CN1712926A - Micro-flow controlling chip for analyzing single cell algae flow - Google Patents
Micro-flow controlling chip for analyzing single cell algae flow Download PDFInfo
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- CN1712926A CN1712926A CN 200510043854 CN200510043854A CN1712926A CN 1712926 A CN1712926 A CN 1712926A CN 200510043854 CN200510043854 CN 200510043854 CN 200510043854 A CN200510043854 A CN 200510043854A CN 1712926 A CN1712926 A CN 1712926A
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Abstract
A mini flow control chip of mono-cell alga flow analysis is prepared by having substrate and cover plate in symmetric structure, setting psi form liquid flow system formed by intermediate sample and sheath channels as well as setting fibre channel on substrate and cover plate, setting fibre channel to lead trigger light to detecting region of separation on chip and setting optical detecting system above detecting region of sample feeding channel to detect fluorescent light and scattering light and then analyzing lights for counting and classifying alga .
Description
Technical field
The present invention relates to a kind of micro-fluidic chip that is applicable to the marine unicellular algae detection that utilizes the flow cytometer principle design to make.
Background technology
Micro-total analysis system (Micro-total analysis system, μ-TAS) proposition of notion has produced significant impact in the 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.Micro-fluidic chip system has efficiently, low consumption, microminiaturization and integrated characteristics, is suitable for all kinds of on-the site analysiss and The real time measure.Nineteen ninety-five Mathies and Woolley adopt little electrophoresis chip to carry out dna sequencing research first, on the passage of effective separation length 3.5cm, and about 150 bases of order-checking in the 10min, accuracy rate 97%.The microchip gene alaysis system of functions such as integrated cell on micro-fluidic chip such as Ramsey in 1998 is cleared up, pcr amplification and electrophoretic separation.
The principle of work of flow cytometer is: will make single cell suspension behind the cell dyeing to be measured, with certain pressure testing sample is pressed into flow chamber, not celliferous phosphate buffer under high pressure sprays from the sheath fluid pipe, sheath fluid tube inlet direction and testing sample stream are angled, like this, sheath fluid just can hold the sample flow at high speed, forms a fluid stream of a circle, cell to be measured single file under the bag quilt of sheath fluid is arranged, and passes through surveyed area successively.
Use flow cytometer to detect miniature phytoplankton and just adopted by more and more researchers, Paau etc. and Yentsch etc. have done correlative study, have promoted the utilization of flow cytometry in the miniature phytoplankton in ocean detects.Flow cytometer usually with laser as light emitting source.Through the light beam after the focusing shaping, vertical irradiation is on sample flow, and pigment and other materials produce scattered light and fluorescence excitation in the cell body under the irradiation of laser beam.The forward angle light scatter light intensity is relevant with the size of cell.The particle properties that the measurement of side scattered light is mainly used to obtain cells involved internal fine structure for information about.The specific wavelength fluorescence signal intensity has been represented the composition and the content of the pigment in the single-cell algae of surveying.But at present, flow cytometer costs an arm and a leg, and complex structure is not suitable for on-the site analysis, and the rig-site utilization that appears as Flow Cytometry of micro-fluidic chip provides may.
Summary of the invention
The purpose of this invention is to provide a kind of miniaturely, portable and be suitable for the micro-fluidic chip of on-the site analysis, it can be counted and the population composition analysis marine unicellular algae.
