Patent Application
20090250345
The present invention is related to the field of electroelution devices and processes, and in particular, microfluidic devices and processes for electroelution with sample collection decoupled from the electrophoretic field.
A fundamental difficulty in biochemistry, genetics, and molecular biology is the ability to reproducibly and efficiently identify and recover electrophoretically separated macromolecules following acrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely utilized as a preparative and analytical technique for the separation of proteins and other macromolecules, e.g., nucleic acids, antigens, and antibodies. Many techniques exist in the art to identify and recover proteins from an acrylamide gel. Most common techniques rely on diffusion or elution to extract the proteins from the acrylamide gel. For example, electroelution is a technique whereby proteins are electrophoretically removed from the acrylamide gel. In electroelution, an SDS-PAGE gel can be treated with a stain, e.g., Coomassie Brilliant Blue or SYPRO orange, to allow the visualization of proteins previously separated by SDS-PAGE. A gel band or spot containing the proteins of interest can be excised from the gel matrix and placed in an elution apparatus. The elution apparatus can create an electrophoretic field across the gel spot such that the proteins are electrophoretically eluted from the gel spot. The electroeluted proteins can be collected for further analysis or sequencing.
However, current electroelution procedures are generally inefficient and non-reproducible for a variety of reasons. For example, most procedures are time consuming because the electroelution apparatus is typically only able to elute a limited number of samples in a given time period. In addition, proteins can adsorb onto the surfaces of the electroelution apparatus or resorb onto the gel during the electroelution process. Furthermore, the overall efficiency and reproducibility of most electroelution procedures are reduced by losses of the sample during extraction and collection, and by contamination of the sample during transport and handling.
However, recent advances in miniaturization have led to the development of microfluidic systems that are designed, in part, to perform a multitude of chemical and physical processes on a microscale level. Microfluidic devices are generally fabricated on a substrate having a system of microstructures, e.g., microchannels and microchambers. Such microfluidic devices can have an internal volume of less than one microliter and the length scale of these channels is typically on the micron or submicron scale, i.e., having at least one cross-sectional dimension in the range from about 0.1 micron to about 500 microns. Microfluidic electroelution devices may offer faster response times and provide precise control over small volumes of fluid by collecting and concentrating many samples in parallel. Such microfluidic devices could enable the development of electroelution devices and processes that increase reproducibility and reliability by reducing sample processing time and sample degradation.
Accordingly, there is a need for microfluidic electroelution devices and processes that reproducibly and efficiently extract electrophoretically separated intact proteins from acrylamide gels.
An embodiment of a microfluidic electroelution module with sample collection decoupled from the electrophoretic field can generally comprise a channel having an inlet and an outlet, a receptacle in fluid communication with the channel intermediate the inlet and outlet, a first port and a second port in fluid communication with the channel, the second port positioned intermediate the receptacle and outlet, the receptacle located between the first and second ports, the first and second ports adapted to receive a first electrode and a second electrode, respectively, such that the electrodes will complete an electrical circuit when fluid is present in the channel to create the electrophoretic field across the receptacle when power is applied to the electrodes, and a flow restricting feature and/or a flow enhancing feature in fluid communication with the channel intermediate the second port and outlet such that fluid flow in the channel towards the outlet is encouraged and fluid flow in the channel towards the second port is discouraged. In further embodiments, the microfluidic modules can further comprise a sorbent material, e.g., a monolith or packed bed, in the channel intermediate the second port and the outlet such that the sorbent material is decoupled from the electrophoretic field created between the electrodes. In still further embodiments, the microfluidic module can be integrated onto a microfluidic chip.
