CN1740779A - Analyzing system and analysing method based on integrated micro-flow control chip - Google Patents
Analyzing system and analysing method based on integrated micro-flow control chip Download PDFInfo
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- CN1740779A CN1740779A CN 200510029876 CN200510029876A CN1740779A CN 1740779 A CN1740779 A CN 1740779A CN 200510029876 CN200510029876 CN 200510029876 CN 200510029876 A CN200510029876 A CN 200510029876A CN 1740779 A CN1740779 A CN 1740779A
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Abstract
On the micro-flow control chip a sample pretreatment channel, an electrophoretic separation channel and an electrophoretic sampling channel are integrated, a solid-phase carrier is fixed in the sample pretreatment channel. The actual sample containing components to be analyzed can be undergone the processes of purification, enrichment and fluorescent derivatization reaction in the pretreatment channel, after the fluorescently-labeled product is eluted from solid-phase carrier, it can be introduced into the separation channel to implement chip electrophoretic separation and laser induced fluorescence detection.
Description
Technical field
The invention belongs to the micro-total analysis technical field, the integrated micro-flow control chip analytic system and the analytical approach of be specifically related to a kind of example enrichment, derive, separating.
Technical background
Micro-total analysis system (micro-total analysis system) is an interdisciplinary technology that has grown up since the nineties, its microminiaturization by chemical analysis apparatus and integrated, to greatest extent the function of assay laboratory is transferred in the portable analytical equipment (as various chips), to realize high flux, chemical analysis cheaply.In micro-total analysis system based on micro-fluidic chip, sample pretreatment, compartment analysis and detection are integrated on the chip of several square centimeters of sizes, experimental provision and operation steps have not only been simplified, reduced the consumption of sample, shorten analysis time, also can realize the on-the-spot real-time analysis of sample and the parallel parsing of multichannel array.Through the tremendous development over 10 years, various sample pretreatments based on micro-fluidic chip, compartment analysis and detection new method continue to bring out.But these operating units are integrated, develop the chip of integrated function and make its analysis that is applied to actual sample be still waiting development.The present invention will combine with the chip electrophoresis isolation technics based on the sample Solid-Phase Extraction-solid phase deriving technology of micro-fluidic chip, cooperate highly sensitive laser-Induced Fluorescence Detection technology, having set up collection sample pretreatment, separation and detection is the integrated micro-flow control chip analytic system of one, carries out the analysis of biological fluid.This chip has integrated, quick, sensitive advantage, can develop into high-throughout array analysis system, very is suitable for the primary dcreening operation of excitant in the biological fluid or other drug.
Summary of the invention
The objective of the invention is to propose a kind of analytic system and analytical approach based on integrated micro-flow control chip, sample pretreatment (comprise enrichment and derive), electrophoretic separation and laser-Induced Fluorescence Detection are integrated on the single micro-fluidic chip, can realize to the materials such as excitant in actual sample such as the biological fluid fast, high throughput testing.
The analytic system that the present invention proposes based on integrated micro-flow control chip, its micro-fluidic chip is made up of sample pretreatment passage 1, electrophoretic separation passage 2, electrophoresis sample intake passage 3 and liquid storage hole 6-10; Its sample pretreatment passage 1 is filled with and is used for the solid phase carrier 4 and 5 that Solid-Phase Extraction and solid phase are derived, and the 1 one-tenth T-shaped in sample intake passage 3 and sample pretreatment road is connected, and sample intake passage 3 is connected with electrophoresis path 2 across; Liquid storage hole 6,9 is positioned at the two ends of sample pretreatment passage 1, can link to each other with external pressure or voltage device, flows through the pre-service passage to drive sample and eluant, eluent; Liquid storage hole 7,8 and 10 lays respectively at the end of sample intake passage 3 and electrophoretic separation passage 2, links to each other with high-voltage power supply, and electric sample introduction and electrophoretic separation required voltage are provided; Micro-fluidic chip places laser induced fluorescence detector, and check point is positioned at tail end of separation channel c.
Among the present invention, the integrated micro-flow control chip material can be quartzy, glass, can be high molecular polymer also, as polymethylmethacrylate, and poly dimethyl silane, polycarbonate etc.Be of a size of 50～150mm * 20～50mm * 0.5～2mm, base plate thickness 0.5～1.5mm, cover sheet thickness 0.2～0.5mm.Passage has the diesis decussate texture, and channel cross-section is for being inverted approximate trapezoid.Pre-service passage 1 length is 5.0-50.0mm, and width is 10-300 μ m, and the degree of depth is 10-300 μ m, and wherein the length of solid phase carrier is 1.0-40.0mm; Split tunnel 2 length are 20.0-100.0mm, and width is 10-100 μ m, and the degree of depth is 10-150 μ m; The distance of T-shaped crossing a and liquid storage hole 5, and the distance of cross junction b and liquid storage hole 7,8 is 20.0-50.0mm; Sample intake passage 3 length are 50.0-80.0mm; Each liquid storage hole internal diameter is 2.0-6.0mm.
