CN102784671A - Centrifugal micro-fluidic chip for detecting pesticide residue and preparation method thereof - Google Patents
Centrifugal micro-fluidic chip for detecting pesticide residue and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a centrifugal micro-fluidic chip for detecting pesticide residue and a preparation method thereof. The centrifugal micro-fluidic chip for detecting the pesticide residue is a disk type chip with a micro structure and a micro channel and consists of multiple layers of chips; the mixing, extracting, reacting, separating and developing process of a sample to be measured and a reagent is realized under the drive of a centrifugal force generated by rotating a centrifugal machine; and finally, the content of the pesticide residue in the sample is quantitatively detected by using an ultraviolet visible light spectrophotometer. The centrifugal micro-fluidic chip for detecting the pesticide residue can be used for directly sampling without pretreating the sample; the consumption of the sample and the reagent is low; a plurality of samples can be simultaneously treated and detected in parallel, and thus the integration, the micromation and the automation of the pesticide residue detection are realized; and the centrifugal micro-fluidic chip has the characteristics of economy, quickness and portability and provides a brand-new analysis technology for quickly detecting the pesticide residue of water bodies, foods and soil.
Description
Technical field
The present invention relates to a kind of centrifugal type microfludic chip that detects residues of pesticides and preparation method thereof; Be based on the micro-fluidic chip analytical technology; This centrifugal type microfludic chip passes through micro-processing technology in surface preparation micro-structural and microchannel; Under the centrifugal force that the centrifuge rotation produces drives; On chip, realize testing sample and each reaction reagent mixing, extraction, react, separate and process color, go out the residues of pesticides content in the sample with the ultra-violet and visible spectrophotometer detection by quantitative at last, be mainly used in association areas such as health supervision, food security, environment measuring.
Background technology
Organic agricultural chemicals is efficient as one type, the pesticide of wide spectrum, is prevention and elimination of disease and pests, improves the important means of crops quality and output; Be widely used in ground insect killing and preventings such as agricultural production, family and storage; Because user's lack of scientific is used the knowledge of agricultural chemicals, the output that covets causes soil, water body, food all to have the residues of pesticides problem to some extent; Many potential harm have been caused, like environmental pollution, poisoning and person poultry poisoning etc.Therefore, easy, to detect organic agricultural chemicals apace residual, have very important significance for association areas such as environmental protection, food security, health protection and strick precaution, customs's detections.
At present, the residual detection of organic agricultural chemicals generally adopts chromatography and enzyme to suppress method.It is accurate, highly sensitive that chromatography detects, but cost is high, program is complicated, minute is long, is not suitable for on-the-spot the detection.Enzyme inhibition method is easy and simple to handle, speed fast, higher selectivity, sensitivity and accuracy are arranged, and is that present China uses maximum fast detecting pesticide residue technology.Yet it is choline vinegar enzyme that enzyme suppresses the needed enzyme of method, can from animals and plants, extract, and the purity of the enzyme that extracts certainly is with active not high enough, and costing an arm and a leg of commercially available enzyme limited enzyme and suppressed the extensive use of method in fast detecting pesticide residue.And in above-mentioned two kinds of detection methods, sample separation and detection are to separate independently process, are prone to cause sample loss and cross pollution, cause the testing result distortion, also are not suitable for field quick detection, and analytical system is microminiaturized and the developing direction of portable.
Therefore, develop a kind of quick, efficient, convenient, low-cost, automation, collection sample pretreatment and detect and be the detection method of one, become the research focus of field detection technique for this reason.In recent years, micro-fluidic chip is as a kind of novel analysis platform, has microminiaturization, automation, advantage such as integrated, convenient and quick, obtained to use widely in the environment measuring association area.Yet; Adopt disk shape chip, be used for analyzing, rely on centrifugal force to drive the sample microfluid in its surface preparation micro-structural and microchannel; Accomplish the centrifugal type microfludic chip analytical technology of several sample detection simultaneously, the application in fast detecting pesticide residue does not have substantial breakthrough as yet at present.