An embodiment of a method of electroelution with sample collection decoupled from the electrophoretic field can generally comprise providing a first fluid pathway in fluid communication with a second fluid pathway, associating a sample having at least one macromolecule species of interest with the first fluid pathway, e.g., positioning a gel spot containing the species of interest in the receptacle, providing an elution liquid in the first and second fluid pathways, creating an electrophoretic field in the first fluid pathway, electrophoretically separating the species of interest from the sample by the electrophoretic field, and causing the species of interest to flow from the first fluid pathway toward the second fluid pathway by using a flow restricting feature and/or a flow enhancing feature. In further embodiments, the method can further comprise collecting the electroeluted species on a sorbent material, e.g., a monolith or packed bed, in the second fluid pathway, and processing, e.g., rinsing, desalting, purifying, and/or concentrating, the species collected on the sorbent material, and/or removing the collected species from the sorbent material.
An embodiment of a microfluidic module for electroelution with sample collection decoupled from the electrophoretic field can generally comprise a channel having a first fluid pathway in fluid communication with a second fluid pathway, the first fluid pathway comprising a first port in fluid communication with a second port, and a receptacle adapted to receive therein a sample containing at least one macromolecule species of interest, the receptacle in fluid communication with the first port, the second fluid pathway comprising an inlet in fluid communication with an outlet, wherein the first port is associated with a first electrode and the second port is associated with a second electrode such that the electrodes will create an electrophoretic field across the receptacle when fluid is present in the channel and power is applied to the electrodes, wherein the channel is configured to create a pressure drop from the first fluid pathway towards the second fluid pathway when fluid is present in the channel, and wherein the pressure drop encourages the electroeluted species of interest to flow from the first fluid pathway toward the second fluid pathway. In further embodiments, the pressure drop can be created by the relatively large volumes and column height of fluid in the reservoirs in the first fluid pathway compared to the volume and column height of fluid in the outlet reservoir.
An embodiment of a method of electroelution with sample collection decoupled from the electrophoretic field can generally comprise providing a first fluid pathway in fluid communication with a second fluid pathway, associating a sample having at least one macromolecule species of interest with the first fluid pathway, providing an elution liquid in the first and second fluid pathways, creating a pressure drop from the first fluid pathway towards the second fluid pathway, creating an electrophoretic field in the first fluid pathway, electrophoretically separating the species from the sample by the electrophoretic field, and wherein the pressure drop causes the species to flow from the first fluid pathway toward the second fluid pathway. In further embodiments, the method can further comprise the step of collecting the species on a sorbent material, e.g., a monolith or packed bed, in the second fluid pathway for further processing, e.g., rinsing, desalting, purifying, and/or concentrating, and/or removing the collected species from the sorbent material.
A more complete understanding of the microfluidic electroelution devices and processes can be obtained by considering the following description in conjunction with the accompanying drawing figures in which like reference numbers refer to like elements, in which:
The term “microfluidic” as used herein describes structures or devices through which a fluid is capable of being passed or directed, wherein one or more of the dimensions is less than about 500 microns, e.g., depth, width, length, diameter, etc. In the devices of the present invention, the microstructures can have at least one cross-sectional dimension between about 0.1 microns and 250 microns, and often between about 0.1 microns and 100 microns.
The term “microstructure” as used herein describes microfluidic structures, e.g., “microchannels” and “microchambers” or any combination thereof. A microchannel can have at least one dimensional feature that is at least about 1 micron but less than about 500 microns in size. The term “channel” as used herein describes a microchannel. During operation, microchannels and microchambers may contain fluids passing therethrough.
The term “microfluidic chip” and “microfluidic device” as used herein refers to at least one substrate having microfluidic structures contained therein or thereon.
The microfluidic electroelution devices of the present invention can be typically constructed using one or more substrates. Substrates are typically made from a transparent material to aid observation, however non-transparent materials can be used. Suitable transparent substrate materials can include glass, polymeric, ceramic, metallic, silica-based, and composite materials, as well as any combination thereof. Examples of polymeric materials typically used include polystyrene, polypropylene, polyethylene, acrylonitrile butadiene styrene, polycarbonate, polymethyl methacrylate, cyclic olefin copolymer, polyester, polyimide, polyamide, or other acrylics, or any combination thereof. In the case of conductive or semi-conductive substrates, a chemical treatment should be applied to the microfluidic structures to provide a substrate with a near-neutral or neutral surface charge and eliminate bulk electroosmotic flow.
