CN1250962C - Preparing method of integrated capillary electrophoresis electrochemical luminescence detecting chip - Google Patents
Preparing method of integrated capillary electrophoresis electrochemical luminescence detecting chip Download PDFInfo
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- CN1250962C CN1250962C CN 200310115822 CN200310115822A CN1250962C CN 1250962 C CN1250962 C CN 1250962C CN 200310115822 CN200310115822 CN 200310115822 CN 200310115822 A CN200310115822 A CN 200310115822A CN 1250962 C CN1250962 C CN 1250962C
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Abstract
The present invention belongs to the fabrication of an integrated capillary electrophoresis electrochemical luminescence detecting chip, which adopts polydimethyl siloxane (PDMS)/glass as chip material and conductive glass with an indium-tin oxide (ITO) coating layer as a working electrode which is manufactured by a photoetching method, wherein the whole chip is formed by the reversible bonding of the working electrode as a bottom board of the chip, and a PDMS layer. The integrated capillary electrophoresis electrochemical luminescence detecting chip has the advantages of simple manufacture process and easy operation, and furthermore, the cost of the chip is reduced. The integrated capillary electrophoresis electrochemical luminescence detecting chip can be widely applied to the detection and the analysis of medicine molecules containing amine, multiple amino acids, alkylamine, oxalic acid and salts thereof, and NADH.
Description
Technical field
The invention belongs to the preparation method of integrated capillary electrophoresis electrochemical light-emitting detection chip.
Technical background
In recent years, micro-total analysis system more and more is subject to people's attention.It is to utilize micro-processing technology to make the analytic system that functional units such as microchannel, micro reaction pool, microreactor constitute on chip, in this system, can carry out chemistry experiment operatings such as the concentrating of sample, separation, reaction and detection, be about to whole chemical analysis process and from the sample introduction to the detection, be integrated into a microsystem, carry out automatic express-analysis.Chip capillary cataphoresis as a pith of micro-analysis system have that analysis speed is fast, quantity of information is high, operation cost is low, sample consumes outstanding advantage such as little and paid close attention to by people always.But, be applicable to that the progress of miniature detection system of chip capillary cataphoresis is slower.At present main use laser-induced fluorescence (LIF) (LIF) detect (Anal.Chem.1997,69,3407-3412), but the required pick-up unit volume of this detection mode is big, cost is high, still is difficult to mate with microchip.And in a large amount of analytes, a lot of materials do not have fluorophor, must just can carry out the LIF detection through behind the deriving of complex operation.It is a kind of very sensitive detection technique based on galvanochemistry and spectral technique that the tris (bipyridine) ruthenium electrochemiluminescence detects, and can be used for the detection of multiple materials such as drug molecule, amino acid, polypeptide, protein and nucleic acid.Its advantage is the detection sensitivity height, and the range of linearity is wide, favorable reproducibility, required instrument and equipment simple economy.Manz once reported chip capillary cataphoresis indirect electrochemical luminous detection (Anal.Chem.2001,73,3282-3288), adopt glass as chip material, platinum is working electrode.But this kind chip and electrode complex manufacturing process, to the equipment requirements height, system stability is poor, is indirect detection in addition, has all limited the application in practice of this system.Fang reported once also that short distance quartz capillary electrophoresis electrochemical light-emitting detected (Anal.Chim.Acta2002,456,167-175), but the capillary electrophoresis chip of real meaning can't be calculated by this system, its working electrode is the platinum electrode that adds, light signal is sent to photomultiplier by the light transmitting fiber that adds and detects, and operating process is loaded down with trivial details, time-consuming, and total system is complicated and do not accomplish integrated.
Summary of the invention
The purpose of this invention is to provide a kind of integrated capillary electrophoresis electrochemical light-emitting detection chip.
Another object of the present invention provides the preparation method of this integrated capillary electrophoresis electrochemical light-emitting detection chip.
The 3rd purpose of the present invention provides the application of this integrated capillary electrophoresis electrochemical light-emitting detection chip.
For achieving the above object, the present invention adopts dimethyl silicone polymer (PDMS)/glass as chip material, and the electro-conductive glass that has indium tin oxide (ITO) coating is a working electrode.Whole chip is positioned in the magazine, and light inlet window is arranged at magazine bottom, and light signal can be caught by photomultiplier by light inlet window, and outputs on the computing machine and handle.