Summary of the invention
The purpose of this invention is to provide a kind of centrifugal type microfludic chip that detects residues of pesticides and preparation method thereof; It is characterized in that this chip is the centrifugal type microfludic chip of disk shape band micro-structural and microchannel; With centrifugal force is the driving force of sample microfluid; With the online use of detector, on chip, realize sample pretreatment and detection simultaneously.Micro-structural and microchannel comprise abstraction pool, inhibitory reaction pond, chromogenic reaction pond (detection cell), microfluidic channel, microflute and micropore through the micro-processing technology preparation.The rotation platform of centrifuge aims at the design of disk shape centrifugal type microfludic chip, and the center is with sucker, draw-in groove or screw-nut fixed wafer shape chip.Disk shape centrifugal type microfludic chip is carved with the micro-structural of micron level by multilayer and the chip and the double-deck adhesive membrane involution of microchannel forms.Disk shape centrifugal type microfludic chip is under the centrifugal force that the centrifuge rotation produces drives; Realize sample and reaction reagent mixing, extraction, react, separate and process color, go out the residues of pesticides content in the sample with the ultra-violet and visible spectrophotometer detection by quantitative at last.
For realizing above-mentioned purpose, the present invention adopts following operating procedure:
1. design and draw the micro-structural and the microchannel figure of each layer chip in the centrifugal type microfludic chip with computer aided design software;
2. on each layer micro-fluidic chip substrate surface and adhesive membrane, process required micro-structural and microchannel through micro-processing technology, comprise abstraction pool, inhibitory reaction pond, chromogenic reaction pond (detection cell), microfluidic flow channels, microflute and micropore;
3. testing sample is added in the abstraction pool;
4. utilize double-deck adhesive membrane, with each leafing core type micro-fluidic chip align, bonding, pressurizing and sealing, form disk shape centrifugal type microfludic chip;
5. extractant is added abstraction pool, add enzyme in the inhibitory reaction pond, (detection cell) adds substrate and developer in the chromogenic reaction pond;
6. startup centrifuge, extractant and testing sample drive down at centrifugal force and mix, and extract residual agricultural chemicals in the sample;
7. change centrifugal speed, extract shift through little valve and get into inhibitory reaction pond, extract and enzyme generation inhibitory reaction;
8. after inhibitory reaction is accomplished, change centrifugal speed again, solution shifts entering chromogenic reaction pond (detection cell) through little valve in the inhibitory reaction pond, with behind the substrate reactions by chromogenic reagent;
9. after the sample chromogenic reaction is accomplished, carry out detection by quantitative with ultra-violet and visible spectrophotometer at last.
Among the present invention, the test sample that detects the centrifugal type microfludic chip of residues of pesticides can be water body, food, also soil.
Among the present invention, the chip substrates that detects the centrifugal type microfludic chip of residues of pesticides can be PMMA, PC, PVC, COC, silicon chip, glass, copper, aluminium, stainless steel disk, also commercially available all kinds of CD CDs.
Among the present invention, detect the chip of the centrifugal type microfludic chip of residues of pesticides and can fix, also can fix through screw-nut through sucker, the draw-in groove of centrifuge.
Among the present invention, the centrifugal type microfludic chip that detects residues of pesticides can be the porose chip in center, center bore 10～30mm, also atresia chip.Chip thickness is 0.5～5mm, and diameter is 60～150mm.
Among the present invention, detect the micro-structural and the microchannel of the centrifugal type microfludic chip of residues of pesticides and can mill quarter, laser ablation, LIGA technology, method of molding, pressure sintering, chemical attack preparation through numerical control, also can be through soft lithographic technique preparation.
Among the present invention, use the adhesive membrane involution between each layer chip of the centrifugal type microfludic chip of detection residues of pesticides, adhesive membrane can be that power causes double-deck adhesive film, also can be common double faced adhesive tape.
