CN1591001A - Laser induced fluorescence and light absorption bifunction detecting microflow controlled electrophoresis ehip - Google Patents
Laser induced fluorescence and light absorption bifunction detecting microflow controlled electrophoresis ehip Download PDFInfo
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- CN1591001A CN1591001A CN 03156108 CN03156108A CN1591001A CN 1591001 A CN1591001 A CN 1591001A CN 03156108 CN03156108 CN 03156108 CN 03156108 A CN03156108 A CN 03156108A CN 1591001 A CN1591001 A CN 1591001A
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Abstract
The invention relates to a laser induced fluorescence and optical absorption double-function detection microfluid control electrophoretic chip. It includes chip base plate and cover plate which are superimposed and bonded into one body and made up by using transparent material. On the base plate the crossed microfluid channels (sample channel and separation channel) are cut; and on the cover plate and in the place correspondent to the tail end position of microfluid channel on the base plate the small-hole type liquid storage cells (sample cell, sample waste liquor cell, buffer cell and waste liquor cell) are set; and on the cover plate or base plate the optical fibre channels are set, these optical fibre channels are close to tail end of the separation channel, perpendicular to said separation channel and positioned at its two sides.
Description
Technical field
The invention belongs to the electrophoresis chip technical field, the particularly a kind of optical fiber coupling micro-fluidic electrophoresis chip that can realize laser-Induced Fluorescence Detection and ultraviolet-visible light absorption detecting simultaneously; This chip both had been applicable to fluorescent material or can be by the separation detection of the biochemical substances sample of fluorescent reagent mark such as amino acid, protein and DNA etc., can be used for again some non-fluorescents but the separation detection of the sample of strong absorption is arranged in the ultraviolet-visible light district.But this chip test sample kind is many, applied widely.
Background technology
Early 1990s micro-total analysis system (Micro-total analysis system, μ-TAS) proposition of notion has produced significant impact in analytical instrument and analysis science field, and the guiding chemical analysis apparatus is towards the trend development of microminiaturized, integrated and portability.It utilizes micro fabrication to make functional units formation very small chemical systems such as little valve, microchannel, microreactor, microflow sensor, little detecting device on chip, desire is integrated in full chemical process such as the pre-treatment of sample, chemical reaction, determinand separation, detection on the chip of heart size and realizes, can be referred to as meaning " laboratory on the chip ".The characteristics of μ-TAS be fast, efficient, the sample reagent dosage is little, simple to operate, is convenient to integrated and microminiaturized.Wherein, micro-fluidic electrophoresis chip is current most active fields and a frontier development among μ-TAS.Detected by the earliest laser-induced fluorescence (LIF) and absorbance with the detection method of micro-fluidic electrophoresis chip logotype and to have developed into multiple detection meanss such as galvanochemistry, chemiluminescence, Mass Spectrometer Method.So far, laser-Induced Fluorescence Detection is the sensitiveest, and uses more optical detecting method, and detection sensitivity generally can reach 10
-9Mol/L～10
-12Mol/L.
At present, in the micro-fluidic electrophoresis chip optical detection, still occupy an leading position by the traditional optical detection system that conventional laser and a series of lens, catoptron, optical filter etc. are formed.This just makes that detection technique versatilities such as laser-induced fluorescence (LIF) and absorbance are poor, bulky, costs an arm and a leg, and the optical texture complexity does not match with the microchannel yardstick of chip, so be difficult to realize the microminiaturization of micro-total analysis system and integrated.So development compact conformation, high performance miniaturization optical detection system become the key subject of chip detection and microfluidic analysis system.Because of semiconductor laser and light emitting diode have stable, the advantages such as price is low, volume is little, long service life of power output, become desirable, the most attractive light source of micro-fluidic electrophoresis chip optical detection analytic system.Along with appearing on the market in succession of each wavelength period semiconductor laser from the ultraviolet to the infrared region and light emitting diode, its application in chip detection is also increasingly extensive, lays a good foundation for chip optical detection system realizes complete microminiaturization.Nearly 2 years, the development of miniature light sources coupled fiber technology, making simple that the complex optical path systems such as beam split, collimation, reflection and convergence that just can be finished by a plurality of traditional optical elements become, is that optical analysis system is realized microminiaturized and integrated another key issue that solved.Beyond any doubt, next step of micro-fluidic electrophoresis chip research is exactly to design a kind of chip that can be coupled with the compact optical detection system, with the overall microminiaturization that realizes micro-fluidic electrophoresis chip and integrated.