The present invention is based on standard photolithography techniques, is the micro-fluidic chip material with glass, makes up microchannel and little liquid storage tank on chip, makes the sample introduction of sample, detection be integrated on the chip and finish.Micro-fluidic chip comprises the substrate and the cover plate of structural symmetry, the optical-fibre channel that is built with liquid fluid system and is used to detect on substrate and cover plate.Liquid fluid system adopts ψ type structure, center-aisle is a sample channel, the subchannel, both sides is called the sheath circulation road, the effect of sheath circulation road is that the frustule in the sample is arranged with single file, sample channel and sheath circulation road consistent size, and the sheath circulation road is 1/4 circular-arc, sample channel is consistent with sheath circulation road end points tangential direction, sheath stream converges in the same way with sample flow like this, avoids liquid stream to converge the generation vortex disturbance, has guaranteed the stability of liquid fluid system; The end of sample channel and sheath circulation road is built with liquid storage tank respectively, and the other end of sample channel also has a waste liquid pool; On chip, make up the optical-fibre channel vertical, make laser instrument coupled fiber level insert this passage, exciting light is incorporated near the surveyed area of split tunnel with sample intake passage.The position parallel with the incident light optical-fibre channel at the opposite side of sample intake passage makes up optical-fibre channel, is used for forward scattering light is introduced the respective detection device.Above chip sample intake passage monitored area, settle the optical detection system, detect fluorescence and side scattered light, when the frustule process detects mouth, be subjected to laser radiation, produce fluorescence (yellow fluorescence, red fluorescence, green fluorescence) and scattered light (forward scattering and lateral scattering), by analysis-by-synthesis, algae is counted and classifies several light signals.This chip has saved three-dimensional collimation, the reflection that just can be finished by a plurality of optical elements and has assembled the complex optical path system, the loss of strength in the optical transmission process is dropped to minimum, has realized microminiaturization, portability and the integrated characteristics of micro-fluidic chip system.
The present invention has embodied the microminiaturization of micro-fluidic chip system and integrated characteristics with the flow cytometer liquid fluid system and the optical detection system integration, is suitable for all kinds of on-the site analysiss and The real time measure.Be widely used in fields such as single-cell algae, cultured cell, blood cell.
The design and the method for making of micro-fluidic chip of the present invention are as described below:
A. will make mask according to the graphics chip of microfluidic chip structure design, 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 gateway.
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, 30 minutes time; Rise to 610 ℃, 30 minutes time with 20 ℃/min from 550 ℃; Rise to 635 ℃, 30 minutes time with 20 ℃/min from 610 ℃; Rise to 650 ℃, 6 hours time with 10 ℃/min from 635 ℃; Naturally cool to room temperature then.
The present invention has made up on the micro-fluidic chip that utilizes the flow cytometer ultimate principle to design and produce and has realized required liquid fluid system and the sense channel of Flow Cytometry, has realized the microminiaturization of single-cell algae flow cytometer showed system, integrated and simplification; Low cost of manufacture is easy to realize standardization and large-scale production.
Description of drawings
Fig. 1 is the structural representation of micro-fluidic chip of the present invention.
Wherein, 1-sample inlet; 2,3-sheath inflow entrance; The 4-waste liquid pool; The 5-sample channel; 6,7-sheath circulation road; 8-passage joint; 9-incident optical passage, 10-outgoing optical-fibre channel.
Fig. 2 is the structural representation of single-cell algae flow cytometer showed chip system.
Wherein, 11,12, the 13-micropump; 14,16-sheath stream; The 15-sample; 17,18, the 19-fluorescence detector; 20-lateral scattering photodetector; The 21-bandpass filter; The 22-dichroic filter; The 23-laser instrument, the 24-detecting device.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and by specific embodiment.