The microfluidic electroelution devices of the present invention typically comprise a system of microstructures, e.g., microchannels and microchambers, to transport fluids into, out of, and onto the various structures within the microfluidic devices, or any combination thereof. The microstructures can be prepared on substrates using standard manufacturing techniques. For example, lithographic techniques may be employed in fabricating glass, quartz or silicon substrates. In addition, photolithographic masking, plasma or wet etching and other semiconductor processing technologies can be used. Alternatively, micromachining methods, such as laser ablation, micromilling, and the like may be employed. Similarly, well known manufacturing techniques may also be used for polymeric substrates, e.g., compression molding, stamp molding, and injection molding, casting or embossing, and the like. For example, microchannels can be prepared by compression molding and microchambers can be prepared by using a diamond tipped drill, such as a microdrill. In order to provide fluid and/or control access to the microstructures, a series of reservoirs or ports in fluid communication with the microstructures can be provided in at least one of the substrates.
In various embodiments of the present invention, microfluidic devices include two substrates, e.g., a cover substrate and a base substrate, which are bonded together. The cover substrate and the base substrate can be bonded together by adhesive bonding, cohesive bonding, thermal bonding, mechanical bonding or any combination thereof. The bonding of the substrates provides regions for containing microstructures, e.g., a system of microchannels and microchambers, in both the base substrate and/or the cover substrate. When bonded together, the spatial arrangement of the microfluidic structures in the cover substrate are typically designed to be in fluid communication with the regions containing the microfluidic structures in the base substrate.
Referring to
In further embodiments, the flow restricting feature can be a branch channel 22 intermediate the second port 50 and the channel 20 such that the branch channel 22 has a smaller diameter than the channel 20 to discourage fluid flow towards the second port 50. The branch channel 22 can connect the second port 50 to the channel 20 at a junction G. A flow restrictor (not shown) can be positioned in the branch channel 22 to further discourage flow thereinto. In certain embodiments, the flow enhancing feature can be a microchamber 90 having at least one cross-sectional dimension or diameter greater than the channel 20 intermediate the branch channel 22 and the outlet 40 such that fluid flow towards the outlet 40 is encouraged.
In further embodiments, the microfluidic electroelution modules 10 can further comprise a first reservoir 55 in fluid communication with the first port 60. The first reservoir 55 can further comprise the receptacle 60. The first electrode 70 can be associated with the first reservoir 55. A sample or gel spot (not shown) containing at least one macromolecule species of interest can be positioned in the receptacle 60. The electrodes 70, 80 can create an electric field across the gel spot from the first electrode 70 towards the second electrode 80 such that the species electrophoretically migrate from the gel spot into the fluid present in the channel 20, e.g., an elution liquid or a buffer solution. The electrodes 70, 80 can be conventional, such as a simple conductor connected to a source of electricity. Furthermore, in accordance with the present invention, the flow restricting features, e.g., branch channel 22, discourage the migration of the electroeluted species towards the second electrode 80 and the flow enhancing features, e.g., microchamber 90, encourage the migration of the electroeluted species towards the outlet 40.
In still further embodiments, the microfluidic electroelution modules 10 can have a sorbent material 95 that collects and/or concentrates the electroeluted species in the channel 20 intermediate the second port 50 and the outlet 40 such that the sorbent material 95 is decoupled from the electrophoretic field created between the electrodes 70, 80. The sorbent material 95 can be a porous polymer monolith, a packed bed, or other suitable materials that act as chromatographic and/or extraction systems. The chromatographic and/or extraction systems provided by the sorbent material 95 can include partition chromatography, adsorption chromatography, ion exchange and ion chromatography, size exclusion chromatography, affinity chromatography, and chiral chromatography. Surprisingly, the collection and/or concentration of the electroeluted species by the sorbent material 95 is substantially improved by decoupling the sorbent material 95 from the electrophoretic field and decoupling the direction of fluid flow from the electrophoretic field.