Among the present invention, the sample pretreatment passage of integrated micro-flow control chip is fixed with solid phase material for 1 li, the carrier of deriving as Solid-Phase Extraction and solid phase.Solid phase carrier can be various chromatograph packing materials (fillers that comprise gas chromatography and liquid chromatography); also can be all solid materials with extracting power; comprise the whole porous bed material of porous polymer polymer beads or high molecular polymer, and the organic and inorganic material is assorted and particle or whole porous bed material.Solid phase carrier can be single homogeneous phase, also can be heterogeneous.
Use analytic system of the present invention, the method for carrying out analyzing and testing is as follows:
The sample and the fluorescent derivatizing agent that will contain measured object, under liquid storage hole 6,9 place's air pressure or driven, enter pre-service passage 1 successively from liquid storage hole 9, the solid phase carrier of flowing through, measured object and fluorescent derivatizing agent are attracted on the solid phase carrier 4,5, and carry out derivatization reaction on this solid phase carrier; Perhaps earlier with fluorescent reagent mark solid phase carrier 4,5, the sample that will contain measured object then enters pre-service passage 1 from liquid storage hole 9 under air pressure or driven, and the solid phase carrier of flowing through carries out derivatization reaction on solid phase carrier.After reaction finishes, by regulating each liquid storage hole place air pressure and voltage, drive eluent with derivative products wash-out, dilution from the solid phase carrier, and be incorporated into sample intake passage 3, carry out electric sample introduction by cross junction b, and then realize electrophoretic separation and laser-Induced Fluorescence Detection.
In the said method, the measured object sample can be biofluids such as urine, blood and sweat, or environmental water sample.The concentration of measured object is controlled to be 1ng/mL-100 μ g/mL in the sample, and once analyzing the specimen in use amount is 10-200 μ L.
In the said method, sample flow is 2.0-20.0 μ L/min through the speed of solid phase carrier, and the fluorescent derivatizing agent consumption is 5-15 μ L, and concentration is 0.01-0.5mM, and the speed of the solid phase carrier of flowing through is 1.0-2.0 μ L/min.
In the said method, can be pacified a kind of of non-its life, methylenedioxyamphetamine, methylenedioxymethamphetamine, ephedrine, pseudoephedrine, demethyl ephedrine, demethyl pseudoephedrine, mefenorex, etilamphetamine, parahydroxyamphetamine, phentermine, phenylpropyl alcohol azanol, veritol, biogenic amine etc. for heptyl amice alcohol, mephentermine, non-its life of peace, methyl by the measured object of fluorescent derivatizing agent mark.
In the said method, used eluant, eluent can be the mixed liquor of organic solvent and buffer solution, also can be any inorganic buffer solution at solid phase carrier absorption-wash-out characteristic.In the eluent, can add adjuvant as required, as surfactant and chirality selective reagent etc.
In the said method, electrophoretic separation can adopt the zone electrophoresis pattern, also can adopt other electrophoretics, as micellar electrokinetic chromatography, electrochromatography, isoelectric focusing and gel electrophoresis.
Among the present invention, carrying out the passage 2 of electrophoretic separation, can be chip material itself, also can be to modify through coating.
The example enrichment that the present invention proposes, the integrated micro-flow control chip analytic system of deriving, separating, being applicable to can be by the analyzing and testing of the measured object of suitable fluorescent reagent institute mark, as non-its the life class of the peace in the excitant, ephedrines stimulant, the polyamines in the biological fluid etc.
The analytic system that the present invention proposes based on integrated micro-flow control chip, have advantages such as easy and simple to handle, that the sample consumption is little, highly sensitive, analysis speed is fast, carry out on this basis realizing the multi-channel parallel analysis, the sample flux can be greatly enhanced, and the chromatographic process that analysis cost is also more general is low.This system provides important foundation for realizing the full chemical analysis on the chip.
Description of drawings
Fig. 1 is an integrated micro-flow control chip synoptic diagram of the present invention.
(1) is the sample pretreatment passage among the figure, (2) be the electrophoretic separation passage, (3) be the electrophoresis sample intake passage, (4), (5) are the solid phase filling material, (6)-(10) be liquid storage hole, (a) for the T-shaped crossing of pre-service passage and electrophoresis sample intake passage, (b) being the cross junction of electrophoretic separation passage and electrophoresis sample intake passage, (c) is check point.
Embodiment
Further specify the present invention below by an embodiment, but be not limited to this embodiment.