The micro-fluidic electrophoresis chip laser-Induced Fluorescence Detection of report has two kinds of patterns at present:
L, common focus type
The optical system of the non-typical case of focus type altogether laser-Induced Fluorescence Detection generally is made of laser focusing lens, catoptron, phosphor collection object lens, optical filter etc.Laser beam is through lens light gathering and mirror reflects, surveyed area with suitable incident angle irradiation chip, excite the fluorescence of generation to collect optically focused, and, detect with photodetectors such as photomultipliers by behind optical filter filtering exciting light and other parasitic lights through object lens.(Jacobson?S.C.，Ramsey?J.M.，Integrated?Microdevice?for?DNA?Restriction?Fragment?Analysis.，Anal.Chem.，1996，68，720-723.；Jacobson?S.C.，Hergenroder?R.，Koutny?L.B.，Ramsey?J.M.，High-SpeedSeparations?on?a?Microchip，Anal.Chem.，1994，66，1114-1118)
2, be total to focus type
The focus type laser-Induced Fluorescence Detection is a most frequently used detection system in the microcurrent controlled capillary tube electrophoresis chip analysis system altogether, its by the confocal fluorescent microscope as optical system, mainly by formations such as dichroic mirror (dichroic mirror), collector lens, interference filters.Laser beam is through beam-expanding collimation, focus on the surveyed area of back vertical irradiation by dichroic mirror reflects and by microcobjective to chip, the fluorescence that excites generation is through the dichroic mirror transmission, and, behind the further filtering interfering light of interference filter, be imaged on the photodetector again and detect by the micro-eyepiece focusing coaxial and confocal with microcobjective.(Jiang?G.F.，Attiga?S.，Ocvirk?G.，Lee?W.E.，Harrison?D.J.，RedDiode?Laser?Induced?Fluorescence?Detection?with?a?Confocal?Microscope?on?aMicrochip?for?Capillary?Electrophoresis.，Biosens.Bioelectron.，2000，14，861-869.)
The micro-fluidic electrophoresis chip ultraviolet-visible absorption detecting pattern of present report:
1, the imaging of ultraviolet-visible absorbance detects
Light source projects chip split tunnel zone with fibre bundle and another condenser lens with incident light equably after bandpass filter optical filtering and lens focus, use the CCD device of a line display that the absorbance of material in the passage is carried out in good time sweep measuring.(Mao?Q.，Pawliszyn?J.，Demonstration?of?Isoelectric?Focusing?onan?Etched?Quartz?Chip?with?UV?Absorption?Imaging?Detection.，Analyst，1999，124，637-641.)
2, the double light path absorbance detects
Usefulness red semiconductor diodes such as Collins are light source, light source place the chip detection zone directly over, the incident light vertical irradiation is to the check point and the adjacent domain of chip split tunnel, transmitted light focuses on through eyepiece, after the bar shaped slit is eliminated interference of stray light, detect with the photoelectric diode display.A display unit of photoelectric diode display detects the light intensity after sample absorbs, another display unit detects near the transmitted intensity of the chip material (glass) of channel part and makes reference, alternately measure sample signal and reference signal, and with both differences of difference processing circuit extraction as the absorbance signal.(Colline?G.E.，Lu?Q.，Microfabricated?CapillaryElectrophoresis?Sensor?for?Uranium(VI).，Anal.Chim.Acta.2001，436，181-189)
Summary of the invention
The object of the invention is: overcome micro-fluidic electrophoresis chip function singleness of the prior art, defectives such as required optical detection system architecture complexity, and the optical fiber coupling that can realize laser-Induced Fluorescence Detection and ultraviolet-visible light absorption detecting simultaneously micro-fluidic electrophoresis chip is provided; This chip both had been applicable to fluorescent material or can be by the separation detection of the biochemical substances sample of fluorescent reagent mark such as amino acid, protein and DNA etc., can be used for again some non-fluorescents but the separation detection of the sample of strong absorption is arranged in the ultraviolet-visible light district, but the test sample kind is many, and is applied widely.