A porous polymer monolith refers to highly cross-linked monolithic porous polymer materials that permit fluid communication through the pores. A porous polymer monolith can be functionalized to have chemical moieties on the surfaces of its pores that are capable of interacting and/or bonding to macromolecules or other analytes contacting or passing through its pores. A functionalized porous polymer monolith can be prepared by including a polymerizable functionalized monomer in a reaction mixture for preparing the porous polymer monolith or post-functionalizing the porous polymer monolith after it is formed. The functionalized monomer can be selected to contain a functional group that directly binds or interacts to a particular analyte or probe compound capable of selectively binding to or interacting with the particular analyte. For example, the functionalized porous polymer monolith can have reversed phase (C4, C8, or C18) or ion exchange chemistry.
The porous polymer monolith can be formed integrally in the channel 20 or microchamber 95 by photoinitiated or thermally initiated in situ polymerization. A method of making a porous polymer monolith within a channel of a microfluidic module can comprise copolymerization of a monomer, a crosslinking agent, a porogenic solvent and an initiator inside a microchannel. For example, a polymerization mixture containing 18% (Wt) butyl, octyl or lauryl acrylate, 12% (Wt) ethylene glycol dimethacrylate (EDMA), 69.5% (Wt) methanol and 2-propanol (porogens), and 0.5% (Wt) benzoin methyl ether (photoinitiator) can be added to the microchannel and exposed to an 8 W ultraviolet-light at 365 nm to form a hydrophobic polymer monolith within the microchannel. The polymerization can be limited to only those portions of the channel that are exposed to ultraviolet-light, i.e., those portions of the channel that are not masked to prevent exposure of ultraviolet-light to the polymerization mixture. The functionalized porous polymer monolith is typically bonded to the microstructure and/or substrate.
Referring to
Referring to
In still further embodiments, the method of electroelution can further comprise collecting the electroeluted species on a sorbent material 95, e.g., a monolith, packed bed, etc., in the second fluid pathway J, e.g., microchamber 95. The collected species on the sorbent material 95 can be further processed, e.g., rinsing, desalting, purifying, and/or concentrating. The collected species can be removed from the sorbent material 95, e.g., flowing a second elution liquid in the channel 20 to elute the collected species from the sorbent material 95. The method of removing the collected species from the sorbent material 95 can be optimized, e.g., providing fluid undulation to create vertical assistance mixing. In embodiments in which the channel 20 is formed from a conductive material, e.g., glass, the method can further comprise coating the first and second fluid pathways with a nonconductive coating to provided a neutral or near-neutral surface change and reduce bulk electroosmotic flow.
Referring to
The pressure drop may be provided by using a column of liquid in fluid communication with the first fluid pathway P having a height greater than a column of liquid, if any, above the second fluid pathway Q. In embodiments of microfluidic modules 200 and chips 300, the liquid column height of the reservoirs in the first fluid pathway, i.e., first 265 and second 255 reservoirs, can be increased above the first port 260 and second port 250, respectively, and/or the liquid column height of the reservoirs in the second fluid pathway, i.e., third 245 and fourth 235 reservoirs, can be decreased above the outlet 240 and inlet 230, respectively. For example, the pressure drop can be created by the relatively large volumes and column height of fluid in the reservoirs in the first fluid pathway 255, 265 compared to the volume and column height of fluid in the outlet reservoir 245.
In still further embodiments, the second fluid pathway Q can comprise at least one microchamber 295 intermediate the first fluid pathway P and the outlet 240, and a first channel segment 270 intermediate the first fluid pathway P and the microchamber 295. The first fluid pathway P can further comprise a second channel segment 275 intermediate the second port 250 and the first channel segment 270, and a third channel segment 280 intermediate the first port 260 and the first channel segment 270.
