in lake, reservoir and river, so that the ecologic structure of water body and function is changed, lead
Cause the distorted proliferation growth of algae particularly cyanophyceae that blue-green alga bloom phenomenon occurs.Cause with the aggravation of body eutrophication
The frequent generation of harmful algal blooms become the environmental problem of common concern.When blue-green alga bloom is serious, not only affect people
Sense organ, destroy aquatic ecosystem in a healthy and balanced way, and the various microcystins discharging after frustule rupture are to people
Constitute serious threat with the drinking water safety of animal.In the various Microcystins having been found that, Microcystin is mesh
The microcystin of frequency of occurrences highest, yield maximum and the most serious that work the mischief in a kind of front known pollution in blue-green alga bloom
Element, the therefore detection to Microcystin are very necessary.
At present, chemical analysis and immunological detection are mainly to the detection method of Microcystin.In chemical detection
In method using more be high performance liquid chromatography (HPLC) technology.HPLC technology typically adopts positive or reversed phase chromatography contratoxin
Carry out separating, then carry out ultraviolet (UV), fluorescence (FL) or chemiluminescence (CL) detection, can be widely used for dividing of Microcystin
From, identification and detection by quantitative.The holdup time of tested toxin and normaltoxin is compared can be identified to tested toxin,
Both peak areas are compared and can carry out accurate quantification to it.But HPLC method and technology content is high, expensive；In addition must
The pure toxin of standard or certain toxin retention under same experimental conditions must be had.Because in our times plastic, plastics add
Plus the extensive application of agent, in the plastic containers of collection water sample, some plastic additives can disturb the inspection to Microcystin for the HPLC
Survey.Immunological detection is mainly enzyme immunoassay (ELISA), and the advantage that the such analysis method of application screens toxin is sensitive
Degree is high, when processing gross sample, analyze speed is fast, operation principle is simple.Possessing microcystin monoclonal antibody, standard
On the premise of pure toxin and relevant reagent, especially with commercialization test kit when, ELISA method be one kind very easy,
Efficiently, quick method.Shortcoming is can not to differentiate toxin very well it may appear that false positive reaction.
Chinese patent 201310712994.5 discloses a kind of quick-check sensor of Microcystin, using photochemistry
Polymerization or electrochemical polymerization carry out molecular engram film synthesis, improve binding capacity, reduce time delay and strengthen spy
Different in nature absorbability；Meanwhile, it is used Lamb wave piezoelectric membrane as the effector of sensor, improve transducer sensitivity, increase
Stability reduces cost.Thus strengthening sensor performance it is achieved that trace detection to Microcystin, establish highly sensitive
The Microcystin detection meanss that degree, quick, inexpensive, low operation require.
Chinese patent 201310713093.8 discloses a kind of Microcystin piezoelectric detection based on molecular imprinting
Sensor, carries out molecular engram film synthesis using photochemical polymerization method or electrochemical polymerization, improves binding capacity, reduction
Time delay simultaneously strengthens specific adsorption ability；Meanwhile, it is used quartz crystal as the effector of sensor, improve sensor
Sensitivity, increase stability reduces cost.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of Microcystin optical detecting sensor, and is used for microcapsule
The sensing chip of Algae toxins optical detecting sensor.
The present invention is to be achieved through the following technical solutions：
A kind of Microcystin optical detecting sensor, including linear polarization light source, 45 °/45 °/90 ° glass prisms, biographies
Sense chip, spectroanalysis instrument, described sensing chip is provided with silica gel circulation groove, and described sensing chip includes sheet glass, is plated in
The layer gold of the sheet glass single side surface and modification Microcystin molecular engram film on layer gold surface, Microcystin divides
Have to Microcystin specific recognition on sub- blotting membrane and equally distributed micropore.
As a kind of scheme, using rubbing method by film modified for Microcystin molecular engram on layer gold surface.Rubbing method is
A kind of main traditional method, prepares grade or nano level molecularly imprinted polymer granule first, then by polymer beads
It is embedded in gel or film, identification layer is obtained using rotary coating or spraying coating, the problem that this method is brought is sensor
Response time extend, to target molecule produce non-specific adsorption and binding capacity low.
As a kind of preferred version, using thermal polymerization Microcystin molecular engram film modified on layer gold surface.This
Method directly can be obtained identification layer in the original location, it is to avoid the drawbacks of traditional method, and reproducible, environmental friendliness.