Embodiment: pacify non-its life by the stimulant that this system detects in the urine.As shown in Figure 1, micro-fluidic chip adopts glass processing to form long 5.0cm, wide 2.5cm, the thick 1.0mm of base plate, the thick 0.5mm of cover plate.The long 3cm of pre-service passage 1, dark 100 μ m, halfwidth 180 μ m.Split tunnel 2 long 4.2cm, dark 100 μ m, halfwidth 125 μ m.Sample intake passage is at the long respectively 5mm in the both sides of cross junction b, and all liquid storage hole internal diameters are 2mm.T-shaped crossing a is to liquid storage hole 6, and cross junction b is respectively 3mm to the distance of liquid storage hole 7.Be filled with the two-phase solid phase carrier in the pre-service passage 1: adopt one section zone preparation porous polymer whole post 4 (multipolymers of vinyl-dimethyl base acrylate and butyl methacrylate) of ultraviolet lighting initiation-situ aggregation method at distance channel end 25mm-15mm, use homogenate method packing gas chromatography filler 5 (Tianjin reagent two factories again in one section zone of distance channel end 15mm-5mm, GDX-502, the 100-120 order).Liquid storage hole 6,9 links to each other by pipette tip and external capillary (15cm is long, internal diameter 250 μ m, external diameter 380 μ m), uses the epoxy sealing interface, by kapillary and syringe pump the pre-service passage is applied air pressure.Insert platinum electrode in the liquid storage hole 7,8,10, link to each other with high-voltage power supply respectively.Laser-Induced Fluorescence Detection point c is positioned on the split tunnel apart from cross junction b 35mm place.
To contain urine (100ng/mL) the 1M NaOH alkalization (pH11) of non-its life of peace, 0.45 behind the μ m filtering with microporous membrane, getting 100 μ L joins in the liquid storage hole 9, apply negative pressure at liquid storage hole 6, make urine sample with the speed of 5 μ L/min by the pre-service passage, again with the borax buffer solution washing pre-service passage (5 μ L/min) of 10 μ L 5mM.Fluorescent reagent FITC (the different sulfuric acid hydrocyanic ester of fluorescein benzene) is become the solution of 0.1mM with the borax buffer preparation of 5mM, get 10 μ L and flow through pre-service passage (5 μ L/min), dry up.Chip is placed micro-wave oven (MK-2270M1 of Haier), under the 540W radiation power, react 5min.Take out chip, (the 20mM borax pH9.5) is full of split tunnel and sample intake passage with electrophoresis buffer solution.With 50 μ L eluents (5mM borax: flushing pretreatment column (5 μ L/min) acetonitrile=60: 40), simultaneously apply small malleation at liquid storage hole 6, liquid storage hole 8 applies voltage-500V, and liquid storage hole 7,10 ground connection make eluted product inflow sample intake passage and do not diffuse into split tunnel.Treat derivative products when cross junction b place concentration reaches maximum, switched voltage, make liquid storage hole 7,10 be respectively+500V and-2000V, liquid storage hole 8 is floating empty.The situation of exerting pressure of each operational phase liquid storage hole sees Table 1.Adopt the borate of 20mM to carry out electrophoretic separation, can obtain the qualitative, quantitative information of non-its life of peace of FITC mark from the spectrogram for electrophoresis buffer solution.
Each liquid storage hole is in the pressure control situation in different operating stage among table 1 embodiment 1
Claims (10)
1, a kind of analytic system based on integrated micro-flow control chip is characterized in that this micro-fluidic chip is made up of sample pretreatment passage (1), electrophoretic separation passage (2), electrophoresis sample intake passage (3) and liquid storage hole (6)-(10); Wherein, product pre-service passage (1) is filled with and is used for solid phase carrier (4) and (5) that Solid-Phase Extraction and solid phase are derived, and sample intake passage (3) becomes T-shaped to be connected with sample pretreatment road (1), and sample intake passage (3) is connected with electrophoresis path (2) across; Liquid storage hole (6), (9) are positioned at the two ends of sample pretreatment passage (1), can link to each other with external pressure or voltage device, flow through the pre-service passage to drive sample and eluant, eluent; Liquid storage hole (7), (8) and (10) lay respectively at the end of sample intake passage (3) and electrophoretic separation passage (2), link to each other with high-voltage power supply, and electric sample introduction and electrophoretic separation required voltage are provided; Micro-fluidic chip places laser induced fluorescence detector, and check point is positioned at tail end of separation channel c.
2, analytic system according to claim 1 is characterized in that described pre-service passage (1) length is 5.0-50.0mm, and width is 10-300 μ m, and the degree of depth is 10-300 μ m, and wherein the length of solid phase carrier is 1.0-40.0mm; Split tunnel (2) length is 20.0-100.0mm, and width is 10-100 μ m, and the degree of depth is 10-150 μ m; The distance of T-shaped crossing (a) and liquid storage hole (5), and the distance of cross junction (b) and liquid storage hole (7), (8) is 20.0-50.0mm; Sample intake passage (3) length is 50.0-80.0mm; Each liquid storage hole internal diameter is 2.0-6.0mm.