Technical scheme of the present invention is as follows:
The difunctional detection micro-fluidic electrophoresis chip of laser-induced fluorescence (LIF) provided by the invention and light absorption, comprise the chip substrate 2 and chip cover plate 1 that stack also bonding all-in-one-piece transparent material making, be carved with the microfluidic channel that is decussation on the chip substrate 2, be respectively sample channel AB and split tunnel CD; Terminal position place corresponding to microfluidic channel on the substrate 2 on the chip cover plate 1 is provided with small through hole type liquid storage tank, is respectively: sample cell A, sample waste liquid pool B, buffer pool C and waste liquid pool D; On chip cover plate 1 or the chip substrate 2 near tail end of separation channel be provided be positioned at split tunnel CD both sides and with perpendicular optical-fibre channel a1 and the a2 of split tunnel, the close end of described optical-fibre channel a1 and a2 and split tunnel CD is the plano-convex lens shape; Optical fiber inserts from optical-fibre channel a1, a2 level; Described chip substrate 2 is chip substrate and the chip cover plate that glass, quartz or organic polymer high grade of transparency material are manufactured with chip cover plate 1; Described organic polymer high grade of transparency material is PMMA, PDMS, polystyrene, Polyvinylchloride, phenolics, epoxy resin, polyurethane, poly-several lactams or any multipolymer between them.
The present invention is based on standard photolithography techniques, superpolymer polymethylmethacrylate (PMMA), dimethyl silicone polymer (PDMS) with the glass and the high grade of transparency are chip material, on chip, make up microchannel and little liquid storage tank, sample introduction, separation and the detection of sample are integrated on the slight chip finish.By on chip, making up and the unlimited close optical-fibre channel of split tunnel surveyed area, make laser instrument coupled fiber level insert this passage after, exciting light is directly coupled near the surveyed area of split tunnel.In addition, in the present invention, the glass between optical-fibre channel front end and the split tunnel or high grade of transparency polymer P MMA and PDMS are designed to the plano-convex lens shape, its light that fiber port is come out has certain convergence converging action.Optical computing and accurate micro-processing technology by strictness, can make the focus of plano-convex lens just drop on the split tunnel, then the light that comes out from optical fiber will be converged to a diameter minimum at split tunnel behind these lens, the hot spot of intensity maximum, this will help obtaining the quite good detecting peak shape, help the raising of degree of separation and detection sensitivity.This chip design is ingenious, make exciting light directly and detection zone closely close, save three-dimensional collimation, the reflection that just can finish by a plurality of optical elements and assembled the complex optical path system, drop to the loss of strength in the optical transmission process minimum, the optical detection that promotes micro-fluidic electrophoresis chip makes the simple of complicated light path operation change towards microminiaturized and the integrated major step that strided forward.
Chip design, making and principle of work that the present invention proposes are as described below:
1. chip design
Make the different of characteristics according to glass-chip with superpolymer chip PMMA and PDMS, chip design is inequality slightly.Because of the optical-fibre channel of glass-chip mainly is to come overstriking by logical hydrofluorite behind substrate and the cover plate for sealing bonding, and single fiber passage and the external world can not form closed fluid circuit, so need interpolation fluid circuit communicates with optical-fibre channel.The optical-fibre channel of superpolymer chip PMMA and PDMS is by the thick burr of etching on force plate, is imprinted onto on the high polymer material then, gets final product so be designed to simple linear pattern.(accompanying drawing 2 is the mask design of chip manufacturing)
2. the method for making of chip
(1) method for making of glass-chip:
A. the micro-fluidic chip figure of design is made mask, graph area is the clear area, and light-transmissive, non-graph area are black region, extinction and can not transmitted light.
B. mask is covered even glue chromium plate (the chromium type: LRC of 63mm * 63mm * 1.5mm; The thick T:145nm glue of chromium class: positive-working photoresist; Glue is thick: 570nm), and exposure under the ultraviolet ray irradiation, photoresists generation photochemical reaction.
C. the chromium plate after the exposure develops in developer solution, and to remove the positive-working photoresist that is exposed, the figure on the mask is copied on the optical cement layer.
D. the chromium film that exposes with chromium film etching liquid (cerous sulfate: perchloric acid: water=50 restrain: 15 milliliters: 300 milliliters) corrosion under the room temperature after high purity water is rinsed well, is dried.Figure on the optical cement layer has been transferred on the substrate, guarantees that simultaneously the optical cement layer of non-graph area and chromium film are not destroyed.
E. wet etching microchannel.With 0.5M HF/0.5M NH
4F is etching agent etching microchannel, and speed is about 10 μ m/h, and getting xsect is trapezoidal recessed microchannel.Remove remaining optical cement layer and chromium film with acetone and chromium film etching liquid successively again, rinse well with high purity water and promptly get totally transparent substrate.