Preparation process is as follows:
Making the glass template of PDMS passage is made by photoetching process, the chromium glass that employing scribbles photoresist is starting material, the transparent film that have channel pattern of mask that photoetching is required for printing, by photoetching with graphic pattern projection to the optical cement layer, then with developing liquid developing and except that dechromising layer, with 1.7%: 1.3% HF/NH
4The F glass erosion liquid to glass corrosion 15-30 minute, is produced the glass template; The PDMS monomer is mixed in 10: 1 ratio with hardening agent, through the vacuum pump degassing, be poured into then on the glass template, heating was solidified in 1-2 hour in 60-80 ℃ of baking oven; After treating full solidification, the PDMS layer is peeled from the glass template; The liquid cell of chip punches on the PDMS layer with stainless-steel tube and makes, and the chip electrophoresis path 10-20 μ m of gained is dark, and 20-50 μ m is wide at the top, and 30-60 μ m is wide in the bottom.
The chip base plate adopts the ITO electro-conductive glass to make, at first on the electro-conductive glass of well cutting, coat optical cement, the optical cement amount is 3-10 μ L, optical cement can apply with roller coating or spin coating mode, rotate 50-100 second down at 1000-4000 rev/min then, with the printed film that has electrode pattern is that mask carries out photoetching, uses developing liquid developing then, with the FeCl of mol ratio＞1: 3
3The HNO of/HCl or mol ratio＜1: 2
3/ HCl mixed solution corrodes 30-60 second to the ITO of glass surface, removes unnecessary optical cement at last, and ITO working electrode and chip base plate have just completed, and working electrode live part width is 50-200 μ m.
Use detersive, deionized water, absolute ethyl alcohol to ready-made battery lead plate and the ultrasonic cleaning of PDMS layer 3-10 minute successively, under hair-dryer, dry up and keep the cleaning surfaces of battery lead plate and PDMS layer then, at microscopically battery lead plate and the PDMS layer mode with reversible keying is bonded together, and the outlet that makes electrode and split tunnel is at a distance of 20-40 μ m.Entire chip as shown in Figure 1.
The chip capillary cataphoresis electrochemiluminescence detects and adopts three-electrode system, with the ITO electrode that is integrated on the chip base plate is working electrode, platinum filament is to electrode, the saturated KCl solution of Ag/AgCl/ is contrast electrode, wherein adopt the polystyrene plastics pipe to be integrated into integral body, detect with the styletable detection mode to the required earthing pole of electrode, contrast electrode and chip electrophoresis.Working electrode voltage 1.2V.It is 200-300V/cm that capillary electrophoresis separation adopts field intensity, and boric acid or phosphoric acid are damping fluid, the tris (bipyridine) ruthenium solution concentration 1-5mM in the detection cell.
This integrated capillary electrophoresis electrochemical light-emitting detection chip of the present invention design has the characteristics such as simple, easy to use, system stability, expense are cheap of making.A lot of materials can directly detect separation and not need to derive, and they comprise various alkyl amines, amino acid, oxalic acid and salt thereof, NADH and multiple nitrogenous medicine etc.
The present invention utilizes electrochemiluminescence to combine with the chip capillary cataphoresis technology as detection technique and the integrated capillary electrophoresis electrochemical light-emitting detection chip that designs.PDMS belongs to a kind of polymeric material, and it can repeat reversible deformation, is easy to carry out Precision Machining and need not super-clean environment with method of molding.PDMS also has good light transmittance, can see through the above ultraviolet light of 300nm.PDMS can also be with even curface reversible or irreversible being bonded together.As adopt reversible manner, when the passage of electrophoresis took place to block, entire chip can be taken cleaning apart, and was very convenient.ITO is a kind of transparent conductive material, is widely used in the liquid crystal display industry owing to have good electrical conductivity, light transmission and stability.Working electrode adopts method such as the photoetching technique, chemical corrosion of standard to be produced on the glass sheet and as the base plate of chip.Entire chip is formed by PDMS layer that has little raceway groove and electrode plate reversible keying.Because ITO is a kind of light transmissive material, be integrated into as working electrode on the base plate of chip and simplified operation greatly, improved the collecting efficiency of electrochemiluminescence signal.Electrochemiluminescence detects the styletable pattern that adopts.
Description of drawings
Accompanying drawing 2 is that ordinate is a signal intensity to continuous six the sample detection electrophoretograms of Ofloxacin, and horizontal ordinate is the time.
Accompanying drawing 3 is the electrophoretogram to continuous six sample detection of proline, and ordinate is a signal intensity, and horizontal ordinate is the time.