In addition, the engineering of the microstructures can be optimized to increase the pressure drop from the first fluid pathway P towards the second fluid pathway Q. For example, the length of the first channel segment 270 can be decreased and the length of the second 275 and third 280 channel segments can be increased. In further embodiments, the magnitude of the pressure drop can be increased by increasing the radius or cross-sectional dimensions of the microchamber 295 and first channel segment 270 and/or decreasing the radius or cross-sectional dimensions of the second 275 and third 280 channel segments. Although the engineering of the microfluidic structures can be optimized to increase the magnitude of the pressure drop from the first fluid pathway P towards the second fluid pathway Q, the pressure drop would be greatly diminished without the presence of the reservoirs 245, 255, and 265. The pressure drop may also be provided by any other means of applying pressure, electroendoosmotic forces, gravitational forces, and surface tension forces.
In yet further embodiments, the microfluidic module 200 can comprise a sorbent material (not shown) in the second fluid pathway Q, e.g., a monolith or packed bed. In some embodiments, the monolith can be a porous polymer monolith formed integrally in the second fluid pathway Q, e.g., the monolith can be a functionalized porous polymer monolith formed integrally in the microchamber 295 in the second fluid pathway Q. The pressure drop from the first fluid pathway P towards the second fluid pathway Q may be disrupted if the sorbent material fills a significant portion of the microchamber 295 and produces back pressure. In the devices of the present invention, the sorbent material can fill between about 5% and 90%, more preferably-between about 10% and 75%, and often between about 25% and 50%.
Referring to
Referring to
Referring to
When a constant voltage of 100-2500V is applied to the microfluidic module 10, typically for less than one hour, the electric current created through the elution liquid establishes an electric field across the gel spot. The electrophoretic field is substantially confined within the first fluid pathway H such that the second fluid pathway J is decoupled from the electrophoretic field. The electroelution voltages drive the proteins from the gel spot into the elution liquid. The principles of gel electrophoresis govern the movement of the proteins out of the gel spot. However, the flow restricting and/or flow enhancing features encourage the electroeluted proteins to migrate toward the outlet 40 instead of the second electrode 80, i.e., the second port 50. After the voltage is turned off, the introduction of hydrodynamic flow of the buffer solution causes the electroeluted proteins to flow onto the sorbent material 95, e.g., a monolith and packed bed. The waste vial rack (not shown) can be removed and replaced with a sample collection vial rack (not shown). A second elution liquid can be introduced at the inlet 30 to elute the proteins collected on the sorbent material 95 into the sample collection vial. Finally, the sample vials can be removed and the proteins can be subjected to subsequent identification and analysis.
Referring to
In addition to the general principles previously described, the pressure drop encourages the electroeluted proteins to migrate toward the second fluid pathway Q instead of the second electrode. The electroeluted proteins can be collected on a sorbent material, e.g., a monolith or packed bed. After the voltage is turned off, the proteins collected on the sorbent material can be removed or eluted from the sorbent material and/or subjected to further processing. The waste vial rack (not shown) can be removed and replaced with a sample collection vial rack (not shown). A solvent delivery system can deliver a second elution liquid at the inlet 230 to elute the proteins collected on the sorbent material into the sample collection vial. Finally, the sample vials can be removed and the proteins can be subjected to subsequent identification and analysis.
The movement of the proteins and other macromolecules in microfluidic electroelution modules and chips is based on the electrophoretic mobility of the proteins in the sample and the principles of capillary electrophoresis that govern the movement and behavior of free proteins in the channel post-electroelution. Referring to
F
Total
=F
1
+F
2
+F
3 . . . etc. (1)
Here,
F
Total
=F
E
+F
HD
+F
HS (2)
where, FE≡electrokinetic force, which is a sum of the forces on the protein due to its inherent electrophoretic mobility and the forces of bulk electroosmotic flow within the fluid pathway, FHD≡hydrodynamic force, and FHS≡hydrostatic force.