F. punch with miniature bench drill place in the liquid storage tank position of substrate, as the chip liquid storage tank.
G. the even glue chromium plate of intercepting and the same size of substrate soaked 2 minutes with acetone, chromium film etching liquid successively, and to remove optical cement layer and chromium film, high purity water is rinsed well and promptly got cover plate.
H. with substrate and cover plate successively at acetone, H
2O-H
2O
2-NH
4OH (5: 1: 1) solution, H
2SO
4: H
2O
2In (4: 1) solution and the high purity water ultrasonic cleaning 5-10 minute, nitrogen dried up, the sealing of in super-clean environment both being alignd then.Bonding under the high temperature, heating schedule is: rise to 550 ℃ with 40 ℃/min from room temperature in 13 minutes, and stopped 30 minutes at 550 ℃; Rise to 610 ℃ with 20 ℃/min from 550 ℃ in 3 minutes, and stopped 30 minutes at 610 ℃; Rise to 635 ℃ with 20 ℃/min from 610 ℃ in 1 minute 15 seconds, and stopped 30 minutes at 635 ℃; Rise to 650 ℃ with 10 ℃/min from 635 ℃ in 1 minute 30 seconds, and stopped 6 hours at 650 ℃; Naturally cool to room temperature then.
I. by hf etching overstriking optical-fibre channel.With peristaltic pump the 2%HF lean solution is expelled to optical-fibre channel continuously by liquid storage tank E, F, waste liquid is discharged from the optical-fibre channel uropore on the chip sides.
(2) method for making of PMMA chip
A. design and produce force plate mask (substrate and cover plate): the black graphics district is microfluidic channel on the substrate mask, and is light tight; The black graphics district is optical-fibre channel on the cover plate mask, and is light tight.
B. make base plate glass force plate and cover-plate glass force plate.The method for making of force plate method for making and glass-chip substrate is basic identical.Mask is covered on the even glue chromium plate, after exposure under the ultraviolet ray irradiation, develop in the developer solution, to remove the positive-working photoresist that is exposed.Use chromium film etching liquid (cerous sulfate: perchloric acid: water=50 grams: 15 milliliters: 300 milliliters) remove exposed chromium film, after the high purity water flushing, dry then.When wetting etching, the used etching liquid concentration of substrate template and cover plate template is different, and the former uses 0.2M HF/0.2MNH
4The etching agent of F, the latter uses 0.5M HF/0.5M NH
4The etching agent of F.After wet etching is finished, remove remaining optical cement layer and chromium film with acetone and chromium film etching liquid successively again, rinse well with high purity water and promptly get the glass mold plungers plate.Wherein, the burr part on the base plate glass force plate is corresponding to the microfluidic channel of chip, and the part of the burr on the cover-plate glass force plate is corresponding to optical-fibre channel.
C. adopt bulk polymerization to make PMMA substrate and cover plate.MMA (methyl-prop diluted acid formicester) after purifying with after initiating agent (benzoyl peroxide) mixes,, is cooled off about 50 minutes 85-90 ℃ of following prepolymerization, after the vacuum outgas, be cast to respectively on base plate glass force plate and the cover-plate glass force plate.45 ℃ of polymerizations 12 hours are warming up to 110 ℃ of polymerizations 2 hours then, slowly cool to the back demoulding about 40 ℃, promptly get water white transparency bright and clean PMMA substrate and cover plate.
D. (error: 0.2 μ m), and the liquid storage tank position on substrate punches with miniature bench drill, as the chip liquid storage tank with DAD321 saw substrate and cover plate to be cut into identical size.
E. substrate and cover plate are cleaned, dried up, alignment is closed up, and pressurization 120 ℃ of bondings 2 hours, is promptly made the PMMA chip.
(3) method for making of PDMS chip
A. the method for making of substrate force plate and cover plate force plate is same as PMMA chip force plate facture.Same, the burr part on the base plate glass force plate is corresponding to the microfluidic channel of chip, and the part of the burr on the cover-plate glass force plate is corresponding to optical-fibre channel.
The making of B.PDMS substrate: PDMS monomer (dimethyl siloxane) is mixed with the part by weight of initiating agent (Sylard184) with 30: 1, water after the degassing and cast from the substrate force plate,, be cooled to room temperature 80 ℃ of polymerizations 1.5 hours, lift-off stencil gets water white transparency PDMS substrate.
