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  Enzyme immobilization for electroosmotic flowUSPTO Application #: 20070108057Title: Enzyme immobilization for electroosmotic flow Abstract: Disclosed herein is a method and apparatus of immobilizing a biocatalyst on a microfluidic biochip for conducting reactions in the presence of electroosmotic flow. The biochip includes a polymer on its microfluidic flow surfaces, wherein the polymer includes a first substituent selected from ionic groups of the same polarity or precursors thereof, a second substituent that is a hydrophobic group, and a third substituent comprising an immobilized biocatalyst-or precursor thereof. The biochip can be used to conduct multiple sequential biocatalyzed reactions in the presence of electroosmotic flow. (end of abstract) Agent: Elmore Patent Law Group, PC - N. Chelmsford, MA, US Inventors: Jonathan S. Dordick, Moo-Yeal Lee, Aravind Srinivasan, Bosung Ku USPTO Applicaton #: 20070108057 - Class: 204601000 (USPTO) Related Patent Categories: Chemistry: Electrical And Wave Energy, Apparatus, Electrophoretic Or Electro-osmotic Apparatus, Capillary Electrophoresis Type The Patent Description & Claims data below is from USPTO Patent Application 20070108057. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATION [0001] This application is a divisional of U.S. application Ser. No. 10/351,976, filed Jan. 24, 2003. The entire teachings of the above application are incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] Microfluidics is the field of microscale fluid flow and control, typically through microscale fluid control features constructed on a substrate such as a glass chip. These devices can be used to manipulate liquid chemical and biological samples in order to conduct analysis, perform synthetic reactions, and the like. [0004] A motive force often used in microfluidics is a phenomenon known as electroosmotic flow (EOF). EOF depends strongly on various aspects of charge mobility in these systems, and in particular, depends on having a high density of ionic groups of the same polarity on the walls or flow surfaces of a microfluidics component. For example, a typical microfluidics feature is a microchannel etched in a glass substrate. The interior surface of the channel is treated to expose free silanol groups, which can then be deprotonated to provide siloxide anions. A voltage is applied across the flow direction of the channel causing solvated counterions of the charged groups on the channel walls to move. Because the dimensions of a microfluidic channel are small, the layer of counterions at the flow surface contact a significant portion of the fluid in the channel. Thus, when the counterion layer moves, the entire volume of the channel moves nearly simultaneously, a mechanism known as "plug flow". For many applications, plug flow is advantageous because it means that the components of a particular volume portion of the flow travel together, and are not spread out as they would be in a conventional pressure driven flow. Thus, on a microfluidics chip, precise volumes can be delivered from one location to another with a high degree of control. [0005] Many features of a macroscale laboratory can thus be miniaturized using microfluidics, allowing significant reductions in the amount of costly, rare, or hazardous materials that are used. For example, microfluidics has the potential to make efficient use of biomolecules such as enzymes or catalytic antibodies, which are typically expensive or difficult to prepare in large quantities. It is particularly desirable that these molecules be reusable or recoverable, for example, by immobilizing them to a solid support, in order to further limit the quantities that are required. [0006] A significant problem that must be solved, however, is the difficulty of immobilizing biomolecules with high biological activity while simultaneously maintaining acceptable EOF capability. [0007] For example, enzymes have been attached to glass chips by covalent attachment to free silanol groups on the glass surface. However, the high pH necessary to provide the charged siloxide anions significantly decreases the stability and catalytic activity of enzymes. Another attempt functionalized the silanol groups with a linker group ending in an amine, which can then be covalently attached to the enzyme. However, this leads to a reduction in the number of available siloxide groups at the surface, and further, shields the groups that are present from the flow, resulting in poor EOF characteristics. [0008] Other attempts have been made to provide polymers that specifically enhance EOF, for example, by using a charged polymer such as dextran sulfate. These polymers, however, are dynamically unstable coatings, i.e., are not substantially adhered, and so are eventually washed away by the flow. Furthermore, they are not easily functionalized with biocatalysts such as enzymes, and they do not maintain the catalytic activity of enzymes. [0009] High enzyme activity can be maintained by encapsulating and/or covalently attaching enzymes to matrices, such as solgels, but such matrices typically fill the entire microfluidic