Abstract:
A microfluidic structure comprising a thermoplastic portion defining a microfluidic recess, a bonding layer on the thermoplastic portion and a siloxane elastomer portion covalently bonded to the bonding layer to seal the microfluidic recess. The microfluidic recess can therefore be formed simply, quickly and cheaply using known injection molding techniques, which are not hampered by the need for a curing step. However, the positive qualities associated with elastomers can be brought to the structure by using this to seal the microchannels. The bonding layer can be formed by silica deposited on the thermoplastic portion using techniques known in the field of optics.

Description:
CROSS-REFERENCE 
     This application is a Continuation Application of U.S. patent application Ser. No. 11/576,182, filed Feb. 28, 2008, which is a National Stage Entry of International Patent Application No. PCT/GB2005/003736, filed Sep. 28, 2005, which claims priority to British Patent Application No. GB 0421529.9, filed Sep. 28, 2004, each of which is incorporated herein by reference in its entirety. 
    
    
     This invention relates to a microfluidic structure and a method for making such a structure. 
     BACKGROUND 
     Microfluidic structures allow liquid volumes to be manipulated on a very small scale, in the nanoliter range. This has many benefits for biological sampling and testing, such as the reduced consumption of samples and reagents, shorter analysis times, greater sensitivity and ease of transportation and disposal. Initially such systems were made using glass or silicon, as methods of manufacturing with these materials were known from the microelectronics industry. Channels were created by, e.g., photo lithography, wet etching or micromachining, after which the channels were sealed by a layer of the same material using anodic bonding, fusion bonding or adhesives. 
     However, glass and silicon are not best suited to the biomedical field as they are expensive, can lack optical clarity, have a low impact strength and poor biocompatibility. Therefore there has been a move away from these materials towards plastics. These offer a wide range of physical and chemical characteristics. A discussion of various methods of manufacture of polymer microfluidic devices can be found in Polymer Microfluidic Devices by Holger Becker and Laurie Locascio (Talanta 56 (2002) 267-287). 
     One of the methods discussed is that of injection moulding using compact disc (CD) manufacturing technology. Here a master is made from silicon using wet chemical etching or deep reactive ion etching. Nickel electroforms are then produced from the silicon master in order to transfer the micro features to a substrate suitable for injection moulding. The nickel electroform is then mounted onto a mould insert and thermoplastic resin is introduced to form the microchannels. These are later sealed to a polymer substrate of the same type or one with a lower glass transition temperature using low temperature thermal annealing. 
     Alternative ways of forming microchannels are by imprinting or hot embossing. 
     Recently elastomers have gained popularity in the field of microfluidic devices due to their flexibility. This allows channels in the elastomer to be closed by the application of pressure to the elastomer, which distorts the shape of the channels. By having a series of layered channels fluid movement in one channel can be controlled by the application of pressurised air to channels positioned above it. This allows microfluidic devices to be fashioned with inbuilt pumps or valves and allows the controlled dispensing or movement of fluid within the device. Poly(dimethylsiloxane) (PDMS) has emerged as a useful elastomer for rapid prototyping of microfluidic structures as it is inexpensive, easy to replicate by moulding and is optically transparent. In addition, PDMS has a high oxygen and carbon dioxide permeability which permits cells located in the microchannels to maintain aerobic metabolism. This is a major difference over conventional plastic, glass and silicon devices which do not allow for gas exchange and are therefore not suited for cell based applications. 
     WO02/43615 discloses several methods of manufacturing a microfluidic device Lasing elastomers, in particular PDMS. 
     First moulds are micromachined Lasing conventional techniques, e.g. photolithography, to create the microchannels in relief. Uncured elastomer is placed over the mould and allowed to cure to form microchannels. The elastomer can them be bonded to a substrate or to another piece of elastomer to seal the channels. 
