CN1563420A - Microchip with multichannel electrodes, preparation method and application - Google Patents
Microchip with multichannel electrodes, preparation method and application Download PDFInfo
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- CN1563420A CN1563420A CN 200410017098 CN200410017098A CN1563420A CN 1563420 A CN1563420 A CN 1563420A CN 200410017098 CN200410017098 CN 200410017098 CN 200410017098 A CN200410017098 A CN 200410017098A CN 1563420 A CN1563420 A CN 1563420A
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Abstract
This invented micro-chip integrates multipath sampling, separation and test and applies an integrated micro-three-electrode test system to realize separation, analysis and test to the sample by testing electrochemical variances generated from special reaction, not necessary to add mark compounds in the test period which eliminates the reduction of accuracy and specificity of target object test resulted from the test of the fluenrenscent or chemical lighting method induced by laser.
Description
Technical field
The present invention relates to the microchip field.Specifically, the present invention relates to a kind of multi-channel electrode microchip that collects separation, analyzes and be detected on one.
Background technology
After the birth of nineteen ninety-five DNA chip, the range gene chip is feature, maps and field such as gene sequencing has obtained conspicuous achievement at discovery, detection in Gene Mutation and polymorphic analysis, the genomic library of medical diagnosis on disease and prediction, drug screening, gene expression spectrum analysis, new gene for distinct advantage with high-throughput, microminiaturization, intellectuality etc. with microelectronic parallel processing and high-density integration technology, comprises omnibearing the developing on an unprecedented scale from academic research to the commercial applications.Technology such as, micro-printing synthetic by the oligonucleotide original position can realize at present two dimension so that three-dimensional differentiate fast, parallel, detect or diagnosis efficiently, the company that many families specialize in DNA chip research and production has all appearred both at home and abroad, and according to estimates will be up to 1,400 hundred million dollars to the global marketing volume of gene chip in 2010.Biochip technology not only makes the Human Genome Project shorten the several years, impels the rapid arrival of genome times afterwards comprehensively and proteomics simultaneously.To be integrated on the micro chip after the microminiaturization of analyzing and testing system, be expected to realize that amount of analysis is few, test speed is fast, highly sensitive, multinomial characteristics such as limit of detection is low, hyperchannel, high-throughput.This analysis chip is integrated responsive unit in a large number in small space, in conjunction with microchip relevant test and data processing means, can further develop and to realize original position, online micro-total analysis system, bring fundamental change to traditional biological analysis and clinical detection.
Microchip is made up of micro-tube that designs for particular analysis (comprising minitype channel, minisize reaction pond etc.) and respective media.The detection method of present microchip mainly is based on induced with laser and produces fluorescence or two kinds of means of chemoluminescence, the luminous intensity that the both need produce by the relevant recognition reaction of spectrophotometer, particularly importantly these two kinds of methods all need to add fluorescent reagent or luminescence reagent and reactant or product are marked just can carry out subsequent detection in system.And this labeling process very easily brings interference to the mensuration of detected object, makes the accuracy of the mensuration of target compound produce error, and produced simultaneously undesired signal can influence the specificity of mensuration.
Summary of the invention
An object of the present invention is to provide a kind of microchip, it is made up of passage lead-in wire and electrode between parting liquid injection port and microelectrode reaction tank, detection liquid injection port, each injection port and the reaction tank; Parting liquid injection port and microelectrode reaction tank are corresponding one by one, are in the two ends of separating pipe respectively, are communicated by separating pipe; Detecting the liquid injection port communicates with each root separating pipe by sample channel; But each injection port links to each other with the electrode of an external voltage operating device respectively; All contain a three-electrode system of forming by counter electrode, working electrode and reference electrode in each microelectrode reaction tank.
Another object of the present invention has provided the preparation method of above-mentioned microchip, and it may further comprise the steps:
1. on a matrix, produce each injection port and reaction tank mouth;
2. on another piece matrix, produce passage and embedding lead-in wire and electrode between each injection port and the reaction tank;
3. merge two matrix.