Accompanying drawing 4 is that ordinate is a signal intensity to the electrophoretogram of the separation detection of tripropyl amine (TPA) (a) and proline (b), and horizontal ordinate is the time.
Embodiment
Embodiment 1: the required mask of photoetching is the transparent film that has channel pattern by the laser printer output of high definition 3000dpi.It is on glass to cover the chromium plate that is coated with optical cement, through the uviol lamp exposure, with developing liquid developing and except that dechromising layer, cleans up then, places 1.7%: 1.3% HF/NH
4In the F glass erosion liquid, the glass part that exposes was corroded 15 minutes, corrosion finishes, and removes unnecessary optical cement and removes the layer that dechromises with 2% sodium hydroxide solution.The glass template is cleaned, and dries.The PDMS monomer is mixed in 10: 1 ratio with hardening agent, through the vacuum pump degassing, be poured into then on the glass template, heating was solidified in 2 hours in 60 ℃ of baking ovens.After treating full solidification, the PDMS layer is peeled from the glass template, with blade PDMS is cut into required chip form then.The liquid cell of chip punches on the PDMS layer with stainless-steel tube and makes.The chip electrophoresis path 10 μ m of gained are dark, and about 20 μ m are wide at the top, and about 30 μ m are wide in the bottom.
The chip base plate adopts the ITO electro-conductive glass to make.Electro-conductive glass is cut into the rectangle fritter of 7 * 2cm, be fixed on the spin coater, coat optical cement in an end distance isolated edge 1.5cm place under 250 rev/mins, the optical cement amount is 3 μ L.After being coated with, rotating 50 seconds down at 4000 rev/mins, is that mask carries out photoetching with the printed film that has electrode pattern, and wherein electrode width is 50 μ m.Using developing liquid developing then, is 1: 3 FeCl afterwards with mol ratio
3/ HCl mixed solution corroded 30 seconds the ITO of glass surface, removed unnecessary optical cement at last, and ITO working electrode and chip base plate have just completed.Working electrode live part width is 50 μ m.
Use detersive, deionized water, absolute ethyl alcohol to ready-made battery lead plate and the ultrasonic cleaning of PDMS layer 10 minutes successively, under hair-dryer, dry up and keep the cleaning surfaces of battery lead plate and PDMS layer then.At microscopically battery lead plate and the PDMS layer mode with reversible keying is bonded together, and the outlet that makes electrode and split tunnel is at a distance of 20 μ m.
The chip capillary cataphoresis electrochemiluminescence detects and adopts three-electrode system, is working electrode with the ITO electrode that is integrated on the chip base plate, and platinum filament is to electrode, and the saturated KCl solution of Ag/AgCl/ is contrast electrode.Wherein adopt the polystyrene plastics pipe to be integrated into integral body, detect with the styletable detection mode to the required earthing pole of electrode, contrast electrode and chip electrophoresis.Working electrode voltage 1.2V.It is 200V/cm that capillary electrophoresis separation adopts field intensity, and the phosphoric acid of 10mM pH8.0 is damping fluid, and the tris (bipyridine) ruthenium solution concentration 1mM in the detection cell, damping fluid are the 50mM phosphoric acid of pH8.0.Detect current potential 1.2V.Sample injection time 5 seconds, sample introduction field intensity are 250V/cm.To continuous six sample detection of 100 μ g/mL Ofloxacins as shown in Figure 2, relative standard deviation RSD＜3.0%.Detection to Ofloxacin is limited to 0.1 μ g/mL, and quality detects and is limited to 10
-17G.This shows that this integrated capillary electrophoresis electrochemical light-emitting detection chip has good sensitivity and reappearance to the detection of drug molecule.
Embodiment 2: the required mask of photoetching is the transparent film that has channel pattern by the laser printer output of high definition 3000dpi.It is on glass to cover the chromium plate that is coated with optical cement, through the uviol lamp exposure, with developing liquid developing and except that dechromising layer, cleans up then, places 1.7%: 1.3% HF/NH
4In the F glass erosion liquid, the glass part that exposes was corroded 20 minutes, corrosion finishes, and removes unnecessary optical cement and removes the layer that dechromises with 2% sodium hydroxide solution.The glass template is cleaned, and dries.The PDMS monomer is mixed in 10: 1 ratio with hardening agent, through the vacuum pump degassing, be poured into then on the glass template, heating was solidified in 1.5 hours in 70 ℃ of baking ovens.After treating full solidification, the PDMS layer is peeled from the glass template, with blade PDMS is cut into required chip form then.The liquid cell of chip punches on the PDMS layer with stainless-steel tube and makes.The chip electrophoresis path 15 μ m of gained are dark, and about 30 μ m are wide at the top, and about 45 μ m are wide in the bottom.
