CN101169403A - Micro-fluidic chip and its preparation and uses - Google Patents
Micro-fluidic chip and its preparation and uses Download PDFInfo
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- CN101169403A CN101169403A CNA2006101340190A CN200610134019A CN101169403A CN 101169403 A CN101169403 A CN 101169403A CN A2006101340190 A CNA2006101340190 A CN A2006101340190A CN 200610134019 A CN200610134019 A CN 200610134019A CN 101169403 A CN101169403 A CN 101169403A
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Abstract
The invention relates to a micro fluidic chip, in particular to a micro fluidic chip and the preparation thereof as well as the application as a solid phase extraction column. The chip is formed by sealedly connecting an upper base chip and a lower base chip, wherein, the upper base chip is provided with a sample inlet, a sample outlet and a microchannel. The microchannel of the upper base chip is provided with a particle guide channel and the inlet of the particle guide channel; dam structures are respectively arranged between the inlet of the particle guide channel and the sample outlet and between the inlet of the particle guide channel and the sample outlet. The two dam structures form a double-cofferdam structure, and the microchannels between the two dam structures form a packed column bed channel. Moreover, the places of the lower base chip corresponding with the dam structures are respectively provided with microchannels, and apertures are reserved between the top surfaces of the dam structures and the microchannel of the lower base chip; wherein, the depth of the apertures is smaller than the depth of the microchannels in the upper base chip. The invention has the advantages of simple and quick production, perfect repeatability, extensive application range and good using effect.
Description
Technical field
The present invention relates to micro-fluidic chip, specifically a kind of micro-fluidic chip and preparation thereof and as the application of solid-phase extraction column.
Background technology
Solid-Phase Extraction (SPE, or claim liquid-solid extraction), be meant the separation principle of utilizing selective adsorption and selectivity desorption, the process of the microcomponent in the purifying or the liquid sample that concentrates.Solid-Phase Extraction (SPE) technology is widely used in separation/purification, concentrate or the enriched with trace measured object, remove chaff interference (as salt etc.), improves the concentration and the detection sensitivity of measured object, is a kind of sample pretreating method commonly used.The microminiaturization of SPE technology, promptly micro-fluidic chip SPE technology is meant control modules such as utilizing the formed Micro Channel Architecture of chip micro-processing technology and little valve/Micropump, finishes a kind of microminiaturized sample preparation technology of SPE operation on the micro-fluidic chip platform.Chip SPE technology has many conventional SPE technology advantage can't be obtained, but that, sample/reagent fast such as speed consumes is low, can be with the integrated array of other operating units etc., is to realize micro-total analysis system (a kind of requisite operating unit of μ-TAS).
At present, the SPE method based on micro-fluidic chip of various ways occurred, not only be applied to micromolecular separation/enrichment, and be applied in the macromolecular analyses such as protein, nucleic acid.According to the different method for makings of solid-phase extraction column, these SPE methods based on micro-fluidic chip can be divided into three types of open tubular columns, packed column, integral post chip SPE method.Such as document [1 Kutter JP, Jacobson S C, Ramsey J M.J Microcolumn Sep.2000,12:93-97.2BroylesB?C，Jacobson?S?C，Ramsey?J?M.Anal.Chem.2003，75：2761-2767。] be reported in the chip channel inwall and be coated with stain C18 silane as open tubular column SPE stationary phase, be used for extraction and enrichment micromolecule.The making of this chip solid-phase extraction column is simple relatively; But less specific surface area causes its stationary phase load capacity on the low side, thereby has limited it to a certain extent in the technical application of chip SPE.For improving the specific surface area of stationary phase, adopt special micro-processing technology, can in the chip manufacturing process, directly in passage, form special microstructure, in order to stop the stationary phase particulate of filling, thereby form filled-type solid-phase extraction column [3 Oleschuk R D, Shultz-Lockyear L L, Ning Y, et al..Anal.Chem.2000,72:585-590.4?Jemere?A?B，Oleschuk?R?D，Ouchen?F，et?al..Electrophoresis?2002，23：3537-3544。]。