Further, described hot polymerization is combined into：Gold-plated sheet glass is used oxygen gas plasma washer to clean 2-4 minute,
Soak more than 12 hours in the ethanol solution of 50-1000mM undecyl mercaptan alkanoic acid, alcohol flushing after taking-up, nitrogen dries up；Soak again
Enter 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 0.1-1M and the N- hydroxysuccinimidyl acyl Asia of 0.01-0.1M
1 hour in the mixed aqueous solution of amine；It is directly immersed in 3 hours in 100-500mM amino-butanamide hydrochloride aqueous solution after taking-up, nitrogen
Air-blowing is done；Take 0.1-200umol Microcystin, 1-2000umol methacrylic acid and 2-2000umol trimethylolpropane tris
Methacrylate adds the mixing of 5-10mL dimethyl sulphoxide solution, nitrogen treatment more than 10 minutes, the good glass of immersion pretreatment
Glass piece, carries out heat polymerization 18 hours about under the conditions of 65 ± 5 DEG C after sealing；Using ethanol and acetic acid equal-volume after taking-up
More than when less than mixed solution flow wash 12, after the completion of alcohol flushing remove surface eluent, nitrogen dries up.
In the present invention, for the needs to specificity, detection speed for the sensor detection, the present invention proposes to use molecular engram
Technology as the sensitive layer of sensor, the principle of molecular imprinting be template molecule with function monomer in disperse medium, according to
By interaction force and space steric effect etc., form the complex of Reversible binding, add cross-linking agent and initiator causes
Polymerization forms porous functional material, and template molecule is regularly wrapped wherein, with ad hoc approach, template is divided after synthesis
Son removes, thus obtain can three-dimensional cavity complementary with template molecule and that there is special identification function, thus quickly, high special
The combination template molecule of property.
In the present invention, for sensor to high sensitivity, easy needs, the present invention proposes using surface plasma altogether
Vibration sensor is transducer, and what this easy, highly sensitive optical pickocff was capable of rapid sensitive detects sensor surface
Variations in refractive index, and device is stable, strong antijamming capability.Micro- by the molecular engram film combination in effector surface modification
Capsule Algae toxins cause the change of surface refractive index, thus causing effector signal to change, using signal acquiring system collection
Tracer signal changes it becomes possible to detect the Microcystin in sample.
Compared with prior art, the invention has the beneficial effects as follows：
1st, employ molecular imprinting as the sensitive layer of sensor, enhance the specificity to Microcystin and know
Not, adsorption efficiency, improves the specificity of detection meanss and shortens detection time；
2nd, employ surface plasma optical detective technology, improve sensitivity, improve the performance of sensor；
3rd, whole sensor operations are simple, quick, operator required low, highly sensitive to Microcystin for realizing
Detection provides strong help.
Brief description
Fig. 1 is the structural representation of Microcystin optical detecting sensor.
Fig. 2 is the structural representation of sensing chip.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and examples：
As depicted in figs. 1 and 2, a kind of Microcystin optical detecting sensor, including linear polarization light source, 45 °/45 °/
90 ° of glass prisms 4, sensing chip 7, spectroanalysis instruments 6, sensing chip 7 is provided with silica gel circulation groove 5, described sensing chip 7
Microcystin molecule on layer gold 9 surface for the layer gold 9 and modification including sheet glass 8, being plated in sheet glass 8 single side surface
Blotting membrane 10, Microcystin molecular engram film 10 has to Microcystin 11 specific recognition and equally distributed micropore
12.
Further, described linear polarization light source is halogen tungsten lamp 1, linear polarizer 2 and is arranged on halogen tungsten lamp 1 and linear
Multimode quartz light pricker 3 between polaroid 2；Described spectroanalysis instrument 6 is visible light wave range spectroanalysis instrument it is seen that optical band light
Spectrometer forms light-path by multimode quartz light pricker 3 and 45 °/45 °/90 ° glass prisms 4.
Embodiment 1
Using thermal polymerization Microcystin molecular engram film modified on layer gold surface：Gold-plated sheet glass is used oxygen etc.