     Several methods of bonding elastomer to elastomer are discussed, including a reference to Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane) by Duffy et al (Anal. Chem. 1998, 70, 4974-4984). This discloses a method for making PDMS microfluidic systems by first creating a master by photolithography of a silicon wafer. Glass posts are then placed in the master to define reservoirs for liquids. Uncured PDMS is cast over the master and cured. This is achieved by heating the PDMS to around 65° C. for 1 hour. After curing, the PDMS is removed from the master and the glass posts are removed. In order to seal the channels a second, flat layer of PDMS is used. Both PDMS elements are oxidised and then brought into contact. The oxidation converts —OSi(CH 3 ) 2 O— groups at the surface to —O n Si(OH) 4-n . This is believed to result in the formation of bridging, covalent siloxane (Si—O—Si) bonds, which forms an irreversible seal between the PDMS layers. 
     Duffy et al also state that PDMS seals irreversibly to glass, silicon, silicon oxide, quartz, silicon nitrate, polyethylene, polystyrene and glassy carbon after cleaning and exposing both surfaces to an oxygen plasma. 
     The method laid out in Duffy proposes a means of manufacture of microfluidic devices in under 24 hours. Reduction in the time taken to make the devices is limited by the time taken to mould and cure the elastomer. 
     In contrast to PDMS devices, thermoplastic microchannels can be made in a matter of seconds due to the high throughput injection moulding techniques available. However, the sealing means for such channels are not so sophisticated. The techniques used (thermal annealing, adhesive tape, solvent bonding) all tend to deform the microstructures and often introduce materials unsuitable for the intended application, such as toxic solvents or highly autofluorescent adhesives. Moreover, as these materials are inflexible, mechanical pumping is not possible and other techniques, such as electro-osmotics, must be relied on. 
     WO02/43615 discloses the bonding of an elastomer to a non-elastomer substrate containing recesses which form microfluidic channels. This can be done by creating microchannels in the substrate using traditional methods, filling the channels with sacrificial material, coating the substrate with uncured elastomer, curing the elastomer and finally removing the sacrificial material. However, this technique still includes a time delay while the elastomer is cured. 
     Other bonding methods disclosed use Van der Waals, covalent and ionic bonds. Covalent bonding is described in relation to the bonding of glass to a silicone elastomer and requires the glass substrate to first be exposed to agents such as vinyl silane or aminopropyltrithoxy silane. The other examples given also relate to glass substrates which, as mentioned previously, are not suited to the field of biomedical applications. 
     Therefore there still exists a need for a cheap, easy to manufacture microfluidic structure which is suited to use in the biomedical and biochemical fields. 
     SUMMARY 
     According to a first aspect the present invention provides a microfluidic structure comprising a thermoplastic portion defining a microfluidic recess, a bonding layer on the thermoplastic portion and a siloxane elastomer portion covalently bonded to the bonding layer to seal the microfluidic recess. 
     By sealing the microfluidic recess a microchannel or series of microchannels are created. 
     Such a microfluidic structure combines the virtues of elastomer technology with those of high-throughput compact disc injection moulding. The microfluidic recess can be formed simply, quickly and cheaply using known injection moulding techniques, which are not hampered by the need for a curing step. However the positive qualities associated with elastomers can be brought to the structure by using this to seal the microchannels. 
     The inventors have realised that a microfluidic device containing these advantageous properties can be created by coating a thermoplastic portion with a bonding layer that is capable of forming a covalent bond with siloxane elastomer when activated. 
     While various siloxane elastomers can be used to form the covalent bond, it is preferable that PDMS is used. 
     The bonding layer can take a number of forms, but preferably the bonding layer is formed from silica. The silica and elastomer can then be oxidised and brought into contact to form an irreversible bond. An advantage of this method of bonding is that no external pressure needs to be applied during the bonding step, which prevents the sagging and structural deformation that can occur in other, more forceful methods of bonding. The bonding between the silica and elastomer is stronger than the tensile strength of the elastomer, such that peel off tests result in the fracture of the elastomer and not the SiO 2 /plastic interface. In tests it has been found that bonding in this manner between silica and PDMS is capable of withstanding a tension of at least 30N/cm 2  before fracture. 
