CN101760428A - Microfluidic chip for enriching microorganisms in air and preparation method thereof - Google Patents
Microfluidic chip for enriching microorganisms in air and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioanalysis and detection, in particular to a microfluidic chip for enriching microorganisms in air and a preparation method thereof. The chip uses optically transparent polydimethylsiloxane, and the like as materials by adopting a molding method, and the chip internally contains a sample enriching channel and a gas channel to realize the collection of biologic grains of bacteria, fungi, viruses, and the like in an air sample. An enriched sample can be combined with a PCR (polymerase chain reaction) microfluidic chip and an immune microfluidic chip to carry out gene analysis or immunoassays so as to achieve the quick detection of the contents and the species of the microorganisms in the air. The invention has the characteristics of high speed, high efficiency, convenient taking, low price and easy automation control, and the integration of the microfluidic chip with other chips can complete automatic signal acquisition, remote transmission and signal analysis, thus the microfluidic chip can be used for the field quick detection and remote control detection within a large range on microorganisms in the air or other gases.
Description
Technical field
The invention belongs to bioanalysis detection technique field, be specifically related to a kind of micro-fluidic chip that is used for on-the-spot fast enriching air microorganism, and this chip production method is provided.
Background technology
The human in recent years stem reality that faces global disease transmission: new transmissible disease constantly occurs, old transmissible disease wreak havoc again and biologically attack, the people is that the transmissible disease that causes takes place and popular.The air aerosol is that the respiratory infectious disease of media has accounted for main positions in the New Development transmissible disease, is emphasis and difficult point problem in the transmissible disease control.Cause that the pathogenic agent that respiratory tract disease infects is the infection that causes the people by the ambient air aerosol transmission, compare easier bamboo telegraph diffusion in the crowd with the infectious disease pathogens that other approach are propagated and be difficult to control, especially densely populated, the city of heavy traffic often becomes the hidden danger that epidemic situation is broken out.The pathogenic agent that causes respiratory tract infection has virus, bacterium and other pathogenic agent (mycoplasma and chlamydozoan) etc.Wherein main pathogenic agent is a virus, has above 20 kinds of viruses and to cause respiratory tract disease in person to person or human-animal's propagation, and bacterial infection less than 25%.Cause the pathogenic agent of seasonal respiratory tract infection except influenza, parainfluenza, RSV and RV etc. are known, also constantly find new Respirovirus in recent years.Since two thousand, from the existing metapneumovirus of the landlocked supervention of patient body, sars coronavirus, novel coronavirus HKU1 and NL63, highly pathogenic human and bird fluenza virus, boca virus and 2 kinds of polyomavirus etc.2003, sars coronavirus was because of the infectivity of its height and pathogenicly cause worldwide extensive epidemic situation to break out, and recent years, and human and bird fluenza frequently takes place and causes high mortality ratio (75%) in the area of contact closely people and animals.The generation of above-mentioned public health event, not only serious threat human life but also cause that to some extent society is panic.Therefore, set up the epidemic situation pre-alerting ability that bioaerosol on-line monitoring technique in the ambient air will greatly improve public health system, for keep social stability, the protection people ' s health has great importance.
Real-time monitoring also is the needs of national security to the bioaerosol that contains pathogenic micro-organism in addition.Calendar year 2001, the U.S. took place after " 911 " incident and the anthrax bacteria incident subsequently, and countries in the world recognize that clear-headedly the extensive attack of terrorism is just threatening normal human society.In diversified attack of terrorism means, scatter high risk on a large scale with the bioaerosol approach, the attack mode of highly infective pathogenic micro-organism (as virus, bacterium etc.), because of its have low, the easy input of cost, destructive strong, influence power big and be easy to characteristics such as propagations, enjoys various countries' concern.Development is carried out early warning to the high risk pathogenic agent Monitoring techniques in the ambient air to the bio-terrorism attack, can effectively take precautions against the generation of biological and chemical attack, and farthest reduce loss.Therefore, many countries begin to establish and improve oneself monitor and early warning system, the communication of reinforcement information with exchange.The U.S. utilizes more than 3000 Detection of Air Quality station of State Bueau of Environmental Protection, the anthrax bacteria that monitoring in China may occur, variola virus etc.After this system starts, can in 12 hours, draw relevant monitoring result the soonest.In addition along with the development of biotechnology, on the high-tech battlefield of 21 century, be that the possibility that occurs of the extensive lethal biological weapon of carrier is also more and more higher with the bioaerosol.Be the protection soldier, the technical equipment of the on-line monitoring that can carry out bioaerosol also is badly in need of in country.