Gas ions washer cleans 2 minutes, soaks more than 12 hours, ethanol after taking-up in the ethanol solution of 50mM undecyl mercaptan alkanoic acid
Rinse, nitrogen dries up；Immerse 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 0.1M and the N- of 0.01M again
1 hour in the mixed aqueous solution of N-Hydroxysuccinimide；It is directly immersed in after taking-up in 100mM amino-butanamide hydrochloride aqueous solution
3 hours, nitrogen dried up；Take 0.1umol Microcystin, 1umol methacrylic acid and 2umol trimethylol propane trimethyl third
Olefin(e) acid ester adds the mixing of 5mL dimethyl sulphoxide solution, nitrogen treatment 10 minutes, the immersion good sheet glass of pretreatment, after sealing
Carry out heat polymerization 18 hours under the conditions of 65 ± 5 DEG C；Using ethanol and the flowing punching of acetic acid equal-volume ratio mixed solution after taking-up
Wash 12 hours, after the completion of alcohol flushing remove surface eluent, nitrogen dries up.
Embodiment 2
Using thermal polymerization Microcystin molecular engram film modified on layer gold surface：Gold-plated sheet glass is used oxygen etc.
Gas ions washer cleans 4 minutes, soaks more than 12 hours, second after taking-up in the ethanol solution of 1000mM undecyl mercaptan alkanoic acid
Alcohol rinses, and nitrogen dries up；Immerse 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 1M and the N- hydroxyl of 0.1M again
1 hour in the mixed aqueous solution of base butanimide；It is directly immersed in after taking-up 3 in 500mM amino-butanamide hydrochloride aqueous solution
Hour, nitrogen dries up；Take 200umol Microcystin, 2000umol methacrylic acid and 2000umol trimethylolpropane tris
Methacrylate adds the mixing of 10mL dimethyl sulphoxide solution, nitrogen treatment 12 minutes, immerses the good sheet glass of pretreatment, close
It is honored as a queen under the conditions of 65 ± 5 DEG C and carry out heat polymerization 15 hours；Using ethanol and acetic acid equal-volume ratio mixed solution after taking-up
Flow wash 18 hours, after the completion of alcohol flushing remove surface eluent, nitrogen dries up.
Embodiment 3
Using thermal polymerization Microcystin molecular engram film modified on layer gold surface：Gold-plated sheet glass is used oxygen etc.
Gas ions washer cleans 3 minutes, soaks more than 12 hours, second after taking-up in the ethanol solution of 200mM undecyl mercaptan alkanoic acid
Alcohol rinses, and nitrogen dries up；Immerse 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 0.2M and 0.05M again
1 hour in the mixed aqueous solution of N-hydroxy-succinamide；It is directly immersed in 200mM amino-butanamide hydrochloride aqueous solution after taking-up
In 3 hours, nitrogen dries up；Take 100umol Microcystin, 200umol methacrylic acid and 300umol trimethylolpropane tris
Methacrylate adds the mixing of 6mL dimethyl sulphoxide solution, nitrogen treatment 13 minutes, immerses the good sheet glass of pretreatment, close
It is honored as a queen under the conditions of 65 ± 5 DEG C and carry out heat polymerization 20 hours；Using ethanol and acetic acid equal-volume ratio mixed solution after taking-up
Flow wash 18 hours, after the completion of alcohol flushing remove surface eluent, nitrogen dries up.
Embodiment 4
Using thermal polymerization Microcystin molecular engram film modified on layer gold surface：Gold-plated sheet glass is used oxygen etc.
Gas ions washer cleans 3 minutes, soaks more than 12 hours, second after taking-up in the ethanol solution of 800mM undecyl mercaptan alkanoic acid
Alcohol rinses, and nitrogen dries up；Immerse 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 0.8M and 0.08M again
1 hour in the mixed aqueous solution of N-hydroxy-succinamide；It is directly immersed in 400mM amino-butanamide hydrochloride aqueous solution after taking-up
In 3 hours, nitrogen dries up；Take 120umol Microcystin, 1500umol methacrylic acid and 1200umol trimethylolpropane
Trimethyl acrylic ester adds the mixing of 8mL dimethyl sulphoxide solution, nitrogen treatment 15 minutes, immerses the good sheet glass of pretreatment,
Carry out heat polymerization 16 hours under the conditions of 65 ± 5 DEG C after sealing；Mixed molten after taking-up using ethanol and acetic acid equal-volume ratio
Liquid flow wash 16 hours, after the completion of alcohol flushing remove surface eluent, nitrogen dries up.
