CA2653038A1 - A microfluidic pipetting system for micro-dosing biological materials and macromolecules - Google Patents

Abstract

A novel microfluidic high throughput screening device is provided. The device is particularly suitable for cell based assays. The device comprisesa nanoporous membrane imprinted with PMDS to block all pores except in specific regions.
The membrane is sandwiched between a gel layer and a microfuid network. Model drug candidates can be pumped into the gel layer from the microchannels below in a controlled manner.

Description

A MICROFLUIDIC PIPETTING SYSTEM FOR MICRO-DOSING BIOLOGICAL
MATERIALS AND MACROMOLECULES

FIELD OF INVENTION

[0001] This paper relates a new microfluidic High Throughput Screening (HTS) device targeted for cell based assays.

BACKGROUND OF THE INVENTION

[0002] In the last decade there has been enormous development in the field of microfluidic systems and related applications. Due to its inherent large-scale, combinatorial and repetitive nature, High Throughput Screening (HTS) for drug discovery offers an excellent opportunity for application of microfluidic technologies. Recently, cell based HTS - where live biological cells (both animal and human) are used to test various compounds to determine their cellular response - have been increasingly used for drug discovery applications [1].
The main advantages of cell-based assays over solution based immunoassays are: a) the cellular response is closer to higher level biological response and b) it allows high-content, multi-parametric studies to elucidate structure-function relationships, binding affinity and pharmacological properties of lead compounds. These provide a better nuanced picture of the compound target interaction in the context of intra- and inter-cellular communication. High-throughput and the ability to deliver precise doses of drug candidates over long testing periods is critical for cell-based HTS [2]. Traditional cell-based HTS uses microwell plates that confine cells in a small volume and deliver non-uniform pulsed doses of drug candidates using robotic arms. The main drawback of this approach is that the cells, dispensed into microwells plates lack contact with neighbors, and screens based on them are of limited physiological significance [3]. Another factor is that pipette based droplet dispensing is not accurate for sub microliter volumes due to surface tension effects. Microfluidic technologies have the ability to precisely handle small volumes and show good promise for application in HTS systems. Several microfabricated fluid handling systems using pneumatic, thermo-pneumatic actuation, electrostatic, electrokinetic, electrohydrodynamic, magneto hydrodynamic and ultrasonic actuation have been demonstrated [4]. Of these, electrokinetic actuation has been found to be the most versatile in accurate metering and delivery of a constant and sustained dose of drug candidates to individual HTS compartments due to electrical control, fast response times, simple fabrication and no moving parts.

[0003] In the recent past, a number of microfluidic devices useful for cell-based HTS have been reported [5, 6]. Lee et. al. [5], fabricated a 10x10 array of microchambers to carryout long-term cell culture and observation for HTS
applications. The cell culture was carried out in an enclosed microchamber where achieving physiologic cell culture environment uniformly in all the microchambers might be difficult. Further miniaturization of this format will lead to space constraints and affect the growth and behavior of the cells. Alternatively, Sabatini et. al. [6] have reported creation of microarrays gel microspots containing cDNA, on which cells were subsequently cultured. This format maintains uniform physiologic environment and does not restrict cell growth. However, dosage of the cDNAs is passive and dependent of slow release based on dissolution of the gel.

[0004] Precise and accurate extraction and delivery of micro to nano amounts of biological materials such as DNA, proteins and cells and other chemical compounds such as drug candidates and macromolecules are critical for a number of applications including high throughput screening (HTS) for drug discovery and biochemical analysis. Microfluidics with electrical control is the most suited method for accurate metering, extraction and delivery.

[0005] Traditional cell based HTS uses microwell plates that confine cells in a small volume and deliver non-uniform pulsed doses of drug candidates using robotic arms. The main drawback of this approach is that the confined cells, without forming contacts with neighbours, are distressed an do not accurately reflect their behaviour in the body. Several microfluidic HTS devices have been reported (1,2) that miniaturize existing microwell plates but they also suffer for the disadvantage mentioned above. Furthermore, they need special instruments, for measurement of cellular response (1,2) or use passive release by spotting drug candidates mixed with gel (3). Thus, there existed a need for an improved microfluidic system for micro-dosing potential agents.