Embodiment 1: the structure of micro-fluidic chip
As shown in Figure 1, microfluidic chip structure divides two parts: liquid fluid system and detection system.Liquid fluid system adopts ψ type structure, center-aisle is a sample channel 5, the subchannel, both sides is sheath circulation road 6 and 7, and the effect of sheath circulation road is to make the frustule in the sample be single file arrangement, sample channel 5 and sheath circulation road 6 and 7 consistent size, the sheath circulation road is 1/4 circular-arc, sheath circulation road 6 and 7 consistent with sample channel 5 directions in the tangential direction of joint 8, like this, sheath stream converges in the same way with sample flow, avoid liquid stream to converge the generation vortex disturbance, guaranteed the stability of liquid fluid system; On chip, make up the optical-fibre channel 9,10 vertical, make laser instrument 23 coupled fiber levels insert this passage, exciting light is incorporated near the surveyed area of split tunnel with sample channel.The position parallel with the incident light optical-fibre channel at the opposite side of sample intake passage makes up optical-fibre channel, is used for forward scattering light is introduced detecting device 24.Above chip sample intake passage surveyed area, settle the optical detection system, fluorescence detector 17,18,19 and lateral scattering photodetector 20 receive the corresponding light signal respectively, when the frustule process detects mouth, be subjected to laser radiation, produce fluorescence (red fluorescence, yellow fluorescence, green fluorescence) and scattered light (forward scattering and lateral scattering), fluorescence and scattered light arrive the respective detection device by the selective transmission and the reflection of dichroic filter 22 and bandpass filter 21.By analysis-by-synthesis, algae is counted and classifies several light signals.This chip has saved three-dimensional collimation, the reflection that just can be finished by a plurality of optical elements and has assembled the complex optical path system, the loss of strength in the optical transmission process is dropped to minimum, has realized microminiaturization, portability and the integrated characteristics of micro-fluidic chip system.
Embodiment 2: the making of micro-fluidic chip
1. the making of glass substrate: the size design of microchannel on the mask film: fluid passage, 30 μ m; Optical-fibre channel, 60 μ m; Between optical-fibre channel and split tunnel apart from d:400 μ m.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, the channel size that records on the chromium plate is: fluid passage, 40 μ m; Optical-fibre channel, 70 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 6 hours more successively, promptly gets substrate.With miniature bench drill punching, drill bit is the diamond bit of 1mm, and the diameter in hole is liquid pool diameter 1mm.Microscopically is measured the microchannel size: microfluidic channel, last bottom width 160 μ m, following bottom width 50 μ m, the degree of depth 60 μ m; Optical-fibre channel, last bottom width 190 μ m, following bottom width 80 μ m, the degree of depth 60 μ m; Between optical-fibre channel and split tunnel apart from d:340 μ m; Other zones except that optical-fibre channel are protected with adhesive tape, continue the etching optical-fibre channel, reach bottom width 600 μ m up to channel size, following bottom width 80 μ m, the degree of depth 270 μ m; Between optical-fibre channel and split tunnel apart from d:120 μ m
2. the making of cover plate: identical with the substrate method for making, construct identically, but do not punch.
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, dry up the sealing of in super-clean environment, both being alignd then, bonding under the high temperature with nitrogen.Heating schedule is: rise to 550 ℃ with 40 ℃/min from room temperature, 30 minutes time; Rise to 610 ℃, 30 minutes time with 20 ℃/min from 550 ℃; Rise to 635 ℃, 30 minutes time with 20 ℃/min from 610 ℃; Rise to 650 ℃, 6 hours time with 10 ℃/min from 635 ℃.Naturally cool to room temperature then.Through microscopically observation, passage does not have distortion behind the bonding, and reaches sealing fully.Chip microchannel ovalize behind the bonding, sample channel major axis are about 190 μ m, and minor axis is about 120 μ m; The about 600 μ m of light channel major axis, the about 540 μ m of minor axis.
Embodiment 3: micro-fluidic chip analyzing and testing system
As shown in Figure 2, connect sampling pump 12 in the import 1 of sample channel 5, both sides sheath circulation road 6 and 7 connects sheath stream 14,16 at its inlet 2 and 3 places.Sample and sheath flow liquid are by pump 11,12 and 13 carry, sheath stream and sample co-flow, it is mobile forward with the form of laminar flow that sheath flow liquid and sample converge the back at point 8, by regulating sheath stream and sample flow rate, making cells in sample be single file arranges, and be advanced through detection zone, in of the laser excitation of surveyed area cell quilt from optical fiber, the optical fiber of passage opposite side is accepted forward scattering light, and it is transferred to fluorescence detector 24, the light of side direction is through the beam split of dichroic filter 22 and bandpass filter 21, filter, respectively by lateral scattering photodetector 20, red fluorescence detecting device 17, yellow fluorescence detecting device 18 and green fluorescence detecting device 19 receiving records, comprehensive all kinds of light signals obtain the information of miniature phytoplankton quantity and structure of community.