     The silica is preferably activated by oxygenation, preferably achieved by oxygen plasma under low pressure or at atmospheric pressure by a corona surface treater. 
     Preferably the layer of silica is approximately 300 nm thick as this allows for the subsequent use of surface modification techniques well established in relation to glass microfluidic structures (such as well known silanisation chemistry). As mentioned above, the first microfluidic devices were manufactured using glass and therefore there is a large body of work detailing how to adapt or treat glass microchannels for use in different applications. By depositing silica over the thermoplastic portion this knowledge can be applied to the microfluidic structures of the present invention. Thinner silica layers of 100 or 200 nm can also be provided. 
     Viewed from another aspect the present invention provides a microfluidic structure comprising a thermoplastic portion defining a microfluidic recess, a layer of silica on the thermoplastic portion, and a PDMS portion covalently bonded to the silica layer such that it seals the microfluidic recess. 
     The microfluidic recess can take any shape necessary in order to carry out the desired function and can be created using any of the known techniques for moulding thermoplastics. 
     Various thermoplastics can be used in the invention, thus allowing the user to select the optimal material for a given application based on, e.g. chemical and optical properties or on price. However, the invention has been found to work particularly well with cycloolefin copolymer (Zeonor), polycarbonate and poly(methylmethacrylate) (PMMA). Zeonor is particularly attractive for fluorescence applications since its autofluorescence levels are similar to those of glass. 
     While the device can simply consist of a thermoplastic portion and an elastomer layer bonded together, it is possible for the layer of elastomer to also be coated onto another material, such as another thermoplastic portion having a microfluidic recess. This results in the elastomer portion forming a flexible membrane separating channels in two thermoplastic portions, e.g. polymer chips. By applying pressurised air to an upper-layer channel, the control channel, the membrane is deflected downwards, influencing the fluid in the lower channel, the flow channel. A single control channel can be used as a valve, to selectively allow and prevent flow in the flow channel, or a number of control channels can be used to pump fluid along the flow channel. Using a microfluidic structure according to the present invention improves the mechanical properties of the actuating channels as only a single wall of each channel is flexible. Undesired wall distortion is thus avoided, and the force from the pressurised air is transferred more efficiently to the membrane than in chips made of elastomer only. 
     According to another aspect the present invention provides a method of manufacturing a microfluidic structure comprising the steps of providing a thermoplastic portion having a microfluidic recess, depositing a layer of silica on the thermoplastic portion, oxidising the silica layer and bringing the oxidised layer of silica into contact with an oxidised portion of PDMS to create a covalent bond. 
     Preferably the bonding step does not involve external pressure, thus preventing distortion of the channels during manufacture. 
     Preferably the master used for injection molding of the thermoplastic portion is fabricated by silicon dry etching. This may be used to make a microfluidic recess or recesses, e.g. channels, with rectangular cross sections. Therefore when the PDMS layer is deflected into the channel only the central part of the flow channel floor is brought into contact with the PDMS layer. Even though the closure is incomplete, the reduction in cross sectional area still achieves the desired pumping effect. However, in order to improve valve closure, it is preferable that the master used for thermoplastic molding is manufactured using isotropical etching techniques in order to create channels with an arched cross section in the thermoplastic portion. 
     Preferably the PDMS is formed by spin coating of uncured polymer onto a backing layer, such as Zeonor support film. This backing layer can be peeled off after curing or left in place depending on the application for which the microfluidic structure will be used. More preferably however, the PDMS layer is created by spraying the uncured elastomer onto a backing layer as this simplifies the process. 
     The manufacture and use of a silica layer on a thermoplastic portion having a microfluidic recess is itself advantageous in that such a thermoplastic portion is then available to have its microfluidic recess sealed to create a microfluidic platform and in addition the channels can be modified in accordance with well known techniques. 