SUMMARY OF THE INVENTION

[0006] The invention provides a microfabricated device capable of continuous, accurate and actively controlled dosing of model drug molecules into an array of individual microspots on a continuous gel layer on which cells can be cultured under normal physiological condition. Complete intercellular communication between cells and hence enhanced physiological significance of screening is possible by having a continuous gel layer on which the cells can be cultured instead of microwells. Secondly, electrophoretic pumping through precisely defined nanoporous regions is used for actively controlled dosage of drug candidates into specific regions in the gel. Thirdly, the gel is used as a convection barrier in preventing mixing of the dosage between neighboring spots.

[0007] The device typically comprises a nanoporous membrane, imprinted with poly-dimethylsiloxane (PDMS) to block all pores except in selected areas (microspots) arranged in an array. This membrane is sandwiched between a gel layer above, for cell culture, and a microfluidic network below, which provides fluidic access to the gel region above the microspots. The nanoporous membrane reduces diffusion significantly while allowing electrophoretic motion.
Electrophoretic pumping of model drug candidates into the gel from the microchannels underneath in a controlled manner was demonstrated. Drug spots of 200pm to 6mm in diameter on 3mm thick gel layer were created. Dosing accuracy of 50Ng was achieved.

[0008] The present invention provides a method for use of precise electrical control of the transport of the target material to create different dose profiles for HTS or to extract small amount of target material for analysis from a gel.

[0009] In one aspect of the invention, a microfluidic device for high throughput screening is provided. The device comprsies a bottom layer having a plurality of microchannels, a middle layer comprising a nanoporous membrane and an upper gel layer. The bottom layer is preferebly made of PDMS elastomer. The middle layer is preferably a thin, porous poly-carbonate membrane and the upper layer is preferably comprises an agarose gel.

[0010] In another aspect of the invention, the device comprises a microfluidic tube with the nanporous membrane at one end. This tube is in contact with a gel layer.
Several microfluidic interfaces can be used to deliver agents to different spots.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows schematic and working principle of the device. A nanoporous membrane is sandwiched between bottom layer containing microchannels and top layer of gel meant for cell culture. Upon application of electric field the drug molecules move into precisely defined microspots on the gel;

Figure 2 shows schematic view of an array of drug spots created. The darker regions are where drug gets pumped by electrophoresis. Lighter blue regions are where drug migrates because of molecular diffusion. A separation between the diffused regions of two adjacent spots is necessary to avoid interaction between them.;

Figure 3 A illustrates a fabrication process flow for the microarray device.
B: 3D
Schematic of the device;

Figure 4 A illustrates a device according to one aspect of the invention; 4B
is an electrical resistance diagram when electrodes are connected to two separate channels.; and 4C is an electrical resistance diagram when electrodes are connected to one channel and gel;

Figure 5 demonstrates the creation of microspots of drug molecule in a number of locations on the gel using simple electrical control. Figure 5A shows this device in initial configuration when the channels are filled. Subsequently, the potentials in the microchannels are changed as depicted in Figure 5B such that electric field lines are established across the gel. The figure also shows microspots of trypan blue created by application of 30 V for 1 min in two locations while there is no pumping in the third spot which has zero potential difference;

Figure 6 shows a demonstration of dominance of Electromigration over diffusion.
A. No field applied, B. No field applied for 1 hour. Electric potential 30V
applied for 30 sec.;

Figure 7 shows a variation of spot size with duration of applied electric potential (30V) application. A. no potential. B. 10 sec C. 30 sec D. 3 min.; and Figure 8 shows diffusion effect on spot size A. 0 min (-2mm dia) B. 25 min (-4mm) C. 60 min (-6mm) D. Variation in compound concentration profile of a single spot due to diffusion.