A further object of the present invention provided above-mentioned microchip application, chip is connected with operating device, add parting liquid and sample, finish separation, analysis and the detection of sample by control voltage.
The invention provides a kind of microchip, it is made up of passage lead-in wire and electrode between parting liquid injection port and microelectrode reaction tank, detection liquid injection port, each injection port and the reaction tank; Parting liquid injection port and microelectrode reaction tank are corresponding one by one, are in the two ends of separating pipe respectively, are communicated by separating pipe; Detecting the liquid injection port communicates with each root separating pipe by sample channel; But each injection port links to each other with the electrode of an external voltage operating device respectively; All contain a three-electrode system of forming by counter electrode, working electrode and reference electrode in each microelectrode reaction tank.
Material as matrix can be silicon, simple glass, high grade quartz and organic polymer etc.In one embodiment of the invention, adopt glass as matrix.
The quantity of the size of this chip and parting liquid injection port, detection liquid injection port, split tunnel, sample intake passage all can be determined according to integrated level and actual needs.
Microchip is connected with electrode by lead-in wire between injection port, detection cell and external operating device.This lead-in wire and electrode can adopt various metallic substance to make.Adopt Chrome gold copper-base alloy to make lead-in wire and electrode in one embodiment of the invention.
Microchip of the present invention can be processed by two matrix, and one contains injection port and test pool hole, is called the A sheet; The electrode that another piece contains the passage between each injection port and the reaction tank and links to each other with injection port is called the B sheet.
The thickness of A sheet and B sheet can be determined according to actual needs.Test through us finds that with regard to glass material, 0.1～0.2mm is thick to be the first-selection of A sheet, and 1～2mm is thick to be the first-selection of B sheet.
A sheet surface tissue comprises parting liquid injection port (1), liquid injection port to be measured (2) and detection cell (3).Wherein: parting liquid injection port (1) is that 1mm, height are the circular hole of A sheet thickness for diameter; Liquid injection port to be measured (2) is that 1mm, height are the circular hole of A sheet thickness for diameter; Detection cell (3) is that 3mm, height are the circular hole of A sheet thickness for diameter.
The B chip architecture comprises various electrodes, lead-in wire, split tunnel (4) and sample intake passage (5).Wherein: is the split tunnel (4) of about 50～80 μ m of diameter from each parting liquid injection port (1) between corresponding detection cell (3); The sample intake passage (5) of about 50～80 μ m of diameter between the joint detection liquid injection port (2) that intersects with split tunnel (4); The alive electrode of electroosmotic flow (6) that is used for parting liquid injection port (1); The alive electrode of electroosmotic flow (7) that is used for liquid injection port to be measured (2); Be used for the alive electrode of detection cell electroosmotic flow (8); Be used for the three-electrode system that sample signal detects.Every electrode is of a size of long 5mm, wide 1mm.
The structure that is used for the three-electrode system that sample signal detects as shown in Figure 2.Working electrode (11) diameter is 0.5～0.8mm, golden film thickness 200-400nm; Counter electrode (12) diameter 0.7～1.1mm, golden film thickness 200～400nm; Silver reference electrode (13) diameter 0.7～1.1mm, bottom gold film thickness 150～350nm adds surperficial 50nm silverskin; Counter electrode (12) outlet line (14), working electrode (11) outlet line (9), reference electrode (13) outlet line (10).The size of three outlet lines is: long 2～3mm, wide 0.2～0.3mm, the golden film of thickness 200～400nm.
Above-mentioned microchip can prepare by following steps:
1. on a matrix, produce each injection port and reaction tank mouth;
2. on another piece matrix, produce passage and embedding lead-in wire and electrode between each injection port and the reaction tank;
3. merge two matrix.
As previously mentioned, the material as matrix can be silicon, simple glass, high grade quartz and organic polymer etc.In one embodiment of the invention, adopt glass as matrix.