     According to another aspect the present invention therefore provides a thermoplastic portion having a microfluidic recess in its upper surface and a layer of silica deposited on the upper surface. 
     Preferably the silica layer is thick enough for standard glass surface modification techniques to be applied. Preferably the silica layer at least 300 nm thick. So that modifications can be applied on all surfaces of the portion it is preferable that a silica layer is deposited on all surfaces of the thermoplastic portion. 
     According to a further aspect the present invention provides a method of preparing a thermoplastic portion, comprising forming a microfluidic recess in its upper surface and depositing a layer of silica on the upper surface. 
     In one preferred method the silica layer is deposited on the thermoplastic portion by electron beam evaporation. This technique is known in the field of optics, for modifying optical properties, but has not previously been used in the field of microfluidics, where the silica is instead used for bonding purposes. Another standard method used in optics that can be used to deposit silica onto the thermoplastic portion is chemical vapour deposition. In another preferred method the silica layer is formed by liquid phase deposition. This reduces the manufacture time of the device as it eliminates the time consuming vacuum pumping of an evacuation chamber. In addition this results in the whole portion being coated with silica, which allows each side of the thermoplastic portion to be covalently bonded. In order to achieve the same effect using electron beam or chemical vapour deposition the deposition step would have to be repeated several times with the thermoplastic portion positioned at different angles. Also, when using liquid phase deposition all the sides of the recess are be coated, which can be beneficial in some applications. 
     Employing the method of liquid phase deposition in the field of microfluidics is inventive in its own right and therefore, viewed from another aspect the invention provides a method of manufacturing a microfluidic structure comprising the steps of forming a substrate having a microfluidic recess and depositing a layer of silica on the substrate by liquid phase deposition. 
     Microfluidic structures in accordance with the present invention can be used in many different applications, but they are particularly suited for use in fluorescence detection (wherein, for example, the fluorescence is provided by labeled cells or concatemeric DNA molecules), cell culture and in the construction of valves and pumps. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
       A preferred embodiment will now be described, by way of example only, with reference to the accompanying drawings, in which: 
         FIG. 1  shows a thermoplastic portion having a microfluidic recess in accordance with the present invention; 
         FIGS. 2A-D  show a process of forming a microfluidic device according to the present invention as shown in relation to a cross section along line A-A of  FIG. 1 ; 
         FIGS. 3A-D  show a process of forming a microfluidic device according to the present invention; 
         FIGS. 4A-C  show Electron Spectography for Chemical Analysis (ESCA) data both before and after disposition of a silica layer on a thermoplastic portion; 
         FIGS. 5A-E  show a further example of a process of forming a microfluidic device according to the present invention; 
         FIG. 6  shows a cross section through the finished microfluidic device created by the process shown in  FIGS. 5A-E ; 
         FIG. 7  shows a side view of a microfluidic chip composition in accordance with the present invention; 
         FIGS. 8A-C  show the construction of a microfluidic pump device in accordance with the present invention; 
         FIGS. 9A  and B show a cross section through the completed device created by the process  FIGS. 8A-C ; 
         FIG. 10  shows optical cross sections through a completed device according to the present invention; and 
         FIG. 11  shows how the support layer can be removed form this chip if required. 
     
    
    
     DETAILED DESCRIPTION 
       FIG. 1  shows a thermoplastic portion  10  after injection moulding to create microfluidic recesses  12 . These consist of a narrow channel  12   a  and larger reservoirs  12   b  for storing liquid. These recesses  12  are formed on the thermoplastic portion  10  by injection moulding using standard compact disc (CD) moulding technology. Numerous chips can be moulded on a single CD after which each chip is diced out. 
     Thermoplastic portion  10  is also shown in  FIG. 2A  in cross section along the line A-A. In order to seal recesses  12 , a layer of silica  14  is deposited on the upper side of thermoplastic portion  10 . This is achieved by electron beam evaporation. 