Because the packed column extraction efficiency is higher, available filling kind is many and filling process is simple relatively, can utilize electroosmotic flow or pressure to introduce and fill particulate, and can go out used filling particulate by back-pressure, thereby can bring in constant renewal in packed column.But this method employing " difference etching " legal system is made the two cofferdam type microstructures in the chip, need be at the double photoetching/etching process that carries out of same chip surface, and manufacture difficulty is big, the cycle is long, cost is high, has still limited its widespread use.By light-initiated [5 Yu C, Davey MH, Svec F, Frechet J M J.Anal.Chem.2001,73:5088-5096.] and thermal induction [6Wolfe K A, Breadmorel M C, Ferrance.J P, et al..Electrophoresis 2002,23:727-733.7?Breadmore?M?C，Wolfe?K?A，Arcibal?I?G，et?al..Anal.Chem.2003，75：1880-1886。] etc. mode polymerization reaction take place in chip channel, form the integral post stationary phase, not only enriched the kind of chip SPE stationary phase, and avoided the use of complicated micro-processing technology.Though this method development is very fast, because polymerization reaction system is limited, the organic polymer integral post is relatively poor to the tolerance of organic solvent simultaneously, and the integral post of heat-induced aggregation is located difficulty in the microchannel, make it be difficult to become a kind of general chip SPE method.
Develop a kind of simple and easy to do and chip SPE column production method that versatility is good, not only for chip SPE technology widely-used provide more may, and be its further integrated laying a good foundation.
Summary of the invention
The object of the present invention is to provide a kind of micro-fluidic chip and preparation thereof and application, its method for making is easy, quick, good reproducibility, need not the chip micro-processing technology of any complexity; Simultaneously, can bring in constant renewal in use when using, thereby further reduce use cost as the SPE post; Applied widely, effect is good.
For achieving the above object, the technical solution used in the present invention is:
A kind of micro-fluidic chip, chip is formed by upper and lower two layers of substrate sealing-in, its upper strata substrate is provided with injection port, outlet and microchannel, on the microchannel of upper strata substrate, be provided with particulate and introduce passage and inlet thereof, introduce between the inlet of passage and injection port and the outlet at particulate and to be respectively equipped with the dam structure, two dam structures have formed a pair of cofferdam type structure, and the microchannel between two dam structures forms packed column bed passage; Be respectively equipped with the microchannel at lower floor's substrate corresponding to the position of dam structure, have the slit between the microchannel of the end face of dam structure and lower floor's substrate; The degree of depth in described slit is less than the degree of depth of microchannel in the substrate of upper strata.
The method for making of described micro-fluidic chip, adopt the chip manufacture method of " two-sided etching/aligning ", the upper and lower two layers of substrate structure that designs is made mask, glass photomask/wet-etching technology according to standard etches required pattern, the upper strata substrate that etching is good punches in desired location with the ultrasonic drilling device, after the cleaning, two glass substrates are aimed at microscopically, the central vertical line of microchannel in dam structure in the substrate of upper strata and the lower floor's substrate is overlapped, thereby form the continuous passage that contains two cofferdam type structures; Then the chip of aiming at is put into vacuum drying oven successively and the temperature programmed control stove is fitted and thermal bonding, can obtain required micro-fluidic chip.
Introduce passage is inserted all size and kind in the packed column bed passage of chip stationary phase particulate by particulate, adopt reversible mode to seal the inlet that particulate is introduced passage, promptly form chip SPE post.
The gap depth of described chip should be less than the minimum diameter of filling stationary phase particulate; Chip material can be silicon, quartz, glass, various polymkeric substance or their complex.
The present invention has following advantage:
1. the present invention produces two cofferdam type structures in chip microchannel, its cofferdam depth controlled and less than the minimum diameter of filling particulate; Two cofferdam type structures can stop particulate loss and be fixed in the packed column bed scope; In the solid-phase extraction chip of the present invention, the inlet of particulate introducing passage adopts the mode of reversible sealing, makes the stationary phase particulate in the SPE post can realize bringing in constant renewal in.