The gaseous sample collection method of domestic and foreign current is mainly collected with special instrument, except using inconvenience, outside efficient is not high, also is difficult to finish the whole process operation of collection analysis.Still do not have practical exemplary gases up to now both at home and abroad and collect also the rapid detection system of monitoring in real time.Following technical development mainly concentrates on the multifunction and microminiaturization of detecting instrument.External multifunction instrument concentrates on and is used for the self-reacting device that the air biological particles is analyzed.Current air sample is collected and the development trend of test set is: (1) successive analysis reaches the purpose of monitoring and early warning; (2) real-time analysis reaches online detection and early warning fast; (3) high-throughput reaches the detection to several samples; (4) increasingly automatedly finish popularizing in nature and public place; (5) portability adapts to the needs of quick layout and military chemical defence.
The microfluid subject that grow up the nineties in 20th century is meant fluidic science and technology in the small network channel of operation (5-500 micron).It is the development and incorporation of various modern technology such as Protocols in Molecular Biology, micro-processing technology, Machine Manufacturing Technology, computer technology.Be based on the micro device of extensive parallel processing biological information molecule principle, have that the information flux is big, an automatization, systematized characteristics.Micro-fluid chip is used for operation, and transmission microlitre (10-6L) is to the fluid of millimicro microlitre (10-15L) magnitude.Some steps of biochemical reaction can be comprised that analysis, washing, detection etc. are integrated on one or a few micro-fluid chip, its aperture, microchannel has only the micron order size, have the effect that concentrates with enrichment, can accelerated reaction shorten the test duration, thereby greatly reduce testing cost.Compare with the experimental technique of routine, the waste liquid that this technology greatly reduced the consumption (at least 3 orders of magnitude) of reagent, analyze to produce simultaneously is few.Transmission ofenergy in small scope, material disperse faster more even, and thermal energy conduction is fast, various the controlling of also easier realization, so reaction is fast, yield is high, pollutes less, cost is low.The micro-fluid chip of a new generation is by macromolecular material, for example silicone polydimethylsiloxane (PDMS) makes (seeing accompanying drawing 1), material cheap (being less than 10 U.S. dollars/sheet), manufacturing cycle short (being less than 24 hours), required equipment is the minority conventional equipment, do not need large-scale secret instrument, be suitable for scale operation, can be used for producing disposable product.The trend of the development of this technology is chip lab, whole Biochemical Lab function can be integrated on the chip piece to finish.
In addition, since the physics elasticity of PDMS material, multiple functional module, for example little valve, fluid pump and fluid mixer can be integrated in the micro-fluid chip.And this valve slightly, can the controlling of functional modules such as fluid pump by computer programming.Like this integrated, digital control type micro-fluid chip just can be finished some complicated operations.For example separate, sample introduction cleans, chemistry or bioanalysis operations such as chromatographic column isolation identification.Based on the instrument of micro-fluid chip pcr amplification instrument for example, albumen crystallization instrument, DNA tests instrument all have been designed to create.Compare with usual manner, its principal feature is:
1) at a low price, because the amount of the reagent that uses is extremely micro-, the expense of finishing the test needs is extremely low;
2) efficient, speed of response is fast in micro-fluid chip, heat and mass efficient height, and test speed is fast;
3) use the manufacturing of masterplate technology, be applicable to scale operation;
4) integrated easily, the test module of different target can be integrated in the chip piece very easily, finishes the multiple goal parallel parsing;
5) level of automation height, it can be easily and the modern electronic technology combination, not only makes the chip analysis test automatically, and signal transmission and the automatic analysis of signal also can be finished automatically.