     In a separate process a layer of PDMS is prepared by spin coating uncured PDMS onto a support film (see  FIG. 5C ). The PDMS is then cured to form a solid layer of PDMS  16 . This can be carried out a location and time remote from the thermoplastic chip manufacture and the cured PDMS stored until such time as it is needed. Therefore the curing time does not interfere with the manufacturing time of the microfluidic structure. 
     When it is desired to join the thermoplastic portion  10  and PDMS layer  16  together, the silica layer  14  and the PDMS layer  16  are oxidised. They are then brought into contact with no external pressure and form a covalent irreversible bond, as shown in  FIG. 2C . 
     If desired a further thermoplastic portion  100  can be attached to the other side of PDMS layer  16 , again using an oxidised silica layer  140  and oxidizing the other side of the PDMS layer  16 . This is shown in  FIG. 2D . By forming the second thermoplastic layer  100  with microfluidic recesses  120  (shown in phantom) and positioning these perpendicular to those of the first thermoplastic portion  10 , valves and pumps can be created. Such a thermoplastic layer  100  can thus act as a control chip, wherein pressurised air pumped into recesses  120  control the flow of fluid in microfluidic recesses  12 . This type of device is described in more detail in relation to  FIGS. 8A-C . 
       FIGS. 3A-C  show this process again in relation to a thermoplastic portion  200  comprising only a single microchannel  220 . Thermoplastic portion  200  is manufactured as previously described. A layer of silica  240  is then deposited on the top surface of the thermoplastic portion  200  using electron beam deposition, although other methods can also be used.  FIGS. 4A-C  show the ESCA data obtained from a surface analysis of the thermoplastic portion  200  before (line A) and after (line B) silica deposition. It can clearly be seen that the carbon of the thermoplastic portion is covered by the silica leaving a surface layer rich in silicon and oxygen. 
     This silica deposition results not only in a layer of silica  240  on the top of the microstructure  200  but also a layer in the microchannel  220  itself. This allows well known glass treatment techniques to be applied to the microchannel  220 . 
     As shown in  FIG. 3D  a lid  250  is created comprising a support film  251  of thermoplastic material (e.g. Zeonor) and a Layer of siloxane elastomer  252  (e.g. PDMS). 
     In order to bond the lid  250  to the thermoplastic portion  200  both components are oxidised and then placed in contact with each other, see  FIG. 3C . Without the need to apply external pressure a covalent bond is created sealing the microchannel  220 . 
       FIG. 5A  shows that the thermoplastic portion  200  is diced from an injection moulded CD  20  created using well known techniques. In  FIG. 5B  the step of silica deposition is shown. This results in a thin coating  240 , approximately 300 nm, being formed on the top surface of the portion  200 . 
       FIG. 5C  shows the creation of the lid  250  using spin or spray coating to create a layer of PDMS  252  on a Zeonor support sheet  251 . This can then be diced into appropriate sizes for attaching to the microstructures. 
     In order to bond the components together a surface of the PDMS layer  252  and the silica coated side of the microstructure  200  are oxidised and then placed in contact with each other. The finished product is shown in  FIG. 5E . A cross section taken along plane A is shown in  FIG. 6  in which the different layers of the structure can clearly be seen. 
       FIG. 7  shows these layers photographically. Again the thermoplastic portion  200  is shown with a thin layer of silica  240  attached to a PDMS lid  252  which in turn is attached to a support film  251 , which can be removed if necessary. 
     One occasion in which the support film  252  is removed is when the device is intended for use as a pump chip. The process of making such a chip is shown in  FIGS. 8A-C . 