2. method for making is simple.Chip is aimed at by the upper and lower two layers of substrate that is carved with different depth and sealing-in forms, last slice contain two dam structures that are not etched, following sheet contains the interface channel of two depth controlled; Two aim at and sealing-in after, the dam structure is positioned at the scope of interface channel fully, forms the continuous passage that contains two cofferdam type structures.
3. the present invention adopts the upper and lower two layers of substrate chip processing method of etching/aligning simultaneously, makes two cofferdam type structures in chip microchannel, and the etching depth of lower floor's substrate is controlled, makes the minimum diameter of its cofferdam degree of depth less than the filling particulate; When introducing passage and fill the stationary phase particulate by particulate, this pair cofferdam type structure can stop particulate loss, thereby be fixed in the packed column bed scope, and form chip SPE post.
The particulate that is used to fill among the present invention be applicable to variety classes and the size chromatographic stationary mutually.
Description of drawings
Fig. 1 is the structural representation of micro-fluidic chip solid-phase extraction column, and wherein 1 is injection port, and 2 is outlet, 3 inlets for particulate introducing passage, and 4 is two cofferdam type structures, 5 is packed column bed passage;
Fig. 2 is respectively the structural representation of upper and lower two layers of substrate;
Fig. 3 is the schematic cross-section after upper strata substrate etching and the punching, wherein 1,2 is respectively injection port and outlet, and 4 is the dam structure that is not etched, and 5 is packed column bed passage;
Fig. 4 is the schematic cross-section after lower floor's substrate etching, and wherein 6 is the interface channel of depth controlled;
Fig. 5 is the schematic cross-section after upper and lower two layers of substrate is aimed at also sealing-in;
Fig. 6 is the sectional view of chip SPE post behind the filling stationary phase particulate;
Fig. 7 is the figure as a result of λ-DNA chip Solid-Phase Extraction gained extraction efficiency;
Fig. 8 is the chip electrophoresis figure with the pcr amplification product of chip SPE method gained DNA sample.
Embodiment
As shown in Figure 1, a kind of micro-fluidic chip, chip is formed by upper and lower two layers of substrate sealing-in, its upper strata substrate is provided with injection port 1, outlet 2 and microchannel, being provided with particulate on the microchannel of upper strata substrate introduces passage and enters the mouth 3, be respectively equipped with the dam structure between the inlet 3 of introducing passage at particulate and injection port 1 and the outlet 2, two dam structures have formed the microchannel formation packed column bed passage 5 between 4, two dam structures of a pair of cofferdam type structure; Be respectively equipped with the microchannel at lower floor's substrate corresponding to the position of dam structure, have the slit between the microchannel of the end face of dam structure and lower floor's substrate; The degree of depth in described slit is less than the degree of depth of microchannel in the substrate of upper strata.
The making of micro-fluidic chip SPE post (with the example that is made as of glass-chip SPE post): the upper and lower two layers of substrate (as shown in Figure 2) that will design is made mask respectively, glass photomask/wet-etching technology according to standard etches required pattern (as shown in Figure 3, Figure 4), wherein upper and lower two etching depth is respectively 70 μ m and 4 μ m, and length and width that particulate is introduced passage are respectively 3mm and 160 μ m; The upper strata substrate that etching is good punches in the relevant position with the ultrasonic drilling device; After strict cleaning process, to aim at microscopically for two, the dam structure in making slice and the central vertical line of the interface channel in the following sheet overlap (as shown in Figure 5) substantially, thereby form the continuous passage that contains pair cofferdam type structures; To aim at then that good chip is put into vacuum drying oven successively and the temperature programmed control stove is fitted and thermal bonding, can obtain required glass-chip.
In the solid-phase extraction chip of the present invention, the cofferdam degree of depth of two cofferdam type structures also can be for other be worth, as long as the control cofferdam degree of depth is less than the diameter of particulate; Length and width that particulate is introduced passage also can be worth for other.