The method of making each sample inlet pool and passage also has multiple, in one embodiment of the invention, adopts the ultraviolet etching method.Before etching A sheet and the B sheet, design, make corresponding mask earlier.Make mask and will consider following factor: the distance of the shape of sample introduction swimming lane and the width that separates swimming lane, length, pipeline (comprising bend, radian, non-specification shape etc.), pipeline etc. is carried out the graphics chip design, and the graphic making that designs is become mask.
1.A the making processes of sheet.The A sheet is made the making that comprises parting liquid injection port (1), liquid injection port to be measured (2) and detection cell (3).With UV-light engraving method etching A sheet the time, except routine techniques, we also fill up the extinction paper of a black and reduce the time shutter to reduce diffuse-reflectance under sheet glass.Concrete making processes is:
(1) cleans: sheet glass was soaked 12 hours in Chrome acid, take out the back and rinse well with deionized water.Under infrared lamp, dry, put into temperature then and be 120 ℃ baking oven baking 15 minutes, remove surface-moisture comprehensively, be unlikely to when making gluing to come unstuck.
(2) gluing.Adopt negative glue, use rotation gluing mode twice, protective glass sheet surface is not corroded by HF as much as possible.
(3) preceding baking is 20 minutes, 75 ℃.
(4) exposure: in general,, do not need to beat again film after cleaning for negative glue; But when doing experiment, we find that negative glue cleans the rear surface and has thin film to cause glass corrosion to be hindered.As seen this film is being scraped the back naked eyes, shows that not being in fact is the residue that needs to beat film after generally removing photoresist.For this reason, we fill up the extinction paper of a black and reduce the time shutter under sheet glass.
(5) develop.Development time is 8 minutes, cleans 1-1.5 minute in scavenging solution then.
(6) post bake.The sheet glass that allows cleaning finish earlier before the post bake dries naturally.The post bake time is 30 minutes.
(7) corrosion: for the better protecting sheet glass, we are chosen in higher temperature, corrode among the bigger HF of concentration.In addition, at the back side that does not have resist coating, we protect with black wax or yellow wax.
We have adopted following two kinds of caustic solutions:
(1) wax+raw material band: coat the sheet glass back side with black wax, take advantage of that it is not dried, superscribe raw material band, and the sheet glass edge protects with raw material band also.Sheet glass front center zone has the place of photoresist material also to do protection with black wax, makes pin hole minimize.
(2) yellow wax: with the yellow wax protective glass sheet back side, the while is protective glass sheet front center area decreases pin hole also.
Glass should take out it etching back reaction product abrim in its hole at once, places it in another smooth sheet glass (perhaps on other smooth dough sheets) after the flushing, scrubs reaction residue with brush, prevents its dried after fixing.Place it in then and remove yellow wax or black wax in the toluene solution.For the photoresist material zone that the yellow wax protection is arranged, be difficult for removing photoresist material, available H
2O
2: H
2SO
4The mixed solution that is about 1: 4 adds heat abstraction, also plays the effect of cleaning glass sheet surface simultaneously.
2.B the making method of sheet and process.The B chip architecture comprises various electrodes, lead-in wire, split tunnel (4) and sample intake passage (5), and making processes is:
The B sheet has 4 * 8+8+6=46 leading foot, adopts 200-400nm Hou De Chrome gold copper-base alloy.Leading foot is respectively the corresponding alive electrode of electroosmotic flow (6) of parting liquid injection port (1), the alive electrode of electroosmotic flow (7) of liquid injection port to be measured (2); Be used for the alive electrode of detection cell electroosmotic flow (8) and be used for the three-electrode system that sample signal detects.Every electrode is of a size of long 5mm, wide 1mm.
The structure of three-electrode system as shown in Figure 2.Working electrode (11) diameter is 0.5～0.8mm, golden film thickness 200-400nm; Counter electrode (12) diameter 0.7～1.1mm, golden film thickness 200～400nm; Silver reference electrode (13) diameter 0.7～1.1mm, bottom gold film thickness 150～350nm adds surperficial 50nm silverskin; Also comprise counter electrode outlet line (14), working electrode outlet line (9), reference electrode outlet line (10).The size of three outlet lines is: long 2～3mm, wide 0.2～0.3mm, the golden film of thickness 200～400nm.Many sample intake passages and many split tunnels have guaranteed the high-throughput performance of this chip.