6) compatible good, other microanalysis technology, microelectrode technology for example, biosensor technology also can be incorporated in the micro-fluid chip technology.
Therefore use the research focus that detection that microflow control technique carries out pathogenic micro-organism in the air becomes present air real time on-line monitoring.But existing gas collector exists volume big, can not effectively abiotic particle be separated with microorganism, the subsequent sample complex disposal process, can not with shortcomings such as miniature device for fast detecting compatibility such as chip.The enrichment chip module that the present invention is based on pathogenic micro-organism in the air of microflow control technique can be good at addressing the above problem.
Summary of the invention
The object of the present invention is to provide a kind ofly have efficiently, at a low price, the characteristics of portable and automatization be used for micro-fluidic chip to the various microorganism fast enrichings of air, and provide this chip production method.
The invention provides a kind of based on the on-the-spot micro-fluidic chip of collecting microorganism in the air fast of being specifically designed to of fluid mechanics principle.This chip is base material with the optically transparent material, is combined to form by example enrichment channel layer and gas passage layers;
Example enrichment passage in the wherein said example enrichment channel layer is 1-100 μ m by width, length is that the continuous parallel arranged level of the arrow shaped groove of 1-4mm becomes, the spacing of adjacent grooves is 100-1000 μ m, depth of groove is 1-100 μ m, and each arrow shaped groove forms a microchamber of collecting microorganism; Gas passage in the described layer is smooth straight groove, and this passage width is 1-4mm, and length is 20-30cm, and the degree of depth is 1-100 μ m; All be communicated with between each microchamber and the gas passage in the described example enrichment passage.
The base-material of micro-fluidic chip adopts elastomeric polymer polydimethylsiloxane (PDMS) among the present invention.
The number of the microchamber of gas enrichment of the present invention can be determined by practical situation, needs to determine as sample size and other experiment.The micro-fluidic chip that has prepared a monocyte sample in concrete enforcement of the present invention can this single passage is in parallel by different way according to practical situation, series connection.
The invention provides the preparation method of above-mentioned micro-fluidic chip, concrete steps are as follows:
(1) substrate is prepared: dry up with nitrogen after silicon chip is put into the deoxidation of Piranha solution, get rid of through spin coater with SU-82050 series and be coated with soft baking on the thermostatically heating plate;
(2) expose and cure: the silicon chip template that designs is placed on gets rid of on the substrate that coats, use the exposure of uv-exposure machine, on hot-plate, cure afterwards;
(3) develop: silicon chip is put into developing solution develop, use Virahol and washed with de-ionized water clean afterwards respectively, and dry up with nitrogen;
(4) hard baking: on hot plate, slowly add heat setting, form mould;
(5) cast: PDMS monomer and solidifying agent mixed by the quality proportioning in 5: 0.8～5: 1.3, and eliminated wherein bubble with vacuum pump, were poured on respectively on the silicon chip mould of example enrichment passage and gas passage, solidified in baking oven, peeled off;
(6) bonding: two-layer PDMS chip calibration bonding is formed chamber, microchannel (being example enrichment passage and gas passage).
Microfluidic control chip of the present invention can be used for the microorganism in the enriched air By, its method is (as pump with the power set of collecting gas, mechanical press device such as aerosol dispenser or artificial propellings movement/absorption air sample device) the collection air sample, will be enriched in microorganism wash-out in each microchamber of example enrichment passage with damping fluid or substratum then; The sample of washing behind the sample directly is communicated with immune microfluidic control chip, perhaps after treatment, connects PCR microfluidic control chip, finishes on-the-spot real-time analysis and detects.
This chip can built-in or external special-purpose micropump, and with control section (computer), operating system (integrated micro-fluid chip, the numerical control interface) and data gathering, data analysis part (computer) is integrated, and to the volume of gaseous sample, the volume of elutriant is controlled.
The microfluidic control chip of the present invention's design can efficient, the airborne various microorganisms of fast enriching.Volume is little, can effectively separate abiotic particle with microorganism, and with other miniature device for fast detecting compatibility.