After making the chip that contains two cofferdam type structures, adopt following method to make chip SPE post: the suspending liquid that will be mixed with stationary phase particulate (the unmodified silica gel of diameter 5 μ m) is added in the inlet 3 that particulate is introduced passage, add vacuum at injection port 1 and outlet 2 then, can fill up the post bed after taking out a few minutes, inlet 3 usefulness syringes at particulate introducing passage apply normal pressure subsequently, with the particulate of closely knit filling; After the post bed was filled evenly, the inlet 3 with the epoxy glue sealing particulate introducing passage that is heating and curing can obtain chip SPE post (as shown in Figure 6).When needing to upgrade the stationary phase particulate, the SPE chip of having filled was placed 100 ℃ of baking ovens about 15 minutes,, with blade it is removed immediately with the epoxy glue of softening sealing usefulness; Add water simultaneously at injection port 1 and outlet 2, and introduce feeder connection 3 usefulness vacuum at particulate and take out, can apply normal pressure at injection port 1 and outlet 2 in case of necessity, like this, can remove the stationary phase particulate of having filled; Repeat above-mentioned filling process again, the SPE post that just can obtain upgrading.
The characteristics of SPE post that the present invention is based on micro-fluidic chip are as follows:
1.SPE the making of chip only need be adopted the lithography/etch process of a step standard, this method is easy, quick, good reproducibility, need not the micro-processing technology of any complexity;
2. the making of chip SPE post is adopted easy and fill method fast, thereby has avoided complicated polymerization technique;
3. particulate is introduced the inlet 3 use reversible manner sealings of passage in the chip SPE post, thereby has realized the repeated use of chip SPE post, has reduced use cost.
4. this method can be applicable to the micro-fluidic chip of different materials, and is applicable to the stationary phase particulate of variety classes and size, thereby has expanded its range of application.
The chip SPE post that embodiment 2 unmodified silica gel (diameter 5 μ m) are filled is used for the extraction of λ-DNA
The sample of using, flushing and elution buffer are respectively the aqueous solution and the TE damping fluid (10mM Tris-1mM EDTA, pH 7.5) of 6M guanidine hydrochloride (be dissolved in the 10mMTris-1mM edta buffer liquid, pH 6.1), 80% isopropyl alcohol in this example; The sample that is used for extracting is the λ-DNA (final concentration is 0.5ng/ μ L) that is dissolved in sample-loading buffer.Adopt homemade chip interface that the SPE chip is linked to each other with syringe pump, the control flow velocity is 3 μ L/min; The volume of the sample of using, flushing and elute soln is respectively 12 μ L, 15 μ L and 15 μ L, and the used time of entire chip extraction process was less than 15 minutes.Collect the DNA sample that wash-out goes out, and carry out the fluorophotometric standard measure, to measure the extraction efficiency and the repeatability of this chip SPE method with embedded fluorescent dye.
From experimental result (as shown in Figure 7) as can be known, when the glass-chip SPE post that utilizes the present invention to make carries out λ-DNA extraction, (average recovery rate is 84.7%, and n=12) (RSD of different chip SPE posts is 1.95%, n=3 with better repeatability can to obtain higher extraction efficiency; The RSD of same chip SPE post is 12.21%, n=4); Simultaneously, the required sample volume of this method little (each 12 μ L samples that only need), rate of extraction fast (less than 15 minutes).
The chip SPE post that embodiment 3 silica gel are filled is used for from the actual sample purified genomic dna
Specimen in use behaviour whole blood sample in this example, its disposal route is: 4.6 μ L whole blood samples are dissolved in 500 μ L sample-loading buffer (6M guanidine hydrochlorides, 10mM Tris-1mM edta buffer liquid, pH 6.1) in, add 10 μ L Proteinase Ks (20mg/mL) again, in 56 ℃ of water-baths, kept 10 minutes then, can obtain the sample solution that is used to extract.The volume of the sample of using, flushing and elute soln is respectively 15 μ L, 20 μ L and 15 μ L, and flow velocity is 3 μ L/min, and the per 5 μ L of the DNA sample that wash-out goes out collect a, get 2.5 μ L for every part and are used for pcr amplification.The primer sequence is:
5 '-AACTGTTGCTTTATAGGATTTT-3 ' (positive-sense strand),
5 '-AGGAGCTTATTGATAACTCAGAC-3 ' (antisense strand);
Amplified production is the 650bp fragment of human genome betaglobulin gene.PCR reaction solution (25 μ L) consists of: 10 * PCR damping fluid, 2.5 μ L, dNTP potpourri (every kind of 10mM) 0.5 μ L, primer (every kind 20 μ M) 0.5 μ L, Taq archaeal dna polymerase (5U/ μ L) 0.2 μ L, dna profiling 2.5 μ L.Pcr amplification is at commercialization PCR instrument (GeneAmp
TM2700, carry out on ABI), reaction conditions is: 95 ℃ * 5 minutes (pre-sex change), 94 ℃ * 30 seconds/55 ℃ * 30 seconds/72 ℃ * 30 seconds (circulate 35 times), 72 ℃ * 10 minutes (back extension).Gained PCR product detects with PMMA chip electrophoresis separation/laser-induced fluorescence (LIF) (LIF), and the electrophoresis sieving media is: add 2% hydroxypropyl methylcellulose (HPMC) and 1 μ M fluorescent dye in the 100mM tbe buffer liquid; Separation condition is: electric field intensity 160V/cm, effectively separation length 3.5cm. the results are shown in Fig. 8.