The basic demand that electrode is made is that electrode and lead-in wire " are inlayed " in substrate, makes to be unlikely to make substrate and tectum loose contact owing to go between in ensuing A, B sheet are bonding.Its main making step comprises: the depression that goes between at cutting, quarter is removed photoresist into lead-in wire with the steaming gold.
Material as substrate can be silicon, simple glass, high grade quartz and organic polymer etc.In one embodiment of the invention, adopted glass and silicon as substrate, wherein silicon substrate is divided into the silicon and the unoxidized silicon of surface band zone of oxidation again.
Be example explanation making processes with the substrate glass sheet below:
(1) cleans.Because plate-making sheet sub-surface has coating, remove coating in the vitriol oil so be placed on.Soaked 12 hours in the same Fang Zhi Zai Chrome acid, take out and in deionized water, rinse well.Be placed on to shine under the infrared lamp and do, put 120 ℃ of baking ovens then into and remove the surface adsorption moisture content.
(2) be coated with negative glue.Preceding baking 20 minutes.
(3) exposure is developed, and cleans post bake.This process is identical with making A sheet.
(4) corrosion: 40 ℃ of following erosion rates are about 100um/10min in 40% dense HF, and this speed can and reduce along with the thickness increase.This corrodes the back side protects with black wax, simultaneously glass substrate is placed smooth forms of corrosion, has eliminated the edge unfairness of the groove that causes because of thermogenetic fluid orientation.
(5) etch after, under the flushing of deionized water, brush the corrosive reaction product with brush, sheet glass is rinsed well, prevent the extra corrosion that remaining HF causes.
(6) Zheng Chrome gold forms electrode and lead-in wire.
At first, clean the sheet glass of having carved groove, mode by hand is coated with the thick photoresist material of last layer in the zone of groove again, thereby has eliminated the accumulation of gold at groove edge.Oven dry is coated with positive glue, preceding baking, and exposure is developed, and cleans post bake.
Secondly, measure glass at H
2O: HF=20: the erosion rate in 1.We measure is 2970A/5min, that is to say to be about 600A/min.And the thickness of the gold that steams generally also is about 500～600A.So we floated the good glass substrate of post bake 1 minute in 20: 1 HF, cleaning, drying Hou Zheng Chrome steams gold.
Then, in toluene solution, photoresist material is removed, thereby lead-in wire has been stayed by vibration.
3. chip join.
This step is a last step of chip manufacturing, also is a step of most critical.For glass and glass bond, general available heat engages, and also can use anodic bonding.We adopt thermal bonding.Thermal bonding generally can be divided into two kinds: water-wetted surface engages and hydrophobic surface engages.Water-wetted surface is that the surface has OH
-, and OH
-Polarization.So,, will be attracted as water molecules for the bipolarity molecule.And there is not the polarity particulate in hydrophobic surface, so water molecules will can not be attracted.
For water-wetted surface, mainly be OH
-Between the hydrogen bond set up impel to engage and form; For hydrophobic surface, mainly be the H atom that exists, it stoped the generation of oxide on surface in several hours; And the Van der Waals force between H atom and the H atom has impelled the formation that engages.
Concrete making step:
(1) washing.At first be placed in the acetone soln and clean, this mainly is a cleaning organic matter impurity.Then can be with methyl alcohol or the residual acetone of ethanol scrub.Then cover glass and glass substrate are placed on H
2SO
4: H
2O
2Be about heated wash in 4: 1 the scavenging solution.Because for the joint of glass and glass, hydrophilic group OH
-And nonessential, so can not carry out the hydrolysis on surface.Certainly, as previously mentioned, in order to help to engage, we have used NH
4The OH heating is as surface hydrolysis, so that it produces hydrophilic group OH
-
(2) vacuum.A spot of moisture content is to help to engage, but too much water molecules can hinder joint.So before adding thermal bonding, vacuumize as much as possible at the moisture content between the glass that will contact and take out.On the one hand being the destruction of anti-sealing, also is to utilize normal atmosphere will need two blocks of glass engaging lumping together tightly on the other hand, and fully contact.