DETAILED DESCRIPTION

[0012] The present invention provides a microfluidic pipetting system for micro-dosing biological materials and macromolecules. In one embodiment, the device consists of a gel layer (where the cells are cultured and into which precisely controlled doses of drug candidates are delivered) supported by a low-porosity nanoporous membrane that is bonded to a network of microchannels underneath it. The gel itself restricts convective transport in it and creates virtual microwells, confining the transported materials pumped from underneath, spatially. The nanoporous membrane is stamped with a thin PDMS prepolymer layer to block all pores except in several micro regions that serve as fluidic interfaces between the microchannel below and the gel above. Low pore density and significant diffusional resistance of individual pores restrict diffusive transport between the microchannels and the gel. However, transport resistance is decreased upon application of electric potential between two microchannels through the nanoporous membrane and the gel and the pores act as electrophoretic pumps.
By selectively switching an array of such micropumps, a number of spots -containing drug molecules - are created simultaneously. Size of each spot can be controlled by pumping potential and duration. Spot sizes ranging from 200um to 6 mm diameter and having inter-spot distances of 1 mm-10mm have been created.

[0013] In another embodiment, the device consists of a microfluidic tube with the nanoporous membrane at one end. The tube is electrified using an electrode which is in contact with the liquid in the tube. The nanoporous membrane has the same function as in its previous manifestation, in preventing diffusional transport while allowing electromigration upon application of electric field. This device coupled with an x-y positioner has been used to spot small molecules in defined location on a gel layer. This device can be extended to a chip with several nanofluidic interfaces that is capable of spotting several spots simultaneously upon electrical control.

[0014] Figure 1 shows the schematic cross-section of a fabricated device. The device has three layers; the bottom layer is made of PDMS and consists of a microfluidic network. The middle layer is composed of a nanoporous membrane with a narrow pore-size distribution. These membranes have been used previously for biomolecule pre-concentration and for studies of transport in nanopores [7]. The nanoporous membrane is patterned using imprint lithography such that pores in region other than an array of 200x200p m2 spots are blocked.
These spots provide fluidic access from the microfluidic network underneath, to specific locations onto the layer above the membrane. The top layer of the device is composed of a hydrogel (agarose) that provides the surface and the medium for cell culture.

[0015] When the microchannels are filled, the difference in concentration of drug molecules between the microchannels and the gel establishes a concentration gradient across the nanoporous membrane. As the membrane pore-size approaches that of the solute, interaction of solute molecule with the walls become significant compared to its interaction with other solute molecules and diffusion kinetics become slower and non-Fickian [8]. Furthermore, the pores become electrified due to the zeta potential of the walls and this substantially reduces the motion of solute of appropriate charge [7]. This combined with the low-density of pore-sites significantly reduces diffusional transport of the drug molecules through the membrane.

[0016] When an electric field is applied either between two channels as depicted in Figure 1 or between a microchannel and the gel layer, the field lines pass through the membrane. Drug molecules, due to their electrophoretic mobility, migrate from the channel into gel through membrane. Electromigration velocity is several orders of magnitude higher than the diffusional velocity [7]. Since the membrane is porous only in certain spots due to microprinting, drug molecules can be pumped quickly and precisely to defined areas on the gel as depicted in Figure 2.

[0017] The gel layer prevents convective transport of the solute molecules and serves as a virtual well, confining the molecules to a local spot. However, diffusive transport still occurs, and the spot starts to grow in size as shown in Figure 2.
Initially the drug molecule is concentrated just above the fluidic access port on the nanoporous membrane as shown by the smaller darker region. As time progresses, diffusion occurs, and the drug molecules spread over a larger area reducing the local concentration. As is seen in Figure 2, a concentration gradient is established across the cross section of the gel which can be maintained, exposing the cells cultured on the top of the gel layer to an electrically controlled concentration of drug molecules. However, since diffusion is isotropic, drug molecules spread laterally as well leading to unwanted interactions between adjacent spots and this limits the maximum spot density of the virtual microwells.
By reducing the thickness of the gel layer, the spots can be brought closer, increasing the spot density.

[0018] The gel layer being un-compartmentalized allows the use of established cell culture equipment and conditions. Furthermore, cells cultured in this manner are not confined and form interactions and establish intercellular communications with other cells and hence reflect a more accurate physiological condition compared to microwells. Cell to cell interaction is very crucial for their proliferation and normal physiological functioning [3] and is critical for obtaining high-content multi-parametric response.

