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Apparatus, system, and method for electrochemical pump-based chromatography separations in microfabricated devices

Apparatus, system, and method for electrochemical pump-based chromatography separations in microfabricated devices description/claims

This application is a continuation-in-part of and claims priority to U.S. Provisional Patent Application No. 60/963,794 entitled “Electrically actuated, pressure-driven liquid chromatography separations in microfabricated devices” and filed on Aug. 6, 2007 for Adam T. Woolley et al., which is incorporated herein by reference.

1. Field of the Invention

This invention relates to microfluidic devices and more particularly relates to monolithic pressure-driven chromatography Microsystems.

2. Description of the Related Art

Miniaturization of analytical techniques holds great potential for performing a variety of sample-limited assays, especially because of the possibility of integrating multiple analysis steps in a single substrate. Since first demonstrated in 1992, capillary electrophoresis in microfabricated devices has seen significant advances, and numerous chemical and biological applications have been reported. Electrically driven methods are well-suited for miniaturization, since electroosmotic flow (EOF) can be controlled without valves or external pumps. However, EOF pumping and fluid transport have inherent issues that may limit their broad application in miniaturized methods. For example, EOF typically requires high voltages (kV) and is affected by Joule heating. Moreover, EOF is sensitive to the solution pH and column surface charge. Finally, electrophoretic techniques are optimal for charged analytes that can be exposed to an electric field.

On the other hand, pressurized separation methods such as liquid chromatography (LC) are more general and broadly used. In 1990, Manz et al. presented advantages of the miniaturization of LC. Even though no experimental data were provided, this paper showed that theoretically, the performance per unit time should be superior in microchip compared to conventional LC. (See A. Manz, Y. Miyahara, J. Miura, Y. Watanabe, H. Miyagi and K. Sato, Sens. Actuators B, 1990, 1, 249.) Manz and others have indicated that there are three critical elements for a fully miniaturized LC system. They are: (i) integration of a pumping mechanism capable of generating pressure and flow compatible with microchannel dimensions; (ii) incorporation of a separation medium inside microchannels; and (iii) a minimal dead volume interface of the separation column with the pumping and injection mechanism. (See Manz et al.; C. M. Harris, Anal. Chem., 2003, 75, 64A; and A. de Mello, Lab Chip, 2002, 2, 48N.) However, the miniaturization of pressure-driven separation methods presents challenges the solution to which is not trivial. Most reports have focused on micromachining a separation column while maintaining an external pumping mechanism. Thus, the full advantages of LC miniaturization have not been realized prior to the discoveries disclosed herein.

Two recent studies have made important progress in the development of miniaturized LC systems with integrated pumping and injection. Lazar et al. fabricated a LC microdevice with integrated EOF micropumps for sample valving and separation. (See I. M. Lazar, P. Trisiripisal and H. A. Sarvaiya, Anal. Chem., 2006, 78, 5513.) However, this approach required the use of relatively high electric fields (500 V/cm), was limited to solutions with low organic solvent concentrations, and was relatively slow (˜40 min elution times). In other work, a hybrid silicon-parylene microfluidic chip with integrated electrochemical micropumps for sample injection and separation was used for the LC analysis of protein digests. This report demonstrated the advantages of having a minimal dead volume between injection and separation, but again suffered from long analysis times (˜1 h). (See J. Xie, Y. Miao, J. Shih, Y.-C. Tai and T. D. Lee, Anal. Chem., 2005,77,6947.)

A variety of micropumps have been constructed for microfluidic applications. Mechanical pumps use moving parts, have relatively complex fabrication and often face compatibility challenges with regard to solutions and samples when integrated with microfluidic systems. Non-mechanical pumps based on electroosmotic, magnetohydrodynamic or electrochemical actuation, are thus appealing alternatives. Electroosmotic pumps, which generate pressure with EOF, are perhaps the most widely used micropumps in microfluidics applications. However, to obtain appropriate flow rates, it is often necessary to apply high voltages (˜kV) and make either packed small-diameter columns or microchannel network arrays, which complicate the fabrication process. Moreover, electroosmotic pumps are only suitable for operation within a certain range of solution pH and conductance values.

Column technology for microchip LC is a key challenge. Packing microchannels with particles as in conventional LC is difficult to achieve on the micro level due to pressure constraints and difficulties in forming frits inside microchannels. In 1998, Regnier et al. demonstrated surface-modified micromachined posts, as a mimic of a packed microcolumn. (See B. He, N. Tait and F. Regnier, Anal. Chem., 1998, 70, 3790.) While this approach is compatible with micromachining techniques, it is hindered by expensive fabrication protocols involving deep reactive ion etching. The possibility of performing separations in monolithic stationary phases or open tubular columns has given new opportunities for the development of miniaturized LC. The fabrication of monolithic structures inside microchannels has been demonstrated and is becoming a promising approach. However, monolithic stationary phases in microchannels require reproducible construction of uniform monoliths with low back pressure, which have not been adequately reduced to practice.