(3) thermal bonding.Make glass produce state of " soft " as much as possible, just suitable will raise temperature a bit, but can not make glass melting again.In one embodiment of the invention, adopted at 615～620 ℃ and annealed half an hour down, naturally cooling presses weight simultaneously and promotes to engage on junction of glass.
On detected result, it is fine totally to engage effect by the chip of above-mentioned thermal bonding method, and all passages and electrode all reach design requirements.The detection system that the present invention adopts is integrated little three-electrode system, the signal of measuring be the electrochemical parameter that produces of specific recognition reaction (as electric current, current potential, electricity lead, electric capacity or impedance etc.) variation, in whole mensuration process, need not to add tagged compound, therefore, the present invention is a kind of unmarked direct detecting method, thereby presented sensitivity and the selectivity higher with comparing of present method, simultaneously this collection multichannel sample introduction, separate and be detected on the microchip analysis system of one can high-level efficiency, high-throughput ground analysis several samples.
The present invention also provides the application of above-mentioned microchip, is about to chip and is connected with operating device, adds parting liquid and sample, and control voltage is finished separation, analysis and the detection of sample.Its principle is, under effect of electric field, utilizes the difference of each composition mobility in damping fluid in the sample and realizes separating, and the measured ion for the treatment of in the sample after the separation detects by the detection cell that is located at microchip one end successively.Many sample intake passages and many split tunnels have guaranteed the high-throughput performance of this chip.
Concrete detection step is as follows:
1) three electrode extraction pole on all chips (14,9,10) is connected to the corresponding work electrode connection of electrochemical analyser (as C8-1040, Shanghai occasion China instrument company), to utmost point wiring and reference electrode Wiring port.
2) the making alive wiring of micro-fluidic chip high-voltage power supply (as CDY-500L, instrument institute of Chemical Inst., Shandong Prov.) is connected the extraction pole (6,7 and 8) of parting liquid injection port, liquid injection port to be measured and detection cell respectively.
3) the method setting and the parameter setting of electrochemical analyser are set on demand.
4) voltage applying mode of micro-fluidic chip high-voltage power supply is set in the sequence of control of CDY-500L on demand.
5) get pre-configured dissociating buffer and go into parting liquid injection port 1 with the micro-creep infusion, open the separation voltage working procedure that CDY-500L sets, parting liquid enters split tunnel d1-d8 with speed slowly.After liquid to be separated is full of passage and enters detection cell (3), with microsyringe analyzed sample is injected liquid injection port to be measured (2), open the sample introduction voltage working procedure that CDY-500L sets, sample will slowly enter sample intake passage and intersect with each sense channel and converge, and separated damping fluid is brought detection cell (3) into.Open the operating software of having set the C8-1040 that finishes, the sample that enters detection cell is measured.
The present invention can be used for separating, analyzing and detect the sample of industries such as chemical industry, biology, medicine.Can pipe blow-through after chip of the present invention uses so that reuse.
Description of drawings
Fig. 1 is multiple tracks sample introduction, separation, detected set moulding analysis chip design diagram.Wherein: 1 is that parting liquid injection port, 2 is liquid injection port to be measured, 3 is detection cell, 4 is that split tunnel, 5 is that sample intake passage, 6 is that the alive electrode of electroosmotic flow, 7 that is used for the parting liquid injection port is that the alive electrode of electroosmotic flow, 8 that is used for liquid injection port to be measured is the counter electrode outlet line that is used for the sample signal detection, 9 for being used for the working electrode outlet line that sample signal detects, and 10 for being used for the reference electrode outlet line that sample signal detects.