[0019] Device is made of three layers assembled and bonded together using spin coated PDMS as an adhesive layer. The fabrication process flow is shown in Figure 3A. The bottom layer is made of PDMS elastomer and contains microchannels (200-im wide and 100-im deep). A SU-8 master mold, containing the structural features of the microfluidic network, is made using soft lithographic technique as described elsewhere [9]. PDMS prepolymer (1:10 mixture of curing agent and base) was cast on this mold, cured at 65 C and the formed elastomer pealed off. Access holes (1 mm in diameter) to the reservoirs in the microchannels are made by cutting PDMS using glass capillaries. Middle layer consists of an 8im thin porous poly-carbonate membrane (GE Osmonics) having uniform pore sizes of 100nm and pore density of 4x104 pores/mm2. The membrane is patterned with PDMS prepolymer using micro imprint method such that pore in regions other than an array of 200imx200im squares (each containing -2400 pores of 100nm diameter) are filled with the prepolymer. This patterned membrane is aligned and attached to the bottom PDMS microchannel layer with a thin layer of spin coated PDMS prepolymer adhesive. The top layer is a 3mm thick PDMS sheet from which the central region (4 cm x 4 cm) is cut and serves as a reservoir for casting of agarose gel layer. Agarose gel (2 wt % agarose) was mixed in 0.1 M KCI solution and cast into the rectangular cavity of the top PDMS
layer. After cooling to room temperature this gel solution forms a 3-mm thick sheet and its top surface can be used for cell culture. Figure 3B shows 3D
schematic structure of this device.

[0020] Methylene blue and trypan blue are chosen as model drug molecules due to their comparable properties to drug molecules (They are complex organic compounds, M.W.: 284.41, 872.9; IogP: 1.16, 4.05; H-bond donors: 0, 8; H-bond accepters: 4, 20 respectively) and ease of visualization. These were purchased from Sigma Aldrich and used as such. Charges on trypan blue and methylene blue are opposite in nature and this is used to verify electrophoretic nature of solute transport.

[0021] Model drug molecules dissolved in 0.5M KCI solution were filled in the microfluidic network through syringes connected to the access holes in the bottom PDMS layer. Keithley 237 was used as source and measurement unit. Figure 4A
shows the actual device and measurement setup. Test and measurement arrangements for two configurations are shown in figure 4B&C respectively. In the first arrangement electric potential is applied between two microchannels and the field passes through the membrane gel combination, while in second arrangement, potential is applied between a microchannel and gel layer and the field passes through the membrane. Both these arrangements lead to electromigration of drug molecules into the gel.

[0022] Once drug molecules are pumped into the gel, the gel layer is separated from the device and concentration measurements were carried out by photometry using Wallac 1420 VICTOR 2 T"' plate reader (PerkinElmer). Recorded absorption measurements were also corrected for background absorption of gel layer. The detection limit of our instrument by photometry for trypan blue was found to be 4.2 micrograms/mm2.

[0023] Several unique features of this device include use of low-porosity nanoporous membrane for diffusion minimization and precise electrical dose control, direct compatibility of the device with existing HTS analysis instruments and most importantly, its capability to perform HTS in uncompartmentalized gel plate that is most conducive for cell culture and tissue simulation.

[0024] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations will be apparent to one skilled in the art. All reference documents referred to herein are hereby incorporated by reference.