Description of drawings
Fig. 1 micro-fluidic chip schema of the present invention.
Number in the figure: 1 is the enriched sample passage, rearranges S by the arrow shaped microchamber, and 2 is gas passage, and 3 is injection port, and 4 is outlet.
Embodiment
1. substrate is prepared.Silicon chip is put into Piranha solution (98% vitriol oil: 30% hydrogen peroxide=7: 3, volume ratio) boil cleaning 15min.Dry up with nitrogen with behind the deionized water rinsing 5 times, and cure 30min. at 200 ℃
2. get rid of and be coated with.The SU-8 glue (down together) of Microchem company is poured on silicon chip central authorities, and the inclination silicon chip makes SU-8 cover the most of zone of silicon chip.Leave standstill 15min, eliminate the bubble that produces in the toppling process simultaneously.(Spin-Coater KW-4A, Chemat Technology Inc.) carry out twice laddering getting rid of and be coated with: 500 commentaries on classics/min spin coating 15s, 3000 commentaries on classics/min spin coating 30s leave standstill 10min and alleviate edge projection effect with spin coater.
3. soft baking.Speed with 5 ℃/min on hot plate progressively is raised to 95 ℃, during keep 3min and 6min respectively at 65 ℃ and 95 ℃.Slowly reduce to room temperature with the speed of 0.5 ℃/min afterwards.
4. exposure.Adopt contact exposure machine (wavelength 365nm).
5. cure.Speed with 5 ℃/min on hot plate progressively is raised to 95 ℃ by room temperature, during keep 1min and 5min respectively at 65 ℃ and 95 ℃.Slowly reduce to room temperature with the speed of 0.5 ℃/min afterwards.
6. develop.Carry out in stink cupboard, the main component of developing solution is 1-Methoxy-2-propyl acetate (PGMEA).Mould is put into the developing solution 6min that develops, use Virahol and washed with de-ionized water clean afterwards respectively, and dry up with nitrogen.
7. hard baking.On hot plate, slowly be heated to 200 ℃, keep 30min, slowly reduce to room temperature again.
8. pour into a mould polydimethylsiloxane (PDMS) polymkeric substance, moulding.To use the ultrasonic successively 5min of acetone, dehydrated alcohol to the microchannel PDMS seal that adopts photolithography to make respectively, remove the moisture on the passage seal, 60 ℃ of baking oven bakings of cleaned seal 4h.PDMS monomer and solidifying agent mix according to 5: 1 quality proportioning, the Ex-all bubble.Be poured on the SU-8 mould that trimethylchlorosilane was handled, 65 ℃ keep 2h to solidify on the horizontal hot plate of adjusting.Form the substrate that the upper strata has the microchannel layer.
9. the PDMS layer that has control channel is made.On silicon chip, get rid of resist coating,, make silica-based smooth formpiston, and, make photoresist material softening in 120 ℃ of annealing 30min through uv-exposure, development.Silica-based optical cement formpiston is handled 7min with trimethylchlorosilane in gas phase, make its surface silicon alkanisation.
10. bonding and interface are made.Gas passage layers is punched.With gas passage layers and enrichment channel layer up and down two careful involutory, 80 ℃ of curing of spending the night.Promptly made the PDMS chip
11. the gas feed of chip is placed on the space that will take a sample, and by pressure assembly, the gas that makes 1-10ml is by chip, the chip internal gas pressure is no more than 20psi.After treating that whole sample gases pass through, according to the feature that will collect microorganism, with suitable buffered soln or special substratum perfusion raw gas channels such as 100-200 μ L phosphoric acid buffers, space size and fluid properties according to the chip microchamber, control suitable flow velocity, wash away the enrichment passage, and the collection elutriant is used for subsequent experimental.