channel, providing a severe impediment to fluid flow. Furthermore, many other examples of polymers exist to immobilize enzymes with high activity, but they are not designed to support EOF. [0010] Therefore, there is a need to immobilize biomolecules on a microfluidics apparatus, while simultaneously maintaining high biological activity and high EOF capability. SUMMARY OF THE INVENTION [0011] It has now been found that certain polymers containing both ionic groups and hydrophobic groups can be substantially adhered to microfluidic channels and can be used to simultaneously immobilize biocatalysts with good catalytic activity while supporting electroosmotic flow. [0012] One embodiment of the invention is method of immobilizing a biomolecule in the presence of electroosmotic flow. One step is providing a microfluidic biochip. The biochip includes a microfluidic component comprising a flow surface; at least two electrodes whereby an electroosmotic flow can be generated at the flow surface; and an immobilizing polymer that is substantially adhered to the flow surface. The polymer includes a first substituent selected from ionic groups of the same polarity and covalent precursors of the ionic groups, wherein the first substituent is optionally a biomolecule immobilizing group. The polymer includes a second substituent that is a hydrophobic group. The polymer optionally includes a third substituent that is a biomolecule-immobilizing group. Between the first substituent and the optional third substituent, the polymer includes at least one substituent that is a biomolecule-immobilizing group. Another step of the method is applying a motive force selected from pressure, electroosmotic force, capillary action, and centrifugal force, thereby generating flow. Yet another step is directing a biomolecule from a source to the polymer by employing the flow. An additional step is reacting the biomolecule with the biomolecule-immobilizing group under suitable reaction conditions, whereby the biomolecule is immobilized. [0013] Another embodiment of the invention is the biochip, wherein the substituents of the immobilizing polymer include a first substituent selected from ionic groups of the same polarity and covalent precursors of the ionic groups; a second substituent that is a hydrophobic group; and a third substituent comprising an immobilized biomolecule. [0014] Another embodiment of the invention is a method for conducting one or more reactions by using the biochip. The biochip additionally includes at least one reservoir, wherein the reservoir contains a starting reactant. All the microfluidic components are in microfluidic communication. The substituents of the immobilizing polymer include ionic groups of the same polarity; a hydrophobic group; a first immobilized biomolecule; and optionally a second, chemically distinct immobilized biomolecule;. A step of the method is applying a voltage to the electrodes, thereby generating electroosmotic flow. Another step is directing the reactant from the reservoir to the first biomolecule by employing the electroosmotic flow, then reacting the first reactant with the first biomolecule under suitable reaction conditions, thereby producing a first reaction product. Another step is optionally contacting the first product with the second biomolecule, if present, and reacting the first product with the second biomolecule under suitable reaction conditions, thereby producing a second reaction product. [0015] The invention can be used to immobilize biomolecules on a microfluidics biochip to conduct reactions. The invention retains the activity of the biomolecules while simultaneously supporting EOF. Also, the invention allows multiple reactions using catalytically distinct biomolecules. Furthermore, the invention allows sequential reactions using catalytically and spatially distinct immobilized biomolecules. Thus, using the invention, a wide array of sequential, stepwise reactions can be conducted with high activity while minimizing the use of costly or dangerous reactants. BRIEF DESCRIPTION OF THE DRAWINGS [0016] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [0017] FIG. 1 graphs enhanced electroosmotic flow versus voltage for PMA-OL (poly (maleic anhydride)-alt-.alpha.-olefin) coated channels of the invention (open circles) compared glutaraldehyde-coated control channels (solid triangles), with reference to glass EOF control channels (solid circles). [0018] FIG. 2A graphs high retained biological activity of PMA-OL immobilized soybean peroxidase (SBP) as shown by H.sub.2O.sub.2 consumption by on the biochip in 2% loading (filled circles) and 10% loading (open circles). [0019] FIG. 2B graphs the kinetics of PMA-OL-immobilized soybean peroxidase. [0020] FIG. 3 graphs the high catalytic activity of two PMA-OL immobilized enzymes for poly(p-cresol) production on the biochip (filled circles) compared to a solution control (open circles). [0021] FIG. 4 graphs the high catalytic activity of three PMA-OL immobilized enzymes for poly(p-cresol) production on the biochip (filled circles) compared to a solution control (open circles). 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