       FIG. 8A  shows a standard thermoplastic portion  300  of the type described in relation to  FIGS. 3A and 4A  having a single flow channel  320 . A second thermoplastic portion  400  is also provided. This is shown in  FIG. 8B  and comprises of a number of separate flow channels  420  which are arranged to be perpendicular to the flow channel  320  of portion  300  in the completed chip. Both of these portions  300 ,  400  have a silica layer deposited on the surface  301 ,  401  in which their respective channels are located. A siloxane membrane  352 , as created in accordance with  FIG. 5C  but with the support film removed, is sandwiched between the two portions  300 ,  400  to create a flexible membrane. The upper and lower surfaces of the siloxane membrane  352  and the silica coated layers  301 ,  401  of thermoplastic portions  300 ,  400  are oxidised and bonded together to create the completed pump chip shown in  FIG. 8C . 
     A cross section of this completed chip taken along plane B is shown in  FIGS. 9A and 9B . In the open mode the pump, or control, channel  421  is not distorted by pressurised air and so the flow channel  320  is left open. When pressurised air is pumped into the pump channel  421  the siloxane layer  352  above the flow channel is distorted into the flow channel  320 , preventing the flow of fluid along this path. By operating the pump channels in sequence material can be pulsed along the flow channel  320 . While the rectangular flow channel  320  can be adequately closed by this method it is also possible to create curved flow paths that allow a more complete closure to be created. 
       FIG. 10  shows a completed microfluidic structure  500  having a number of channels and reservoirs. Optical cross sections were taken through the structure  500  at the positions indicated (I, II, III). The channels were filled with 10 micromolar of the fluorescent substance Cy5 followed by washing with distilled water. Confocal z-sectioning was performed using a Carl Zeiss LSM 5 META confocal microscope (10×/0.45 NA Carl Zeiss Plan-apochromat objective). Twenty slices spaced 7.8 micrometer apart in the z-axis was scanned generating a 156 micrometer thick stack of images, the pinhole was set to 50 resulting in an optical slice of &lt;5.8 micrometer. A HeNe laser of 633 nm was used for excitation and emission was collected using a 650 nm long pass filter. 
     This resulted in an x/z image of 1300×156 micrometer covering the top and bottom of the microchannel. The results show an upper thin line corresponding to the elastomer lid and a lower line representing the thermoplastic chip. In the first cross section I the lines are broken, indicating the different microchannels. In each of these cross sections it can be seen that the lines are parallel. indicating that no sagging of the lid has occurred. This is true even in the middle of the channel (seen at cross section III). This result is achieved as no external pressure is required to bond the lid to the chip. 
       FIG. 11  provides an illustration of how the support film  551  used during the creation of the PDMS layer can be removed if necessary, for example in order to create the pump device shown in  FIG. 8C . Alternatively this layer can be retained on the chip, as for example in optical detection. 
     Example 1 
     System Fabrication 
     Microstructures were designed in L-Edit Pro (Tanner EDA, Pasadena, Calif., USA). Master fabrication and compact disc injection molding were performed by Åmic AB (Uppsala, Sweden). Connection holes were drilled and the desired structures were diced out. The plastic chips were rinsed in ethanol and blown dry with nitrogen. SiO 2  was deposited on the chips in an Edwards 306 ED 3 electron beam evaporation chamber, resulting in an approximately 300 nm thick layer (filament current: 60 mA, acceleration voltage: 5.58 kV, pressure 3.6×10−6 mbar). The chamber temperature during deposition is well below the glass transition temperature for most thermoplastics. An atomic force microscope (Nanoscope III, Digital Instruments) operated in contact mode was used for surface topology imaging. PDMS (Elastosil RT 601, Wacker) prepolymer and curing agent were mixed at 10:1 and degassed. The uncured PDMS was spin-coated onto an 85 μm thick Zeonor support film (Nippon Zeon Co, Japan). A rotational speed of 1000 rpm resulted in an approximately 110 μm thick PDMS film, followed by curing at 70° C. for 1 hour. A Corona surface treater (Model ED-20, Electo-Technic Products, Illinois, USA) was used to oxidize the surfaces to be bonded. The electrode was swept back and forth 3 mm above the SiO 2  coated surface of the plastic chips for one minute, and over the PDMS film for 30 sec. The two surfaces were brought in contact and left for 10 min at 60° C. with no external pressure. Tensile tests were performed by an Instron 5544 load frame (Instron, Canton, Mass.). The support film was left in place for optical applications, but peeled off for cell culture and actuator applications. 