The making of C.PDMS cover plate: PDMS monomer (dimethyl siloxane) is mixed with the part by weight of initiating agent (Sylard184) with 3: 1, water after the degassing and cast from the cover plate force plate,, be cooled to room temperature 80 ℃ of polymerizations 1.5 hours, lift-off stencil gets water white transparency PDMS cover plate.
D. with the DAD321 saw substrate and cover plate are cut into identical size (error: 0.2 μ m).Punch with miniature bench drill in liquid storage tank position at substrate, as the chip liquid storage tank.
E. substrate is alignd with cover plate and closes up, 80 ℃ of following bondings 1.5 hours, cover plate and substrate just combine securely, the PDMS chip.
3, chip separation detection principle
Between sample cell and sample waste liquid pool, add appropriate voltage, under electroosmotic flow drives, sample is moved to sample intake passage by sample cell, enter into the crossing of sample intake passage and split tunnel when sample after, add separation voltage between buffer pool and waste liquid pool, sample cell and sample waste liquid pool float or add appropriate voltage.Sample component difference owing to rate travel in split tunnel obtains separating, and when the surveyed area on the pairing split tunnel of arrival optical-fibre channel, is detected from the optical fiber rayed.
Fig. 1 and Fig. 3 are respectively the schematic diagram that glass-chip and POLYMER PMMA chip (or PDMS chip) are used for laser-Induced Fluorescence Detection: laser instrument coupled fiber (single mode or multimode) inserts chip by the optical-fibre channel level of a side; the exciting light that comes out from fiber port is radiated at the surveyed area of split tunnel after the plano-convex lens of optical-fibre channel front end is assembled.Sample be excited and the fluorescence signal that produces by photodetectors such as photomultiplier with the perpendicular direction of incident light on detect.
Fig. 2 and Fig. 4 are respectively glass-chip and POLYMER PMMA chip (or the PDMS chip) fundamental diagram when being used for light absorption and detecting: according to Beer law, absorption peak strength is directly proportional with the absorption light path, because of general chip split tunnel mean breadth is about ten times of the degree of depth, so be that light path can improve detection sensitivity with the channel width.After incident light shone detection zone by the optical-fibre channel of split tunnel one side, transmitted light was accepted by the optical-fibre channel optical fiber of opposite side again, and light path promptly is the mean breadth of split tunnel.The transmitted light that comes out from optical fiber is detected by photodetectors such as photoelectric diode displays.For light absorption detects, the collimation of incident light and transmitted light and to aim at be to realize the key that accurately detects, operation easier is bigger, and the present invention makes alignment function be easy to realization by the optical-fibre channel of the alignment of etching phase on chip.
The present invention compares with existing optics micro-fluidic electrophoresis chip, and following advantage is arranged:
The present invention is combined in same micro-fluidic electrophoresis chip with laser-induced fluorescence (LIF) and light absorption detection, accomplishes that a slice is dual-purpose.Make optical-fibre channel on chip, by optical fiber exciting light (or incident light) is directly coupled to surveyed area, avoided numerous and diverse long light path alignment system, reduced the light loss in the light transmission, it is simple that whole optical path becomes.In addition, the optical-fibre channel Front-end Design becomes the plano-convex lens shape, and its light that optical fiber is come out has certain convergence converging action, makes hot spot more concentrated, helps the raising of degree of separation and detection sensitivity like this.This micro-fluidic electrophoresis chip low cost of manufacture is easy to produce in batches, is complementary with miniature light sources such as fiber coupled laser diode or light emitting diodes, has realized the microminiaturization of chip optical detection, integrated and simplification.