Among the present invention, quarter, laser ablation preparation can be milled through numerical control in the micro-structural of the adhesive membrane of the centrifugal type microfludic chip of detection residues of pesticides and microchannel, also can be through common carving machine preparation.
Among the present invention; Between the abstraction pool and inhibitory reaction pond of the centrifugal type microfludic chip of detection residues of pesticides; The inhibitory reaction pond is 380 μ m with the width that is connected the microchannel and the degree of depth between the detection cell; The quartz capillary of inserting external diameter 375 μ m is as little valve, is used for sample microfluid flowing in chip microchannel.The internal diameter of quartz capillary can be 25,50,75,100 μ m, also 150 μ m, and the internal diameter of quartz capillary is more little, and the sample microfluid just needs higher centrifugal rotational speed to produce bigger centrifugal force to come through quartz capillary.
Among the present invention; The different testing processes that detect the centrifugal type microfludic chip of residues of pesticides adopt different centrifugal rotational speeds; The centrifugal rotational speed of extractant and sample mix extraction is 50～100 rev/mins; Extraction solution is 300～600 rev/mins through the centrifugal rotational speed that quartz capillary gets into the inhibitory reaction pond, and reaction solution is 800～2000 rev/mins through the centrifugal rotational speed that quartz capillary gets into the chromogenic reaction pond.
Among the present invention, the extractant that detects the centrifugal type microfludic chip of residues of pesticides is the alkalescent PBS, and enzyme is a second phthalein choline vinegar enzyme, and substrate is acetate-1-naphthalene ester, and developer is solid blue B salt.
The disk shape band micro-structural of the detection residues of pesticides that the present invention proposes and centrifugal type microfludic chip of microchannel and preparation method thereof; Be a kind of analytical method of novel detection residues of pesticides, parallel detection simultaneously is a plurality of or organize sample more, and sample and reagent dosage are little; Sample pretreatment is simple and efficient; Be prone to and the detector coupling, detection time is short, have portable, economical, fast, characteristics accurately and efficiently; Meet modern analysis detection technique and instrument portable, microminiaturization, integrated developing direction, have a good application prospect in the association area of fast detecting pesticide residue.
Description of drawings
Fig. 1. the five layers of chip of the centrifugal type microfludic chip of detection residues of pesticides and the structural representation of one group of sample analysis detecting unit.
The structural representation of the centrifugal type microfludic chip of the detection residues of pesticides of the integrated 10 groups of sample analysis detecting units of figure i..
The structural representation of five layers of chip in the centrifugal type microfludic chip of figure ii. detection residues of pesticides.
1) the 1st layer (the upper strata chip has sample holes and passage), 2) the 2nd layer (double-deck adhesive membrane, bonding usefulness), 3) the 3rd layer (the intermediate layer master chip has micro-structural and microchannel), 4) the 4th layer (double-deck adhesive membrane, bonding usefulness), 5) the 5th layer (bottom chip, involution with).
The structural representation of one group of sample analysis detecting unit of figure iii..
1) abstraction pool, 2) the inhibitory reaction pond, 3) colour developing pond (detection cell), 4) quartz capillary, 5) quartz capillary, 6) passage and ventilation microchannel.