EXAMPLES

[0025] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of microbiology, molecular biology and chemistry referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1. Craation of microspots [0026] Figure 5 demonstrates creation of microspots of drug molecule in a number of locations on the gel using simple electrical control. Figure 5A
shows this device in initial configuration when the channels are filled.
Subsequently, the potentials in the microchannels are changed as depicted in Figure 5B such that electric field lines are established across the gel. The figure also shows microspots of trypan blue created by application of 30 V for 1 min in two locations while there is no pumping in the third spot which has zero potential difference.
Example 2. Effect of electromigration [0027] Dominance of electromigration over diffusional transport is demonstrated in Figure 6. The microchannels are filled with trypan blue and the allowed to diffuse through the nanoporous membrane for 1 hour. There is virtually no diffusional transport (less than visual and working limit of instruments i.e. 4.2 micrograms/mm2) through the nanoporous interface in the absence of electric field even after one hour after establishment of the concentration gradient as is seen in fig 6B. But, application of electric potential for 30 sec creates electromigratory transport of drug molecule into the gel as shown in Fig 6C.
Example 3. Effect of pumping potential and duration [0028] Size of each spot and amount of drug molecule pumped can be controlled by varying either the pumping potential and duration (Figure 7). Spot sizes ranging from 200im to 6mm diameter and having inter-spot distances of 1 mm-10mm have been created. Doses between 50-3000 g of typan blue were pumped into the gel.

Example 4. Effect of diffusion on spot size [0029] Molecular diffusion of drug molecule in the gel subsequent to the end of electromigration results in the increase of the original spot size with time.
Figure 8 A,B,C shows one such spot growing in size with increasing time. As shown in figure 8D the radial direction concentration profile of model drug in a single spot is narrow at time of spot creation but with increasing time grows in size while reducing in peak concentration. The number of spots that can be created in a given area is thus limited by this diffusion. Spot densities of 5.5 spots per cm2 were achieved in this study which compares favorably to 3.5 well/cm2 for standard 384-well plates.

[0030] The mechanism of pumping is verified by using oppositely charged molecules (trypan blue and methylene blue) dissolved in 0.5M KCI solution.
Electroosmotic pumping direction is dependent on the properties of solvent and nanopore surface charge (i.e. zeta potential) while for electrophoretic pumping it is depends upon the charges on molecules. The transport direction for trypan blue and methylene blue was opposite verifying pumping mechanism to be electrophoretic.

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Claims (15)

1. A microfluidic device for high throughput screening, said device comprising a bottom layer having a plurality of microchannels, a middle layer comprising a porous membrane and an upper gel layer.

2. A microfluidic device according to claim 1 wherein the bottom layer is made of PDMS elastomer.

3. A microfluidic device according to claim 1,wherein the microchannels are about 200 um wide and about 100 um deep.

4. A microfluidic device according to claim 1 wherein the porous membrane is a nanoporous membrane.

5. A microfluidic device according to claim 1 wherein the middle layer is a thin, porous poly-carbonate membrane.

6. A microfluidic device according to claim 1 wherein the nanoporous membrane comprises pores having a diameter of about 5 nm to 5um.

7. A microfluidic device according to claim 1 wherein the pores have a diameter of about 100 nm.

8. A microfluidic device according to claim 1 wherein the upper layer comprises a medium suitable for cell growth.

9. A microfluidic device according to claim 1 wherein the medium suitable for cell growth is a gel selected from the group consisting of hydrogel, polyacrylamide gel agarose gel and the like.

10.A microfluidic device according to claim 1, wherein the upper layer comprises an agarose gel.

11.A method of making a microfluidic device according to claim 1, said method comprising: preparing a first layer comprising PDMS elastomer containing microchannels; applying to said first layer, a second layer comprising a porous membrane and applying a third layer of a gel to the second layer.

12.A method of delivering a test substance to cells, said method comprising obtaining a device as defined in claim 1, establishing growth of cells on the gel layer, filling the microchannels with a test substance and electrophoretically pumping the test substance from the microchannels to the gel in predetermined spots.

13.A microfluidic device for nanoscale pipetting, said device comprising a top layer and a bottom layer wherein the top layer comprises a plurality of microchannels, and a reservoir and the bottom layer comprises a nanoporous membrane having electrodes embedded in the top layer.

14.A device according to claim 13 wherein the reservoir is a pipette or container.

15.A method of depositing biomolecules, cells and macromolecules, said method comprising obtaining a device as defined in claim 11, filling the microchannels, a pipette or a container with a biomolecule or cells or macromolecuels and electrophoretically pumping the test substance from the top layer to a electrified surface, gel, liquid layer, metal in predetermined spots.