Description of drawings
Fig. 1 the present invention's (glass-chip) structural representation also is the schematic diagram that is used for laser-Induced Fluorescence Detection;
Fig. 2 is the structural representation of the present invention's (glass-chip), also is the schematic diagram that is used for the ultraviolet-visible light absorption detecting;
Fig. 3 is the structural representation of the present invention's (POLYMER PMMA chip or PDMS chip), also is the schematic diagram that is used for laser-Induced Fluorescence Detection;
Fig. 4 is the structural representation of the present invention's (POLYMER PMMA chip or PDMS chip), also is the schematic diagram that is used for the ultraviolet-visible light absorption detecting;
Wherein: chip substrate 2 chip cover plates 1 sample channel AB
Split tunnel CD sample cell A sample waste liquid pool B
Buffer Pool C waste liquid pool D optical-fibre channel a1, a2
Liquid storage tank E passage b1 liquid storage tank F passage b2
Optical-fibre channel a1, a2 apart from split tunnel CD apart from d≤200 μ m
Embodiment
Embodiment 1: the making of the optical fiber coupling micro-fluidic electrophoresis chip of glass material
1, the making of chip substrate 2:
The size design of microchannel on the mask film: the width of fluid passage is 40 μ m; The width of optical-fibre channel is 60 μ m; Between optical-fibre channel and the split tunnel CD is 200 μ m apart from d;
The mask film is placed on the even glue chromium plate of 63mm * 63mm * 1.5mm, ultraviolet exposure 180 seconds (wavelength 365nm) after developing 100 seconds in the developer solution, is dried half an hour down for 100 ℃;
At room temperature use chromium film etching liquid (cerous sulfate: perchloric acid: water=50 grams: 15 milliliters: 300 milliliters) corrode the chromium film, rinse oven dry then with high purity water well;
By the digit microscope shooting, record the channel size on the chromium plate: fluid passageway width is 60 μ m; The optical-fibre channel width is 90 μ m;
With 0.5M HF/0.5M NH
4The exposed Pyrex of F etching agent corrosion, speed is about 10 μ m/h, and etching is removed residual light glue-line and chromium film with acetone, chromium film etching liquid after 2.5 hours more successively, promptly gets chip substrate 2;
With miniature bench drill punching, drill bit is the diamond bit of 2mm, and the diameter in hole is liquid pool diameter 2mm.Microscopically is measured:
Sample intake passage AB and split tunnel CD size: go up bottom width 110 μ m, following bottom width 60 μ m, the degree of depth 20 μ m;
Optical-fibre channel a
1, a
2: go up bottom width 140 μ m, following bottom width 110 μ m, the degree of depth 26 μ m;
Distance between optical-fibre channel and split tunnel: 120 μ m.
2, the making of chip cover plate 1:
The even glue chromium plate of intercepting and chip substrate 2 same sizes soaked 2 minutes with acetone, chromium film etching liquid successively, to remove optical cement layer and chromium film, promptly got chip cover plate 1.
3, chip substrate 2 and chip cover plate 1 are successively at acetone, H
2O-H
2O2-NH
4OH (5: 1: 1) solution, H
2SO
4: H
2O
2In (4: 1) solution and the high purity water ultrasonic cleaning 5-10 minute, dry up with nitrogen, seamless bonding is carried out in the sealing of in super-clean environment both being alignd then under the high temperature.Heating schedule is: rise to 550 ℃ with 40 ℃/min from room temperature in 13 minutes, and stopped 30 minutes at 550 ℃; Rise to 610 ℃ with 20 ℃/min from 550 ℃ in 3 minutes, and stopped 30 minutes at 610 ℃; Rise to 635 ℃ with 20 ℃/min from 610 ℃ in 1 minute 15 seconds, and stopped 30 minutes at 635 ℃; Rise to 650 ℃ with 10 ℃/min from 635 ℃ in 1 minute 30 seconds, and stopped 6 hours at 650 ℃; Naturally cool to room temperature then.Through microscopically observation, passage does not have distortion behind the bonding, and reaches sealing fully.
4, hf etching overstriking optical-fibre channel.With peristaltic pump passage b1 and the b2 of 2%HF lean solution by liquid storage tank E, F is expelled to optical-fibre channel a1 and a2 continuously, waste liquid is discharged through the optical-fibre channel uropore of chip sides, and optical-fibre channel is carried out overstriking; Etching time is 4-5 hour, the optical-fibre channel degree of depth 180 μ m, last bottom width 280 μ m, following bottom width 110 μ m.
The making of the difunctional detection micro-fluidic electrophoresis chip of embodiment 2:PMMA laser-induced fluorescence (LIF) and light absorption
1, the design of glass mold plungers plate mask film (substrate and cover plate): the microfluidic channel width is 120 μ m on the chip substrate force plate mask; The optical fiber channel width is 250 μ m on the chip cover plate force plate mask, and the distance between two optical-fibre channels is 240 μ m.