Specific embodiments
Detect the micro-structural and the microchannel figure of the centrifugal type microfludic chip of residues of pesticides with computer aided design software design and drafting.On three strata carbonic ester (PC) chips, prepare micro-structural and microchannel respectively through numerical control CNC micro-machining system, clean each layer chip with running water, distilled water respectively, and with spots such as the residual fingerprint of ethanol wiping chip surface, oil stains.Cause on the adhesive membrane in double-deck power, prepare required micro-structural and microchannel with carving machine.The vegetable sample that 40mg is shredded is positioned in the abstraction pool, with 3 layers of PC chip and the alignment of 2 layers of adhesive membrane, bonding, pressurizing and sealing, processes the centrifugal type microfludic chip that detects residues of pesticides.In abstraction pool, add 120 μ L phosphate buffers (pH=7.71); Add 60 μ L second phthalein choline vinegar enzyme solutions (40U/mL) in the inhibitory reaction pond; The developer that adding 20 μ L equal-volumes mix in the chromogenic reaction pond (detection cell) (Gu blue B salt, 0.08%) and substrate (acetate-1-naphthalene ester, 0.1%).Be fixed in centrifugal type microfludic chip on the rotation platform of centrifuge through sucker; Behind 80rpm Centrifugical extraction 10min,, solution is all shifted get into and suppress pond (adopting the quartz capillary of internal diameter 100 μ m) again with the centrifugal 30s of 300rpm; Behind the inhibitory reaction 10min; With the centrifugal 30s of 1000rpm, reaction solution is all shifted get into colour developing pond (adopting the quartz capillary of internal diameter 50 μ m), chromogenic reaction 10min.At last, centrifugal type microfludic chip is moved to the absorption detecting system of ultra-violet and visible spectrophotometer,, detect the content of residues of pesticides in the sample in the absorbance of 525nm place working sample.
Detect the micro-structural and the microchannel figure of the centrifugal type microfludic chip of residues of pesticides with computer aided design software design and drafting.On three strata methyl methacrylate (PMMA) chips, prepare micro-structural and microchannel through numerical control CNC micro-machining system, clean each layer chip with running water, distilled water respectively, and with spots such as the residual fingerprint of ethanol wiping chip surface, oil stains.On the double faced adhesive tape film, with carving machine processing and preparing required micro-structural and microchannel.Claim that the 40mg pedotheque is positioned in the abstraction pool,, process centrifugal type microfludic chip 3 layers of PMMA chip and 2 layers of double faced adhesive tape film alignment, bonding, pressurizing and sealing.In abstraction pool, add 120 μ L phosphate buffers (pH=7.71); Add 60 μ L second phthalein choline vinegar enzyme solutions (40U/mL) in the inhibitory reaction pond; The developer that adding 20 μ L equal-volumes mix in the chromogenic reaction pond (detection cell) (Gu blue B salt, 0.08%) and substrate (acetate-1-naphthalene ester, 0.1%).Centrifugal type microfludic chip is fixed on the centrifuge rotation platform through draw-in groove; Behind 80rpm Centrifugical extraction 10min,, extraction solution is all shifted get into and suppress pond (adopting the quartz capillary of internal diameter 100 μ m) with the centrifugal again 30s of 600rpm; Behind the inhibitory reaction 10min; With the centrifugal 30s of 1500rpm, reaction solution is all shifted get into colour developing pond (adopting the quartz capillary of internal diameter 50 μ m), chromogenic reaction 10min again.At last, centrifugal type microfludic chip is moved to the absorption detecting system of ultra-violet and visible spectrophotometer,, detect the content of residues of pesticides in the sample in the absorbance of 525nm place working sample.
Claims (10)
1. one kind is detected centrifugal type microfludic chip of residues of pesticides and preparation method thereof; It is characterized in that; This chip is the centrifugal type microfludic chip of disk shape band micro-structural and microchannel; With centrifugal force is sample microfluid driving force, with the online use of detector, on chip, realizes sample pretreatment and detection simultaneously.Micro-structural and microchannel comprise abstraction pool, inhibitory reaction pond, chromogenic reaction pond (detection cell), microfluidic channel, microflute and micropore through the micro-processing technology preparation.The rotation platform of centrifuge aims at the design of disk shape centrifugal type microfludic chip, and the center is with sucker, draw-in groove or screw-nut fixed wafer shape chip.Disk shape centrifugal type microfludic chip is carved with the micro-structural of micron level by multilayer and the chip and the double-deck adhesive membrane involution of microchannel forms.Disk shape centrifugal type microfludic chip is under the centrifugal force that the centrifuge rotation produces drives; Realize sample and reaction reagent mixing, extraction, react, separate and process color, go out the residues of pesticides content in the sample with the ultra-violet and visible spectrophotometer detection by quantitative at last.