Claims (4)
1. micro-flow controlling chip for analyzing single cell algae flow, it is characterized in that it comprises the substrate and the cover plate of structural symmetry, on substrate and cover plate, be built with the liquid fluid system of the ψ type structure that comprises middle sample passage and both sides sheath circulation road and the optical-fibre channel that is used to detect.
2. micro-fluidic chip according to claim 1 is characterized in that described sheath circulation road is 1/4 circular-arc, and sample channel is consistent with sheath circulation road go side point tangential direction.
3. micro-fluidic chip according to claim 1 is characterized in that described optical-fibre channel and sample intake passage are orthogonal.
4. micro-fluidic chip according to claim 1 is characterized in that it is material with glass.
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| CN 200510043854 CN1712926A (en) | 2005-06-19 | 2005-06-19 | Micro-flow controlling chip for analyzing single cell algae flow |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7561267B2 (en) | 2006-09-30 | 2009-07-14 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Flow cytometer |
| CN100552422C (en) * | 2006-12-30 | 2009-10-21 | 清华大学 | Microfluidic chip device for multifunctional detection of single particulate matter |
| US7706476B2 (en) | 2006-11-27 | 2010-04-27 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Real-time digital quadrature demodulation method and device for ultrasonic imaging system |
| CN101226132B (en) * | 2007-11-15 | 2010-09-01 | 重庆大学 | Microchip and method for measuring rapidly cell size |
| CN102087198A (en) * | 2010-11-18 | 2011-06-08 | 苏州生物医学工程技术研究所 | Flow cytometry |
| CN102128776A (en) * | 2010-12-20 | 2011-07-20 | 无锡荣兴科技有限公司 | Method for detecting alga and coloured dissolved organic matters by adopting flow cytometry |
| CN102128777A (en) * | 2010-11-24 | 2011-07-20 | 西安交通大学 | 3D (Three Dimensional) micro-fluidic structure for cell detection and preparation method thereof |
| CN102128779A (en) * | 2010-12-20 | 2011-07-20 | 无锡荣兴科技有限公司 | Method for analyzing algae and chormophoric dissolved organic matters on line by non-external sheath fluid flow cytometry |
| CN102998234A (en) * | 2012-12-14 | 2013-03-27 | 江苏苏净集团有限公司 | Micro liquid grain counter chip |
| CN103105352A (en) * | 2013-01-28 | 2013-05-15 | 大连海事大学 | Device and method for rapidly detecting surviving unicellular organisms in ship ballast water |
| CN103616356A (en) * | 2013-11-25 | 2014-03-05 | 大连海事大学 | Device and method for classifying microalgae in ship ballast water |
| CN104677789A (en) * | 2015-03-05 | 2015-06-03 | 江苏苏净集团有限公司 | Nanoparticle counting detection device and method |
| CN104677788A (en) * | 2015-03-05 | 2015-06-03 | 江苏苏净集团有限公司 | Liquid particle counting detection device and method |
| CN105717047A (en) * | 2016-01-28 | 2016-06-29 | 中国科学院重庆绿色智能技术研究院 | Flowing sample pool device for optically detecting cell monolayer |
| CN106104254A (en) * | 2013-12-18 | 2016-11-09 | 汉迪恩公司 | In order to characterize the chip assembly of granule, flow chamber and flow cytometer |
| CN108627448A (en) * | 2018-06-05 | 2018-10-09 | 江苏卓微生物科技有限公司 | The method of counting micro particles |
| CN110186836A (en) * | 2019-06-21 | 2019-08-30 | 山东师范大学 | The optofluidic flow cytometer of circulating tumor cell separation analysis and Classification Count |
| CN113941377A (en) * | 2021-10-11 | 2022-01-18 | 北京理工大学 | All-glass microfluidic chip and processing method |