Fig. 2 is used for the three-electrode system synoptic diagram that sample signal detects.11 are working electrode: diameter is 0.5～0.8mm, golden film thickness 200-400nm; 12 is counter electrode: diameter 0.7～0.9mm, golden film thickness 200～400nm; 13 is silver-colored reference electrode: diameter 0.7～0.9mm, and bottom gold film thickness 150～350nm adds surperficial 50nm silverskin; 14 is the counter electrode outlet line, and 9 is the working electrode outlet line, and 10 is the reference electrode outlet line.Three outlet lines are of a size of: long 2～3mm, wide 0.2～0.3mm, the golden film of thickness 200～400nm.
Fig. 3 is the floor map of B sheet surface leads and passage.Wherein, 4 is that split tunnel, 5 is that sample intake passage, 6 is that the alive electrode of electroosmotic flow, 7 that is used for the parting liquid injection port is that the alive electrode of electroosmotic flow, 14 that is used for liquid injection port to be measured is the counter electrode outlet line that is used for the sample signal detection, 9 for being used for the working electrode outlet line that sample signal detects, 10 for being used for the reference electrode outlet line that sample signal detects, and 15 is connection electrode and passage lead-in wire.
Embodiment
The making of embodiment 1A sheet
The A sheet is made the making that comprises parting liquid injection port, liquid injection port to be measured and detection cell, and making processes is:
(1) clean: get a 4cm*3.5cm, thickness is 0.1 millimeter sheet glass, soaks 12 hours in Chrome acid, takes out the back and rinses well with deionized water.Under infrared lamp, dry, put into temperature then and be 120 ℃ baking oven baking 15 minutes, remove surface-moisture comprehensively, be unlikely to when making gluing to come unstuck.
(2) gluing.Adopt negative glue, use rotation gluing mode twice, protective glass sheet surface is not corroded by HF as much as possible.
(3) preceding baking is 20 minutes, 75 ℃.
(4) exposure: in order to guarantee the quality of exposure imaging, we fill up the extinction paper of a black and have reduced the time shutter under sheet glass before common step of exposure.
(5) develop.In developing solution, developed 8 minutes, in scavenging solution, cleaned 1.5 minutes then.
(6) post bake.Allow earlier before the post bake and clean the sheet glass that finishes and dry naturally, avoid water evaporation when carrying out the high temperature post bake and make photoresist material come unstuck.The post bake time is 30 minutes.
(7) corrosion: we are chosen among the 40% dense HF corroded 10 minutes under 40 ℃.In addition, at the back side that does not have resist coating, we protect with black wax or yellow wax.
A sheet glass surface tissue comprises parting liquid injection port, liquid injection port to be measured and detection cell.Comprising: 8 parting liquid injection ports (1), being respectively diameter is that 1mm, height are the circular hole of A sheet thickness; 6 liquid injection ports to be measured (2), being respectively diameter is that 1mm, height are the circular hole of A sheet thickness; 8 detection cells (3), being respectively diameter is that 3mm, height are the circular hole of A sheet thickness.
The making of embodiment 2B sheet
(1) cleans.Get a 4cm*3.5cm, thickness is 1 millimeter substrate glass, is placed on earlier and removes coating in the vitriol oil; Soaked 12 hours in the Fang Zhi Zai Chrome acid then, take out and in deionized water, rinse well; Subsequently, be placed on to shine under the infrared lamp and do, put 120 ℃ of baking ovens again into and remove the surface adsorption moisture content.
(2) be coated with negative glue.Preceding baking 20 minutes.
(3) exposure is developed, and cleans post bake.This process is identical with making A sheet.
(4) corrosion: 40 ℃ corroded 4 minutes down in 40% dense HF.
(5) etch after, under the flushing of deionized water, brush the corrosive reaction product with brush, sheet glass is rinsed well, prevent the extra corrosion that remaining HF causes.
(6) Zheng Chrome gold forms electrode and lead-in wire.Its detailed process is as follows:
At first, clean the sheet glass of having carved groove, mode by hand is at the thick photoresist material of the zone of groove coating last layer again.Oven dry is coated with positive glue, preceding baking, and exposure is developed, and cleans post bake.