The use of capillaries for open tubular liquid chromatography (OTLC) was first proposed by Jorgenson et al. A key advantage of OTLC is lower back pressure than packed or monolithic columns, leading to faster analysis times. The main disadvantages of OTLC relative to packed column LC are slower mass transfer into the stationary phase and reduced sample capacity due to lower stationary phase volume. However, micromachined systems can have small channel cross sections, which increases the mass transfer to the stationary phase. Theoretical work on OTLC has shown that band dispersion is lowest for microchannels with high aspect ratios. Jacobson et al. reported the use of high aspect ratio microchannels to perform open channel electrochromatography. Later, the same group determined that 5 μm channel depths were a good compromise between efficiency and ease of operation. (See S. C. Jacobson, R. Hergenroder, L. B. Koutny and J. M. Ramsey, Anal. Chem., 1994, 66, 2369; and J. P. Kutter, S. C. Jacobson, N. Matsubara and J. M. Ramsey, Anal. Chem., 1998, 70, 3291.) However, these devices were not tested for pressure-driven separations.

In recent years, great interest has arisen in developing electrochemical systems for microchip pumping, resulting in devices for valve actuation and dosing systems for applications in biology and medicine. We have shown that the pressure caused by the build-up of electrolysis gases in an enclosed chamber can pump liquids in fluidic microchannels. (See J. W. Munyan, H. V. Fuentes, M. Draper, R. T. Kelly and A. T. Woolley, Lab Chip, 2003, 3, 217.) Our electrochemical micropumps are integrated easily with microfluidics and can pump liquids with rates as high as ˜10 μL/min. More recently, electrochemical actuation was demonstrated for sample delivery and solvent gradient generation in electrospray ionization mass spectrometry analysis. Only one report has appeared on the use of electrolysis-based micropumps in LC. While this initial work demonstrated feasibility, the separation time achieved was approximately one hour, which is similar to the separation times in conventional LC. Moreover, the electrolysis voltage was applied directly to the sample and eluent. (See J. Xie, Y. Miao, J. Shih, Y.-C. Tai and T. D. Lee, Anal. Chem., 2005, 77, 6947.)

A need exists for an apparatus, system, and method that integrates an electrochemical pump and a chromatography microcolumn in a single chip. Beneficially, such an apparatus, system, and method would provide short retention times and relatively high resolutions.

The present invention has been developed in response to the present state of the art, and in particular, in response to the problems and needs in the art that have not yet been fully solved by currently available chromatography systems including microcolumns and existing pumps for microfluidic systems. Accordingly, embodiments of the present invention have been developed to provide an apparatus, system, and method for electrochemical pump-based chromatography separations in microfabricated devices that overcome many or all of the shortcomings in the art.

In a simple form a microfluidic device in accordance with one embodiment of the invention includes a constant volume electrochemical pump formed in at least one layer of the microfluidic device. The microfluidic device includes a fluid reservoir coupled with the electrochemical pump for receiving pressure from the electrochemical pump during actuation. The microfluidic device also has a chromatographic microcolumn fluidically coupled to the fluid reservoir for receiving a fluid from the reservoir during actuation.

In another embodiment, the electrochemical pump may be a first injection pump and the fluid reservoir may be a first sample reservoir. In this case, the microfluidic device may further include at least one additional constant volume electrochemical pump formed in at least one layer of the device for pumping eluent. The microfluidic device may also include at least one additional reservoir fluidly coupled with the at least one additional electrochemical reservoir for receiving pressure from the additional electrochemical pump. The additional reservoir is also fluidically coupled to the chromatographic column.

In another embodiment, the microfluidic device may be formed of a plurality of layers. Electrodes may be disposed on or in at least one of the layers of the microfluidic device. In this embodiment, the electrodes form part of the electrochemical pump.

In one embodiment, the layers include a top layer having port holes therethrough in alignment with the electrochemical pump(s) and fluid reservoir(s). The port holes may be configured to receive an electrolyte and at least one of a sample and an eluent therethrough. The microfluidic device may further include a waste reservoir in the at least one of the layers. In any case, the waste reservoir is fluidically coupled to the chromatographic microcolumn for receiving at least a portion of the fluid after it passes through the microcolumn.

In another emobodiment, at least one layer is an intermediate layer and the microfluidic device further includes a top layer having port holes therethrough in alignment with the pump(s) and reservoir(s). The layers may further include a bottom layer. Thus, the intermediate layer may have the top layer and the bottom layer bonded thereto to form a monolithic pressure-driven liquid chromatography device. In still another embodiment, the microfluidic device may include a plurality of intermediate layers such that the pump(s) and reservoir(s) are formed by the plurality of intermediate layers.

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