-
2005
- 2005-06-19 CN CN 200510043854 patent/CN1712926A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7561267B2 (en) | 2006-09-30 | 2009-07-14 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Flow cytometer |
| US7706476B2 (en) | 2006-11-27 | 2010-04-27 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Real-time digital quadrature demodulation method and device for ultrasonic imaging system |
| US8179997B2 (en) | 2006-11-27 | 2012-05-15 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd | Real-time digital quadrature demodulation method and device for ultrasonic imaging system |
| CN100552422C (en) * | 2006-12-30 | 2009-10-21 | 清华大学 | Microfluidic chip device for multifunctional detection of single particulate matter |
| CN101226132B (en) * | 2007-11-15 | 2010-09-01 | 重庆大学 | Microchip and method for measuring rapidly cell size |
| CN102087198A (en) * | 2010-11-18 | 2011-06-08 | 苏州生物医学工程技术研究所 | Flow cytometry |
| CN102128777A (en) * | 2010-11-24 | 2011-07-20 | 西安交通大学 | 3D (Three Dimensional) micro-fluidic structure for cell detection and preparation method thereof |
| CN102128776A (en) * | 2010-12-20 | 2011-07-20 | 无锡荣兴科技有限公司 | Method for detecting alga and coloured dissolved organic matters by adopting flow cytometry |
| CN102128779A (en) * | 2010-12-20 | 2011-07-20 | 无锡荣兴科技有限公司 | Method for analyzing algae and chormophoric dissolved organic matters on line by non-external sheath fluid flow cytometry |
| CN102998234B (en) * | 2012-12-14 | 2015-03-25 | 江苏苏净集团有限公司 | Micro liquid grain counter chip |
| CN102998234A (en) * | 2012-12-14 | 2013-03-27 | 江苏苏净集团有限公司 | Micro liquid grain counter chip |
| CN103105352A (en) * | 2013-01-28 | 2013-05-15 | 大连海事大学 | Device and method for rapidly detecting surviving unicellular organisms in ship ballast water |
| CN103616356A (en) * | 2013-11-25 | 2014-03-05 | 大连海事大学 | Device and method for classifying microalgae in ship ballast water |
| CN103616356B (en) * | 2013-11-25 | 2015-12-30 | 大连海事大学 | The sorter of micro-algae and sorting technique in a kind of ballast water for ship |
| CN106104254B (en) * | 2013-12-18 | 2019-06-07 | 深圳市芯凯瑞生物科技有限公司 | To characterize the chip assembly, flow chamber and flow cytometer of particle |
| CN106104254A (en) * | 2013-12-18 | 2016-11-09 | 汉迪恩公司 | In order to characterize the chip assembly of granule, flow chamber and flow cytometer |
| CN104677789A (en) * | 2015-03-05 | 2015-06-03 | 江苏苏净集团有限公司 | Nanoparticle counting detection device and method |
| CN104677788A (en) * | 2015-03-05 | 2015-06-03 | 江苏苏净集团有限公司 | Liquid particle counting detection device and method |
| CN104677788B (en) * | 2015-03-05 | 2017-05-03 | 江苏苏净集团有限公司 | Liquid particle counting detection method |
| CN105717047A (en) * | 2016-01-28 | 2016-06-29 | 中国科学院重庆绿色智能技术研究院 | Flowing sample pool device for optically detecting cell monolayer |
| CN108627448A (en) * | 2018-06-05 | 2018-10-09 | 江苏卓微生物科技有限公司 | The method of counting micro particles |
| CN110186836A (en) * | 2019-06-21 | 2019-08-30 | 山东师范大学 | The optofluidic flow cytometer of circulating tumor cell separation analysis and Classification Count |
| CN110186836B (en) * | 2019-06-21 | 2022-04-01 | 山东师范大学 | Optofluidic flow cytometer for separating, analyzing and typing counting circulating tumor cells |
| CN113941377A (en) * | 2021-10-11 | 2022-01-18 | 北京理工大学 | All-glass microfluidic chip and processing method |
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Open date: 20051228 |