3, analytic system according to claim 1; it is characterized in that described solid phase carrier is various chromatograph packing materials; perhaps be porous polymer polymer beads or high molecular polymer integral bed material, perhaps for the organic and inorganic material assorted and particle or whole porous bed material.
4, analytic system according to claim 1 is characterized in that described solid phase carrier is single homogeneous phase, perhaps is heterogeneous.
5, analytic system according to claim 1 is characterized in that described electrophoretic separation passage has coating to modify.
6, a kind of method of using the described analytic system of claim 1 to carry out analyzing and testing, it is characterized in that concrete steps are as follows: the sample and the fluorescent derivatizing agent that will contain measured object, locate under air pressure or the driven in liquid storage hole (6), (9), enter pre-service passage (1) successively from liquid storage hole (9), the solid phase carrier of flowing through, measured object and fluorescent derivatizing agent are attracted on solid phase carrier (4), (5), and carry out derivatization reaction on this solid phase carrier; Perhaps earlier with fluorescent reagent mark solid carrier (4), (5), the sample that will contain measured object then enters pre-service passage (1) from liquid storage hole (9) under air pressure or driven, and the solid phase carrier of flowing through carries out derivatization reaction on solid phase carrier; After reaction finishes, by regulating each liquid storage hole place air pressure and voltage, drive eluent with derivative products wash-out, dilution from the solid phase carrier, and be incorporated into sample intake passage (3), (b) carries out electric sample introduction by cross junction, and then realizes electrophoretic separation and laser-Induced Fluorescence Detection.
7, analyzing detecting method according to claim 6 is characterized in that the concentration of measured object is controlled to be 1ng/mL-100 μ g/mL in the sample, and once analyzing the specimen in use amount is 10-200 μ L.
8, analyzing detecting method according to claim 6, it is characterized in that sample flow is 2.0-20.0 μ L/min through the speed of solid phase carrier, the fluorescent derivatizing agent consumption is 5-15 μ L, and concentration is 0.01-0.5mM, and the speed of the solid phase carrier of flowing through is 1.0-2.0 μ L/min.
9, analyzing detecting method according to claim 6 is characterized in that described measured object is a kind of of non-its life of heptyl amice alcohol, mephentermine, peace, non-its life of methyl peace, methylenedioxyamphetamine, methylenedioxymethamphetamine, ephedrine, pseudoephedrine, demethyl ephedrine, demethyl pseudoephedrine, mefenorex, etilamphetamine, parahydroxyamphetamine, phentermine, phenylpropyl alcohol azanol, veritol, biogenic amine.
10, analyzing detecting method according to claim 9 is characterized in that used eluant, eluent is the mixed liquor of organic solvent and buffer solution, or at any inorganic buffer solution of solid phase carrier absorption-wash-out characteristic.
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| CN102033127A (en) * | 2009-12-30 | 2011-04-27 | 复旦大学 | Microfluidic chip for cotinine quick detection and preparation method thereof |
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| CN101690855B (en) * | 2009-10-12 | 2012-04-18 | 中国检验检疫科学研究院 | Microfluidic chip for integrated solid phase micro extraction and detection method |
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- 2005-09-22 CN CN 200510029876 patent/CN1740779A/en active Pending
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| CN101690855B (en) * | 2009-10-12 | 2012-04-18 | 中国检验检疫科学研究院 | Microfluidic chip for integrated solid phase micro extraction and detection method |
| CN102033127A (en) * | 2009-12-30 | 2011-04-27 | 复旦大学 | Microfluidic chip for cotinine quick detection and preparation method thereof |
| CN102236003A (en) * | 2010-04-27 | 2011-11-09 | 复旦大学 | Capillary solid-phase microreactor and preparation method thereof |
| CN102527306A (en) * | 2010-12-28 | 2012-07-04 | 中国科学院化学研究所 | Array type continuously-flowing microfluidic chip device and manufacture method and application thereof |
| CN102527306B (en) * | 2010-12-28 | 2014-01-29 | 中国科学院化学研究所 | Array type continuously-flowing microfluidic chip device and manufacture method and application thereof |
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| CN104297327A (en) * | 2014-10-29 | 2015-01-21 | 邓杏飞 | Method for analyzing fine sub-fractions of serum lipoprotein subtype by adopting micro-fluidic chip |
| CN106596699A (en) * | 2016-11-28 | 2017-04-26 | 大连大学 | Method for detecting biogenic amine by using micro fluidic chip and nanoliter electrospray ion mobility spectrometry combination technology |
| CN106807468A (en) * | 2017-01-18 | 2017-06-09 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Micro-fluidic chip clamp and droplet preparation system |
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