2, the making of substrate force plate: chip substrate mask film is placed on the even glue chromium plate of 63mm * 63mm * 1.5mm, ultraviolet exposure 5 minutes (wavelength 365nm), after developing 100 seconds in the developer solution, 100 ℃ are dried by the fire half an hour down.(cerous sulfate: perchloric acid: corrosion chromium film water=50 grams: 15 milliliters: 300 milliliters), water is rinsed well then at room temperature to use chromium film etching liquid.With 0.2M HF/0.2M NH
4The F etching agent carries out etching, and speed is about 4 μ m/h, etching time 8 hours.Remove residual light glue-line and chromium film with acetone, chromium film etching agent successively again, promptly get the substrate force plate.After measured, the burr of split tunnel and sample intake passage is of a size of on the glass substrate force plate: go up bottom width 50 μ m, following bottom width 120 μ m, height 30 μ m;
3, the making of glass cover-plate force plate: will cover on the even glue chromium plate that bar mask film places 63mm * 63mm * 1.5mm, ultraviolet exposure 6 minutes (wavelength 365nm), after developing 120 seconds in the developer solution, 100 ℃ of baking half an hour down.(cerous sulfate: perchloric acid: corrosion chromium film water=50 grams: 15 milliliters: 300 milliliters), water is rinsed well then at room temperature to use chromium film etching liquid.With 0.5M HF/0.5M NH
4The F etching agent carries out etching, and speed is about 10 μ m/h, etching time 20 hours; After removing remaining optical cement layer and chromium film, the optical-fibre channel burr is of a size of on the mensuration glass cover-plate force plate: go up bottom width 190 μ m, following bottom width 250 μ m, height 180 μ m;
4, PMMA chip substrate 2 and chip cover plate 1 are made in bulk polymerization: the MMA after will purifying (methyl-prop diluted acid formicester) mixes with 1000: 5 ratios with initiating agent (benzoyl peroxide), 85-90 ℃ of prepolymerization about 50 minutes, be cooled to room temperature rapidly, after the vacuum outgas 30 minutes, be cast to respectively on above-mentioned substrate force plate and the cover plate force plate, 45 ℃ of polymerizations 12 hours, be warming up to 110 ℃ of polymerizations 2 hours then, slowly cool to the back demoulding about 40 ℃, get the chip substrate 2 and the chip cover plate 1 of PMMA material, the recessed channel size is corresponding to the burr on the force plate.Split tunnel and sample intake passage are of a size of on the PMMA chip substrate 2: go up bottom width 120 μ m, following bottom width 50 μ m, the degree of depth 30 μ m; Optical-fibre channel is of a size of on the PMMA chip cover plate 1: go up bottom width 250 μ m, following bottom width 190 μ m, the degree of depth 180 μ m;
5, with the DAD321 saw chip substrate 2 and chip cover plate 1 are cut into identical size (error: 0.2 μ m).Punch with miniature bench drill in liquid storage tank position at chip substrate 2, as the chip liquid storage tank.
6, thermal bonding: chip substrate 2 alignd with chip cover plate 1 close up, 120 ℃ of following bondings 2 hours are promptly made the optical fiber coupling micro-fluidic electrophoresis chip of the PMMA material of present embodiment.By the digit microscope shooting, the distance that records between optical-fibre channel and split tunnel is 120 μ m.
The making of the laser-induced fluorescence (LIF) of embodiment 3:PDMS material and the difunctional detection micro-fluidic electrophoresis chip of light absorption
1, the design of glass mold plungers plate mask film (substrate and cover plate): the microfluidic channel width is 120 μ m on the substrate force plate mask; The optical fiber channel width is 250 μ m on the cover plate force plate mask, and the distance between two optical-fibre channels is 240 μ m;
2, the making of glass substrate force plate: substrate mask film is placed on the even glue chromium plate of 63mm * 63mm * 1.5mm, ultraviolet exposure 5 minutes (wavelength 365nm), after developing 100 seconds in the developer solution, 100 ℃ are dried by the fire half an hour down.(cerous sulfate: perchloric acid: corrosion chromium film water=50 grams: 15 milliliters: 300 milliliters), water is rinsed well then at room temperature to use chromium film etching liquid.With 0.2M HF/0.2M NH
4The F etching agent carries out etching, and speed is about 4 μ m/h, etching time 8 hours.Remove residual light glue-line and chromium film with acetone, chromium film etching liquid successively again, promptly get the substrate force plate.After measured, the burr of split tunnel and sample intake passage is of a size of on the glass substrate force plate: go up bottom width 50 μ m, following bottom width 120 μ m, height 30 μ m;