Claims (3)
1. a kind of Microcystin optical detecting sensor, including linear polarization light source, 45 °/45 °/90 ° glass prisms, sensings
, it is characterised in that being provided with silica gel circulation groove on described sensing chip, described sensing chip includes glass for chip, spectroanalysis instrument
Glass piece, it is plated in the layer gold of sheet glass single side surface and modifies Microcystin molecular engram film on layer gold surface, microcapsule
Have to Microcystin specific recognition on Algae toxins molecular engram film and equally distributed micropore；Will be micro- using thermal polymerization
Capsule Algae toxins molecular engram film modified on layer gold surface；Described hot polymerization is combined into：Gold-plated sheet glass is used oxygen gas plasma clear
Wash device cleaning 2-4 minute, soak more than 12 hours in the ethanol solution of 50-1000mM undecyl mercaptan alkanoic acid, ethanol after taking-up
Rinse, nitrogen dries up；Immerse 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and the 0.01- of 0.1-1M again
1 hour in the mixed aqueous solution of the N-hydroxy-succinamide of 0.1M；It is directly immersed in 100-500mM aminobutyryl amine salt after taking-up
3 hours in acid salt aqueous solution, nitrogen dries up；Take 0.1-200umol Microcystin, 1-2000umol methacrylic acid and 2-
2000umol trimethylol-propane trimethacrylate adds the mixing of 5-10mL dimethyl sulphoxide solution, nitrogen treatment 10 minutes
More than, the good sheet glass of immersion pretreatment, carry out heat polymerization under the conditions of 65 ± 5 DEG C 18 hours after sealing；Make after taking-up
With ethanol and acetic acid equal-volume ratio mixed solution flow wash more than 12 hours, after the completion of alcohol flushing remove surface eluent,
Nitrogen dries up.
2. a kind of Microcystin optical detecting sensor according to claim 1 is it is characterised in that described linear polarization
Light source is halogen tungsten lamp, linear polarizer and is arranged on the multimode silica fibre between halogen tungsten lamp and linear polarizer；Described light
Spectrometer be visible light wave range spectroanalysis instrument it is seen that optical band spectroanalysis instrument pass through multimode silica fibre and 45 °/45 °/
90 ° of glass prisms form light-path.
3. a kind of sensing chip for Microcystin optical detecting sensor is it is characterised in that including sheet glass, being plated in glass
The layer gold of glass piece single side surface, the Microcystin molecular engram film modified on layer gold surface using thermal polymerization method；Microcapsule
Have to Microcystin specific recognition on Algae toxins molecular engram film and equally distributed micropore；Described hot polymerization is combined into：Will
Gold-plated sheet glass uses oxygen gas plasma washer to clean 2-4 minute, in the ethanol solution of 50-1000mM undecyl mercaptan alkanoic acid
Middle immersion more than 12 hours, alcohol flushing after taking-up, nitrogen dries up；Immerse 1- (3- the dimethylamino-propyl) -3- of 0.1-1M again
1 hour in the mixed aqueous solution of the N-hydroxy-succinamide of ethyl-carbodiimide hydrochloride and 0.01-0.1M；After taking-up directly
3 hours in immersion 100-500mM amino-butanamide hydrochloride aqueous solution, nitrogen dries up；Take 0.1-200umol Microcystin,
1-2000umol methacrylic acid and 2-2000umol trimethylol-propane trimethacrylate add 5-10mL dimethyl sub-
Sulfolane solution mixes, nitrogen treatment more than 10 minutes, the good sheet glass of immersion pretreatment, carries out after sealing under the conditions of 65 ± 5 DEG C
Heat polymerization 18 hours；Using ethanol and acetic acid equal-volume ratio mixed solution flow wash more than 12 hours after taking-up, complete
Alcohol flushing removes surface eluent afterwards, and nitrogen dries up.
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| CN104792852B (en) * | 2015-04-09 | 2018-01-16 | 宁波大学 | A kind of Algae toxins molecular engram chemoreceptor sensor and its preparation method and application |
| CN106405080A (en) * | 2016-03-07 | 2017-02-15 | 天津科技大学 | Intelligent detection system for microcystins |
| CN105866044A (en) * | 2016-06-23 | 2016-08-17 | 湖北出入境检验检疫局检验检疫技术中心 | Quick detector for microcystic toxins |
| CN106568745A (en) * | 2016-10-27 | 2017-04-19 | 暨南大学 | Microcystic toxin biological chip and preparation method and application thereof |
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| CN103760052B (en) * | 2013-12-21 | 2016-06-29 | 中国科学院苏州生物医学工程技术研究所 | A kind of Microcystin piezoelectric detection sensor based on molecular imprinting |
| CN103698242B (en) * | 2013-12-21 | 2016-09-28 | 中国科学院苏州生物医学工程技术研究所 | A kind of quick-check sensor of Microcystin |
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