Secondly, the glass substrate that post bake is good has been floated in 20: 1 HF 1 minute, cleaning, drying Hou Zheng Chrome steams gold.
Then, in toluene solution, photoresist material is removed, thereby lead-in wire has been stayed by vibration.
The B sheet that obtains at last as shown in Figure 3.The B sheet has 4 * 8+8+6=46 leading foot, adopts 300nm Hou De Chrome gold copper-base alloy.Leading foot is respectively the alive electrode of electroosmotic flow (6) of parting liquid injection port correspondence, the alive electrode of the electroosmotic flow of liquid injection port to be measured (7); Be used for the alive electrode of detection cell electroosmotic flow (8) and be used for the three-electrode system that sample signal detects.Every electrode is of a size of long 5mm, wide 1mm.The B chip architecture also comprises various electrodes, lead-in wire, split tunnel (4), sample intake passage (5).Transverse passage-way is for being the test sample transfer passage, and perpendicular passage is the damping fluid transfer passage.Be used for three-electrode system that sample signal detects as shown in Figure 2.11 are working electrode: diameter is 0.6mm, golden film thickness 300nm; 12 is counter electrode: diameter 0.9mm, golden film thickness 300nm; 13 is silver-colored reference electrode: diameter 0.9mm, and bottom gold film thickness 250nm adds surperficial 50nm silverskin; 14 is the counter electrode outlet line, and 9 is the working electrode outlet line, and 10 is the reference electrode outlet line.Three outlet lines are of a size of: long 3mm, wide 0.2mm, the golden film of thickness 300nm.
The joint of embodiment 3A sheet and B sheet
(1) washing.At first, be placed in the acetone soln and clean.Then, can be with methyl alcohol or the residual acetone of ethanol scrub.Subsequently, cover glass and glass substrate are placed on H
2SO
4: H
2O
2Be about heated wash in 4: 1 the scavenging solution.In order to help to engage, we have used NH
4The OH heating is as surface hydrolysis.
(2) vacuum.A spot of moisture content is to help to engage, but too much water molecules can hinder joint.So before adding thermal bonding, vacuumize as much as possible at the moisture content between the glass that will contact and take out.
(3) thermal bonding.Employing is annealed half an hour down at 615～620 ℃, and naturally cooling presses weight simultaneously and promotes to engage on junction of glass.
The application of embodiment 4 microchips: measure plumbum ion concentration the time in many swimming lanes
(1) three electrode extraction pole on the chip (promptly 14,9,10) is connected to corresponding 8 the working electrode wiring, 8 of 8 passage electrochemical analysers (as C8-1040, Shanghai occasion China instrument company) to utmost point wiring and 8 reference electrode Wiring ports.The extraction pole (6,7 and 8) that the making alive wiring (totally 22) of micro-fluidic chip high-voltage power supply (as CDY-500L, instrument institute of Chemical Inst., Shandong Prov.) is connected parting liquid injection port, liquid injection port to be measured and detection cell respectively.
(2) the method setting and the parameter setting of 8 passage electrochemical analysers are set on demand, the pre-concentration voltage Vi (0.5～-0.8V vs.Ag/AgCl) that comprises working electrode galvanic deposit lead ion in each detection cell, reverse stripping sweep velocity v (0.01V/s 0.05V/s), sweep stopping point voltage Vh (0.2V～-0.1V) voltage scan range, the sweep trigger amplitude (30～60mV), scanning pulsewidth (0.03～0.07s); The voltage applying mode of micro-fluidic chip high-voltage power supply set in the sequence of control of CDY-500L on demand (be generally 200～300V); Get pre-configured dissociating buffer and go into parting liquid injection port (1) with 8 passage micro-creep infusions, open the separation voltage working procedure that CDY-500L sets, parting liquid enters split tunnel (4) with speed slowly.
(3) after liquid to be separated is full of passage and enters detection cell (3), with microsyringe analyzed sample (the lead ion standardized solution of different concns) is injected liquid injection port to be measured (2), open the sample introduction voltage working procedure that CDY-500L sets, sample slowly enters sample intake passage and intersects with each sense channel and converges, and separated damping fluid is brought detection cell (3) into.Open the operating software of having set the C8-1040 that finishes, the sample that enters 8 detection cells is measured.