The chip base plate adopts the ITO electro-conductive glass to make.Electro-conductive glass is cut into the rectangle fritter of 7 * 2cm, be fixed on the spin coater, coat optical cement in an end distance isolated edge 1.5cm place under 250 rev/mins, the optical cement amount is 10 μ L.After being coated with, 3000 rev/mins of down rotations 100 seconds, being that the film of 200 μ m is that mask carries out photoetching with the printed electrode pattern width that has, using developing liquid developing then, is 1: 2 HNO afterwards with mol ratio
3/ HCl mixed solution corroded 30 seconds the ITO of glass surface, removed unnecessary optical cement at last, and ITO working electrode and chip base plate have just completed.Working electrode live part width is 200 μ m.
Use abluent, deionized water, absolute ethyl alcohol to ready-made battery lead plate and the ultrasonic cleaning of PDMS layer 5 minutes successively, under hair-dryer, dry up and keep the cleaning surfaces of battery lead plate and PDMS layer then.At microscopically battery lead plate and the PDMS layer mode with reversible keying is bonded together, and the outlet that makes electrode and split tunnel is at a distance of 40 μ m.
The chip capillary cataphoresis electrochemiluminescence detects and adopts three-electrode system, is working electrode with the ITO electrode that is integrated on the chip base plate, and platinum filament is to electrode, and the saturated KCl solution of Ag/AgCl/ is contrast electrode.Wherein adopt the polystyrene plastics pipe to be integrated into integral body, detect with the styletable detection mode to the required earthing pole of electrode, contrast electrode and chip electrophoresis.Working electrode voltage 1.2V.It is 300V/cm that capillary electrophoresis separation adopts field intensity, and the boric acid of 10mM pH9.2 is damping fluid, the tris (bipyridine) ruthenium solution concentration 5mM in the detection cell, and damping fluid is the boric acid of 50mM pH9.2. detect current potential 1.2V.Sample injection time 5 seconds, sample introduction field intensity are 200V/cm.To continuous six sample detection of 100 μ M prolines as shown in Figure 3, relative standard deviation RSD＜2.2%.Detection to proline is limited to 1.2 μ M/L, and absolute detection limit is 10
-19M.This shows that this integrated capillary electrophoresis electrochemical light-emitting detection chip has good sensitivity and reappearance to the detection of amino acid molecular.
Embodiment 3: the required mask of photoetching is the transparent film that has channel pattern by the laser printer output of high definition 3000dpi.It is on glass to cover the chromium plate that is coated with optical cement, through the uviol lamp exposure, with developing liquid developing and except that dechromising layer, cleans up then, places 1.7%: 1.3% HF/NH
4In the F glass erosion liquid, the glass part that exposes was corroded 30 minutes, corrosion finishes, and removes unnecessary optical cement and removes the layer that dechromises with 2% sodium hydroxide solution.The glass template is cleaned, and dries.The PDMS monomer is mixed in 10: 1 ratio with hardening agent, through the vacuum pump degassing, be poured into then on the glass template, heating was solidified in 1 hour in 80 ℃ of baking ovens.After treating full solidification, the PDMS layer is peeled from the glass template, with blade PDMS is cut into required chip form then.The liquid cell of chip punches on the PDMS layer with stainless-steel tube and makes.The chip electrophoresis path 20 μ m of gained are dark, and about 50 μ m are wide at the top, and about 60 μ m are wide in the bottom.
The chip base plate adopts the ITO electro-conductive glass to make.Electro-conductive glass is cut into 7 * 2cm rectangle fritter, be fixed on the spin coater, get 5 μ m optical cement roller coating at one end apart from edge 1.5cm place.After being coated with, 1000 rev/mins of down rotations 80 seconds, being that the film of 100 μ m is that mask carries out photoetching with the printed electrode pattern width that has, using developing liquid developing then, is 2: 1 FeCl afterwards with mol ratio
3/ HCl mixed solution corroded 60 seconds the ITO of glass surface, removed unnecessary optical cement at last, and ITO working electrode and chip base plate have just completed.Working electrode live part width is 100 μ m.
Use detersive, deionized water, absolute ethyl alcohol to ready-made battery lead plate and the ultrasonic cleaning of PDMS layer 3 minutes successively, under hair-dryer, dry up and keep the cleaning surfaces of battery lead plate and PDMS layer then.At microscopically battery lead plate and the PDMS layer mode with reversible keying is bonded together, and the outlet that makes electrode and split tunnel is at a distance of 30 μ m.