     Fluid Pumping 
     Chips for fluid pumping ware fabricated by sandwiching the oxidized elastomer film (approximately 110 μm) between two oxidized silica coated Zeonor chips, one representing a flow channel chip and the other one a control channel chip. All channels had a depth of 15 μm. Four control channels (width 200 μm) were oriented perpendicular to the flow channel (width 1800 μm). Pumping was also performed using 200 μm wide channels, resulting in similar flow velocities. Pressurized air (35 psi), modulated by computer-controlled three-way switch valves (LHDA 1211111H; Lee Valve, Westbrook, Conn., USA), was connected to the channels of the control chip. A 16-channel relay output board (PCLD 785, ELFA, Sweden), driven by a digital in/out board (Advantec PCI 1751, ELFA, Sweden), was employed for valve control. Dedicated software, written in Visual Basic 6 (Microsoft), was developed for hardware management. Pressurized air was applied sequentially to the control channels in each pump cycle. A typical cycle time was 500 ms with 100 ms lag time between adjacent control channels and 200 ms inter-cycle time. A suspension of fluorescent microbeads (Molecular probes, Eugene, Oreg.) was pumped through the channel and the particle movement was observed by fluorescence microscopy (Zeiss Axiovert). The volumetric flow rate was calculated from the linear velocity of the fluorescent microbeads. 
     Cell Culture 
     Sealed chips with the support film removed were sterilized in ethanol and thoroughly rinsed with cell culture medium. Chinese hamster ovary (CHO-KI) cells tranfected with an enhanced green fluorescent protein plasmid (Clontech) were removed from the tissue culture vessels by trypsin treatment. The resuspended cells were washed in cell culture medium (RPMI 1640 with 10% FBS and penicillin/streptomycin), and injected in the microchannels using a syringe. The chips were immersed in a Petri dish containing cell culture medium and incubated at 37° C. with 5% CO 2 . Chips were transferred to a Petri dish containing fresh medium after 72 hrs. Cell viability, adhesion and growth were monitored for seven days by removing one chip every other day, for examination by fluorescence microscopy. 
     Cell Counting 
     Mononucleated cells were purified from peripheral blood by Ficoll separation and resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4). The cells were stained by anti-CD19 PE+anti-CD3 FITC, anti-CD4 PE+anti-CD8 FITC, or with anti-CD20 FITC+anti-CD4 PE (Dako). The three different stained lymphocyte samples were pumped through three parallel microchannels (50×40 μm cross section.) with a syringe pump PHD-2000 (Harvard instruments) at a rate of 1 μL/min. A confocal microscope (Zeiss LSM 5 META) operated in line-scanning mode, across all three channels perpendicular to the direction of liquid flow, was used to detect the cells. The microscope focus was set to the center of the microchannels and the pinhole was set to 400 μm corresponding to a calculated optical slice of &lt;5 μm. Typically 10000 lines of 512 pixels were recorded, with a voxel time of 1.6 microseconds, resulting in a data acquisition time of approximately 10 seconds. The line scan data were stored in an 8-bit/channel rgb-TIF-file and analyzed by dedicated software written in MATLAB 6.5 (MathWorks, Mass.). The data obtained was verified by flow cytometry using a FacSORT instrument (Becton Dickinson). 
     Therefore the present invention provides an efficient means for large scale, inexpensive production of disposable microfluidic chips with integrated actuators. The microfluidic structures of the present invention support mammalian cell growth and have excellent optical properties for fluorescence detection.