(4) through a series of optimization, the experiment condition of detection cell is: electroplated 120 seconds under control operating voltage-0.7v condition, after static 15 seconds, reverse scan is to-0.2v.Sweep velocity 0.02V/s wherein, pulse amplitude amp=0.05V, pulsewidth pw=0.05s.At the lead ion of liquid injection port to be measured (2) sample introduction different concns, can obtain 8 groups of good standardized solution stripping peaks in 8 detection cells.Measure the peak current at these stripping peaks, can set up the pairing linear dependence relation of peak current and plumbum ion concentration, thereby reach the concentration of lead ion to be measured in a plurality of swimming lanes of rapid detection.The result shows, sample introduction 5 * 10
-5The lead ion of the normal concentration of mg/L, the average detected concentration that obtains of 8 detectors is 4.97 ± 0.15 * 10
-5Mg/L, relative standard deviation is 3.1%; Sample introduction 8 * 10
-4The lead ion of the normal concentration of mg/L, the average detected concentration that obtains of 8 detectors is 7.96 ± 0.14 * 10
-5Mg/L, relative standard deviation is 1.8%.
Claims (9)
1. a microchip is characterized in that, it is made up of passage lead-in wire and electrode between parting liquid injection port and microelectrode reaction tank, detection liquid injection port, each injection port and the reaction tank; Parting liquid injection port and microelectrode reaction tank are corresponding one by one, are in the two ends of separating pipe respectively, are communicated by separating pipe; Detecting the liquid injection port communicates with each root separating pipe by sample channel; But each injection port links to each other with the electrode of an external voltage operating device respectively; All contain a three-electrode system of forming by counter electrode, working electrode and reference electrode in each microelectrode reaction tank.
2. microchip as claimed in claim 1 is characterized in that, it by glass as matrix.
3. microchip as claimed in claim 1 is characterized in that, links to each other with electrode by lead-in wire between injection port, detection cell and external operating device.
4. microchip as claimed in claim 2 is characterized in that, it is to be formed by two glass bond, and one contains injection port and test pool hole, the electrode that another piece contains the passage between each injection port and the reaction tank and links to each other with injection port.
5. microchip as claimed in claim 4 is characterized in that, the thickness of glass that contains injection port and test pool hole is the 90-200 micron, and contains the passage between each injection port and the reaction tank and the thickness of the glass of the electrode that links to each other with injection port is the 1-2 millimeter.
6. preparation method of microchip according to claim 1 is characterized in that it may further comprise the steps:
(1) on a matrix, produces each injection port and reaction tank mouth;
(2) on another piece matrix, produce passage and embedding lead-in wire and electrode between each injection port and the reaction tank;
(3) merge two matrix.
7. the preparation method of microchip according to claim 1 is characterized in that, it by glass as matrix.
8. preparation method as microchip as described in the claim 7, it is characterized in that, when making each injection port, reaction tank mouth and passage, carry out photoetch with ultraviolet ray, and on the glass sides pad relative with ultraviolet light source a black light-absorbing paper, to reduce diffuse-reflectance.
9. the application of microchip according to claim 1 is characterized in that, chip is connected with operating device, adds parting liquid and sample, and control voltage is finished separation, analysis and the detection of sample.
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| CN1991356B (en) * | 2005-12-31 | 2010-11-10 | 博奥生物有限公司 | Multiple-pass capillary tube electrophoresis chip and voltage control method thereof |
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| CN101343656B (en) * | 2008-08-22 | 2013-03-06 | 重庆大学 | Cell separation microchip based on silicon structure on insulators |
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| CN103424447B (en) * | 2013-08-22 | 2015-05-13 | 中国科学院上海微系统与信息技术研究所 | Nano-particle enhancement detection device based on non-modified monolayer graphene being used as working electrode and application thereof |
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| CN1243109C (en) | 2006-02-22 |
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