Claims (4)
1. micro-fluidic chip that is used for the enriched air By microorganism; It is characterized in that this chip is base material with the optically transparent material, is combined to form by example enrichment channel layer and gas passage layers;
Example enrichment passage in the wherein said example enrichment channel layer is 1-100 μ m by width, length is that the continuous parallel arranged level of the arrow shaped groove of 1-4mm becomes, the spacing of adjacent grooves is 100-1000 μ m, depth of groove is 1-100 μ m, and each arrow shaped groove forms a microchamber of collecting microorganism; Gas passage in the described layer is smooth straight groove, and this passage width is 1-4mm, and length is 20-30cm, and the degree of depth is 1-100 μ m; All be communicated with between each microchamber and the gas passage in the described example enrichment passage.
2. micro-fluidic chip according to claim 1 is characterized in that described optically transparent material is an elastomeric polymer: polydimethylsiloxane.
3. the preparation method of micro-fluidic chip according to claim 1 is characterized in that adopting moulding method, and concrete steps are as follows:
(1) substrate is prepared: dry up with nitrogen after silicon chip is put into the deoxidation of Piranha solution, get rid of through spin coater with SU-82050 series and be coated with soft baking on the thermostatically heating plate;
(2) expose and cure: the concrete example enrichment passage that will design and the silicon chip template of gas passage are placed on respectively gets rid of on the substrate that coats, and uses the exposure of uv-exposure machine, cures on hot-plate afterwards;
(3) develop: substrate is put into developing solution develop, use Virahol and washed with de-ionized water clean afterwards respectively, and dry up with nitrogen;
(4) hard baking: on hot plate, slowly add heat setting, form mould;
(5) cast: polydimethylsiloxane monomer and solidifying agent mixed by the quality proportioning in 5: 0.8～5: 1.3, were poured on respectively on the silicon chip mould of example enrichment passage and gas passage, solidified in baking oven, peeled off;
(6) bonding: two-layer polydimethylsiloxanechip chip calibration bonding is formed the chamber, microchannel.
4. micro-fluidic chip as claimed in claim 1 application in the microorganism in enriched air By, it is characterized in that collecting air sample, will be enriched in microorganism wash-out in each microchamber of example enrichment passage with damping fluid or substratum then with the power set of collecting gas; The sample of washing behind the sample directly is communicated with immune microfluidic control chip, perhaps after treatment, connects PCR microfluidic control chip, finishes on-the-spot real-time analysis and detects.
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| CN103189747A (en) * | 2010-07-02 | 2013-07-03 | 米克罗森斯医疗技术有限公司 | Capture of micro-organisms |
| CN104893963A (en) * | 2015-05-26 | 2015-09-09 | 大连理工大学 | Microfluidic chip for capturing fungal spores in air and preparation method of microfluidic chip |
| CN105486571A (en) * | 2015-11-09 | 2016-04-13 | 上海海洋大学 | Microfluidic chip used for enriching microbial aerosol and preparation method thereof |
| CN106676004A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院微生物研究所 | Micro-fluidic culture device and method for culturing cells or microorganisms by applying micro-fluidic culture device |
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| CN103189747A (en) * | 2010-07-02 | 2013-07-03 | 米克罗森斯医疗技术有限公司 | Capture of micro-organisms |
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| CN110793956A (en) * | 2019-11-13 | 2020-02-14 | 无锡物联网创新中心有限公司 | Micro-fluidic device integrating functions of trace gas enrichment and detection and preparation and detection methods thereof |
| CN111912697A (en) * | 2020-08-14 | 2020-11-10 | 南京原码科技合伙企业(有限合伙) | Rapid concentration device and method for pathogenic microorganisms |
| CN111912697B (en) * | 2020-08-14 | 2023-03-07 | 南京原码科技合伙企业(有限合伙) | Rapid concentration device and method for pathogenic microorganisms |
| CN115364911A (en) * | 2021-05-18 | 2022-11-22 | 重庆大学 | Aerosol microorganism sampling enrichment chip and preparation method thereof |
| CN115364911B (en) * | 2021-05-18 | 2023-12-05 | 重庆大学 | Aerosol microorganism sampling enrichment chip and preparation method thereof |
| CN114395468A (en) * | 2021-12-24 | 2022-04-26 | 西湖大学 | Device for collecting pathogens of human respiratory diseases |
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