In embodiments of the microfluidic electroelution modules 10 and chips 100, the substrate may be designed such that by chemical treatment or natural properties it has a near-neutral or neutral surface charge, thereby eliminating bulk electroosmotic flow (EOF). If a negative surface charge is present on the substrate, then a bulk EOF flow will be established towards the first electrode 70; conversely, if a positive surface charge is present on the substrate, then a bulk EOF flow will be established towards the second electrode 80. Since EOF is near zero in the fluidic channel network H, FE can be approximated by the electrophoretic force on the proteins as produced by their inherent electrophoretic mobilities in the applied electric field. Therefore, in the case of conductive or semi-conductive substrates, the microstructures should be chemically treated with an insulating layer.
The hydrodynamic force is due to back pressure from the channel 20 size restrictions and the solvent delivery system connected to the inlet 30. The hydrostatic force is due to the relatively large volumes of fluid and column heights of buffer associated with the second port 50 and first reservoir 55 as compared to the volume and column height of the outlet reservoir (not shown). Prior to intersection K, FE dominates Equation 2 such that FTotal≈FE. The microfluidic structures on the device 10, in particular, the flow restricting and/or flow enhancing features, can be designed such that the hydrodynamic force balances the hydrostatic force, i.e., the vector sum of FHS and FHD is approximately zero. However, after intersection K, the proteins experience the new unbalanced forces FHS and FHD deriving from both directions of the second port 50 and first reservoir 55 such that FHD+FHS>>FE.
At intersection L, the proteins experience a strong hydrodynamic force that inhibits movement of the proteins toward the inlet 30. The solvent delivery system connected to the inlet 30 provides strong hydrodynamic resistance such that the vector sums of all forces, FTotal, experienced by the proteins in the channel 20 directs the movement of the proteins past intersection M and into the microchamber 90. Therefore, electroeluted and free sample proteins are directed from the receptacle 60, past intersections K and L, into the microchamber 90, and toward the sorbent material 95. A low hydrodynamic flow can be introduced at intersection L via a syringe to further assist the movement of the proteins toward and onto the sorbent material 95.
The movement of proteins and other macromolecules in microfluidic modules 200 and chips 300 are governed by similar principles of capillary electrophoresis as described above. Referring to
P=ρ·g·h+P
a (3)
where, P≡hydrostatic pressure, ρ≡liquid density, g≡gravitational acceleration, h≡height of liquid relative to the fluid within channel, and Pa≡atmospheric pressure.
The dynamics of fluid movement in microfluidic modules 200 and chips 300 are generally governed by the diameter and length of the microchannel structure according to Poiseuille's Law. The magnitude of the pressure drop along each section of the channel 220 can be estimated using Poiseuille's Law given in Equation 4
where, Q≡volumetric flow rate, ΔP≡pressure drop, Π≡pi, r≡radius of channel, η≡viscosity, L≡length of channel. Poiseuille's equation is only strictly valid for circular flow channels. The channels of this invention can have cross-sections of various shapes, e.g., circular, wedge-shaped and substantially rectangular. Thus, in embodiments with non-circular cross-sections, Poiseuille's equation can be considered only as an approximate relation between the variables represented. According to Poiseuille's equation, the pressure drop is directly proportional to the length of the microchannel structure and the radius or diameter of the microchannel structure has a fourth power effect on the pressure drop. Therefore, the pressure drop can be increased by, e.g., decreasing the length of the first channel segment 270, increasing the length of the second 275 and third 280 channel segments, increasing the radius or cross-sectional dimensions of the microchamber 295 and first channel segment 270, and/or decreasing the radius or cross-sectional dimensions of the second 275 and third 280 channel segments.
Therefore, what has been described above includes exemplary embodiments of microfluidic electroelution modules, devices, and processes utilizing an extraction and/or a collection and recovery scheme that is decoupled from the electrophoretic field. It is, of course, not possible to describe every conceivable combination of components or methodologies for purposes of this description, but one of ordinary skill in the art may recognize that further combinations and permutations are possible in light of the overall teaching of this disclosure. Accordingly, the description provided herein is intended to be illustrative only, and should be considered to embrace any and all alterations, modifications, and/or variations that fall within the spirit and scope of the appended claims.