The chip capillary cataphoresis electrochemiluminescence detects and adopts three-electrode system, is working electrode with the ITO electrode that is integrated on the chip base plate, and platinum filament is to electrode, and the saturated KCl solution of Ag/AgCl/ is contrast electrode.Wherein adopt the polystyrene plastics pipe to be integrated into integral body, detect with the styletable detection mode to the required earthing pole of electrode, contrast electrode and chip electrophoresis.Working electrode voltage 1.2V.It is 250V/cm that capillary electrophoresis separation adopts field intensity, and the boric acid of 10mM pH9.2 is damping fluid, and the tris (bipyridine) ruthenium solution concentration 5mM in the detection cell, damping fluid are that the boric acid of 50mM pH9.2 detects current potential 1.2V.Sample injection time 5 seconds, sample introduction field intensity are 200V/cm.To the separation detection of 25 μ M tripropyl amine (TPA)s (a) and 50 μ M prolines (b) as shown in Figure 4, illustrate that this chip can finish the not separation detection of material of the same race at short notice.
Claims (3)
1, a kind of integrated capillary electrophoresis electrochemical light-emitting detection chip, chip structure comprises 1) the dimethyl silicone polymer layer; 2) base plate; 3) sample cell; 4) buffer pool; 5) detection cell; 6) working electrode; 7) split tunnel, it is characterized in that adopting dimethyl silicone polymer/glass as chip material, the electro-conductive glass that has the indium tin oxide coating is a working electrode, and working electrode adopts photoetching process to process on the chip base plate also and dimethyl silicone polymer layer reversible keying formed entire chip together.
2, according to the method for making working electrode on the integrated capillary electrophoresis electrochemical light-emitting detection chip base plate of claim 1, it is characterized in that the chip base plate adopts the electro-conductive glass that has the indium tin oxide coating to make, at first on the electro-conductive glass of well cutting, coat optical cement, the optical cement amount is 3-10 μ L, optical cement applies with roller coating or spin coating mode, rotate 50-100 second down at 1000-4000 rev/min then, with the printed film that has electrode pattern is that mask carries out photoetching, use developing liquid developing then, with the FeCl of mol ratio＞1: 3
3The HNO of/HCl or mol ratio＜1: 2
3/ HCl mixed solution corrodes 30-60 second to the indium tin oxide coating of glass surface, removes unnecessary optical cement at last, and working electrode live part width is 50-200 μ m.
3, the described integrated capillary electrophoresis electrochemical light-emitting detection chip of claim 1 is applied to contain the detection of amine drug molecule or is used for check and analysis to several amino acids, alkyl amine, oxalic acid and salt thereof or NADH.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1295508C (en) * | 2005-02-06 | 2007-01-17 | 中国科学院上海微***与信息技术研究所 | Low temperature binding method for glass microflow control chip |
| CN100425973C (en) * | 2006-03-30 | 2008-10-15 | 湖南大学 | Nano biological sensor for detecting NADH concentration and its detecting method |
| CN101148324B (en) * | 2007-09-14 | 2010-05-19 | 中国科学院上海微***与信息技术研究所 | Preparation method for cell cultivation chip based on ITO glass substance and application thereof |
| CN101424660B (en) * | 2007-10-31 | 2012-01-04 | 南京大学 | Design and production of portable highly effective capillary pipe electrophoresis chip sampling by chink |
| CN101716485B (en) * | 2009-11-05 | 2012-07-04 | 浙江大学 | Tapered quartz capillary tube-based micro-reactor |
| CN101819078B (en) * | 2010-04-30 | 2012-04-18 | 重庆大学 | Laminar flow based multichannel pressure detection chip and production and pressure detection method thereof |
| CN103499571B (en) * | 2013-10-14 | 2016-03-23 | 南京大学 | A kind of electrochemiluminescdetection detection device |
| CN107064116B (en) * | 2017-03-01 | 2019-05-31 | 浙江大学 | A kind of electrochemical luminescence biochemical detection system and method based on mobile phone USB-OTG interface |
| CN113289701A (en) * | 2021-05-21 | 2021-08-24 | 合肥工业大学 | Pump-free micro-fluidic chip for electrochemical detection and preparation method thereof |
| CN114414568A (en) * | 2022-01-07 | 2022-04-29 | 大连理工大学 | Microfluidic liquid crystal sensor capable of detecting organophosphorus pesticide in real time and detection method thereof |
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