2. by the described centrifugal type microfludic Detecting Pesticide of claim 1 chip, it is characterized in that its making step is following:
1) with computer aided design software design with draw the micro-structural and the microchannel figure of each layer chip in the centrifugal type microfludic chip;
2) on each layer micro-fluidic chip substrate surface and adhesive membrane, process required micro-structural and microchannel through micro-processing technology, comprise abstraction pool, inhibitory reaction pond, chromogenic reaction pond (detection cell), microfluidic flow channels, microflute and micropore;
3) testing sample is added in the abstraction pool;
4) utilize double-deck adhesive membrane, with each leafing core type micro-fluidic chip align, bonding, pressurizing and sealing, form disk shape centrifugal type microfludic chip;
5) extractant is added abstraction pool, add enzyme in the inhibitory reaction pond, (detection cell) adds substrate and developer in the chromogenic reaction pond;
6) start centrifuge, extractant and testing sample mix under centrifugal force drives, residual agricultural chemicals in the extraction sample;
7) change centrifugal speed, extract shifts through little valve and gets into inhibitory reaction pond, extract and enzyme generation inhibitory reaction;
8) after inhibitory reaction is accomplished, change centrifugal speed again, solution shifts entering chromogenic reaction pond (detection cell) through little valve in the inhibitory reaction pond, with behind the substrate reactions by chromogenic reagent;
9) after the sample chromogenic reaction is accomplished, carry out detection by quantitative with ultra-violet and visible spectrophotometer at last.
3. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; The Core Feature device of this Detecting Pesticide chip is a disk shape centrifugal type microfludic chip; With centrifugal force is the driving force of sample microfluid, and micro-fluidic chip base material wide material sources can be produced in batches, clean simply, repeatedly utilization, flexible design and assembling.
4. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; The micro-structural on this Detecting Pesticide chip and the size of microchannel are micron levels, and the micro-processing method that mills quarter, laser ablation, LIGA technology, method of molding, pressure sintering, chemical attack, soft lithographic technique through numerical control is in the chip substrates surface preparation.
5. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chip is formed by stacking multilayer chiop; Constitute the micro-structural and the microchannel network of 3 D stereo, on chip, directly accomplish sample pretreatment and analyzing and testing.
6. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chip can be made many group micro-structurals and microchannel on chip piece; Constitute many group sample analysis detecting units, improve the parallel detectability of unit interval.
7. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chip directly adopts micro-example, need not the sample introduction pre-treatment, and integrated form is accomplished the whole process of analyzing and testing on chip.
8. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chip is embedded into the little valve of formation in the microchannel with the quartz capillary of different inner diameters, regulates and control the flow regime of sample microfluid through the internal diameter of control quartz capillary.
9. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chip need not the flow regime that external driving pump and valve are controlled the sample microfluid, controls the centrifugal force of generation through the rotating speed that changes centrifuge, under centrifugal force drives; Make extract be transferred to the inhibitory reaction pond by abstraction pool and react, liquid gets into chromogenic reaction pond and developer generation chromogenic reaction in the reaction inhibitory reaction pond.
10. press the centrifugal type microfludic chip and the preparation method of claim 1 or 2 described detection residues of pesticides; It is characterized in that; This Detecting Pesticide chipset is simple, be easy to carry, Direct Sampling, need not that sample introduction pre-treatment, sample and reagent dosage are little, the parallel analysis detectability high, be prone to be connected with detector, sample pretreatment and testing process is accomplished on same chip, sample need not transfer, sample cross pollution probability is little; Satisfy the needs of micro-total analysis system development well, have very application prospects in the association area of fast detecting pesticide residue.
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