3, the making of glass cover-plate force plate: cover plate mask film is placed on the even glue chromium plate of 63mm * 63mm * 1.5mm, ultraviolet exposure 6 minutes (wavelength 365nm), after developing 120 seconds in the developer solution, 100 ℃ are dried by the fire half an hour down.(cerous sulfate: perchloric acid: corrosion chromium film water=50 grams: 15 milliliters: 300 milliliters), water is rinsed well then at room temperature to use chromium film etching liquid.With 0.5M HF/0.5M NH
4The F etching agent carries out etching, and speed is about 10 μ m/h, etching time 20 hours.After removing remaining optical cement layer and chromium film, the optical-fibre channel burr is of a size of on the mensuration glass cover-plate force plate: go up bottom width 190 μ m, following bottom width 250 μ m, height 180 μ m;
4, the chip substrate 2 of the synthetic PDMS material of bulk polymerization: PDMS monomer (dimethyl siloxane) is mixed with the part by weight of initiating agent (Sylard184) with 30: 1, water after the degassing and cast from the substrate force plate, 80 ℃ of polymerizations 1.5 hours, be cooled to room temperature, lift-off stencil gets water white transparency PDMS substrate 2.Spill microchannel size is corresponding to the burr on the force plate.Split tunnel and sample intake passage are of a size of on the PDMS chip substrate 2: go up bottom width 120 μ m, following bottom width 50 μ m, the degree of depth 30 μ m;
5, the making of the chip cover plate 1 of PDMS material: PDMS monomer (dimethyl siloxane) is mixed with the part by weight of initiating agent (Sylard184) with 3: 1, water after the degassing and cast from the cover plate force plate, 80 ℃ of polymerizations 1.5 hours, be cooled to room temperature, lift-off stencil, the chip cover plate of water white PDMS material.Spill optical-fibre channel size is corresponding to the burr on the force plate.The optical fiber channel size is on the PDMS chip cover plate 1: go up bottom width 250 μ m, following bottom width 190 μ m, the degree of depth 180 μ m;
6, the DAD321 saw is cut into identical size (error: 0.2 μ m) with chip substrate and chip cover plate.Punch with miniature bench drill in liquid storage tank position at substrate, as the chip liquid storage tank.
7, chip substrate is alignd with the chip cover plate close up, 80 ℃ of following bondings 1.5 hours, cover plate and substrate just combine securely, the optical fiber coupling micro-fluidic electrophoresis chip of PDMS material of present embodiment, the distance between optical-fibre channel and split tunnel is 120 μ m.
Claims (3)
1, the difunctional detection micro-fluidic electrophoresis chip of a kind of laser-induced fluorescence (LIF) and light absorption, it is characterized in that, comprise the chip substrate (2) and chip cover plate (1) that stack also bonding all-in-one-piece transparent material making, chip substrate is carved with the microfluidic channel that is decussation on (2), is respectively sample channel (AB) and split tunnel (CD); Chip cover plate (1) is gone up corresponding to substrate (2) and upward is provided with small through hole type liquid storage tank in the terminal position place of microfluidic channel, is respectively: sample cell (A), sample waste liquid pool (B), buffer pool (C) and waste liquid pool (D); Chip cover plate (1) or chip substrate (2) are gone up and to be provided with the both sides that are positioned at split tunnel (CD) and with the perpendicular optical-fibre channel (a1) of split tunnel and (a2) near tail end of separation channel, described optical-fibre channel (a1) and (a2) and the close end of split tunnel (CD) be the plano-convex lens shape; Optical fiber inserts from optical-fibre channel (a1), (a2) level.
2, by described laser-induced fluorescence (LIF) of claim and the difunctional detection micro-fluidic electrophoresis chip of light absorption, it is characterized in that wherein said chip substrate (2) is chip substrate and the chip cover plate that glass, quartz or organic polymer high grade of transparency material are manufactured with chip cover plate (1).
3, by described laser-induced fluorescence (LIF) of claim and the difunctional detection micro-fluidic electrophoresis chip of light absorption, it is characterized in that wherein said organic polymer high grade of transparency material is PMMA, PDMS, polystyrene, Polyvinylchloride, phenolics, epoxy resin, polyurethane, poly-several lactams or any multipolymer between them.
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| CNB03156108XA CN100425983C (en) | 2003-08-29 | 2003-08-29 | Laser induced fluorescence and light absorption bifunction detecting microflow controlled electrophoresis ehip |
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