From chip electrophoresis result (as shown in Figure 8) as can be seen, utilize the genomic DNA of chip SPE post of the present invention purifying from people's whole blood sample can successfully carry out pcr amplification, and the amount of amplified production (650bp) and conventional method (positive control) are suitable, show that this chip SPE post is higher to the purifying and the extraction efficiency of genomic DNA, thereby lay a good foundation for the application of the present invention in foranalysis of nucleic acids.
Claims (5)
1. micro-fluidic chip, chip is formed by upper and lower two layers of substrate sealing-in, its upper strata substrate is provided with injection port (1), outlet (2) and microchannel, it is characterized in that: on the microchannel of upper strata substrate, be provided with particulate and introduce passage and inlet (3) thereof, introduce between the inlet (3) of passage and injection port (1) and the outlet (2) at particulate and to be respectively equipped with the dam structure, two dam structures have formed a pair of cofferdam type structure (4), and the microchannel between two dam structures forms packed column bed passage (5); Be respectively equipped with the microchannel at lower floor's substrate corresponding to the position of dam structure, have the slit between the microchannel of the end face of dam structure and lower floor's substrate; The degree of depth in described slit is less than the degree of depth of microchannel in the substrate of upper strata.
2. the method for making of the described micro-fluidic chip of claim 1, it is characterized in that: the chip manufacture method that adopts " two-sided etching/aligning ", the upper and lower two layers of substrate structure that designs is made mask, glass photomask/wet-etching technology according to standard etches required pattern, the upper strata substrate that etching is good punches in desired location with the ultrasonic drilling device, after the cleaning, two glass substrates are aimed at microscopically, the central vertical line of microchannel in dam structure in the substrate of upper strata and the lower floor's substrate is overlapped, thereby form the continuous passage that contains two cofferdam type structures; Then the chip of aiming at is put into vacuum drying oven successively and the temperature programmed control stove is fitted and thermal bonding, can obtain required micro-fluidic chip.
3. the application of the described micro-fluidic chip of claim 1, it is characterized in that: introduce passage by particulate and in the packed column bed passage (5) of chip, insert the stationary phase particulate, adopt reversible mode to seal the inlet (3) that particulate is introduced passage, can form chip SPE post.
4. according to the application of the described micro-fluidic chip of claim 3, it is characterized in that: the gap depth of chip described in the claim 1 should be less than the minimum diameter of filling stationary phase particulate.
5. according to the application of the described micro-fluidic chip of claim 3, it is characterized in that: described chip material is silicon, quartz, glass, various polymkeric substance or their complex.
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| CN111019804B (en) * | 2019-12-17 | 2024-04-05 | 东莞市东阳光诊断产品有限公司 | Nucleic acid amplification device, system and method |
| WO2022062897A1 (en) * | 2020-09-25 | 2022-03-31 | 中国科学院青岛生物能源与过程研究所 | Microfluidic chip for particle screening and separation |
| CN112973814A (en) * | 2021-03-03 | 2021-06-18 | 北京理工大学 | Interlayer automatic alignment bonding device and method for multilayer microfluidic chip |
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