This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.

A simple two-step method for fabricating poly(ethylene glycol) (PEG) hydrogel-based microreactors and microsensors within microfluidic channels is described. The intrachannel micropatches contain either a dye, which can report the pH of a solution within a fluidic channel, or enzymes that are able to selectively catalyze specific reactions. Analytes present within the microfluidic channel are able to diffuse into the micropatches, encounter the enzymes, and undergo conversion to products, and then the products interact with the coencapsulated dye to signal the presence of the original substrate. The micropatches are prepared by photopolymerizing the PEG precursor within the channel of a microfluidic system consisting of a poly(dimethylsiloxane) mold and a glass plate. Exposure takes place through a slit mask oriented perpendicular to the channel, so the size of the resulting micropatch is defined by the channel dimensions and the width of the slit mask. Following polymerization, the mold is removed, leaving behind the micropatch(es) atop the glass substrate. The final microfluidic device is assembled by irreversibly binding the hydrogel-patterned glass slide to a second PDMS mold that contains a larger channel. Multiple micropatches containing the same or different enzymes can be fabricated within a single channel. The viability of this approach is demonstrated by sensing glucose using micropatches copolymerized with glucose oxidase, horseradish peroxidase, and a pH-sensitive dye.

We report a novel technique for manufacturing polymeric microparticles containing biocatalysts by the behavior of immiscible liquids in microfluidic systems and in situ photopolymerization. The approach utilizes a UV-polymerizable hydrogel/enzyme solution and an immiscible oil solution. The oil and hydrogel solutions form emulsions in pressure-driven flow in microchannels at high values of the dimensionless capillary number (Ca). The resultant hydrogel droplets are then polymerized in situ via exposure to 365 nm UV light. This technique allows for the generation of monodisperse particles whose size can be controlled by the regulation of flow rates. In addition, both manufacturing microparticles and immobilizing biocatalysts can be performed simultaneously and continuously.

We report a novel strategy for efficiently mixing solutions and carrying out multistep catalytic reactions in microfluidic systems. The approach involves immobilizing catalysts on microbeads, placing the beads into well-defined microreactor zones, and then passing reactants through one or more of the reactor zones to yield products. The catalyst-modified beads mix reactants and increase the effective surface area of the channel interior, both of which improve reaction velocities compared to open channels. In addition to providing a general route to chemical synthesis within microfluidic systems, this design strategy may also be applicable to modeling reaction pathways within cells and for bio/chemical sensing applications. [1][2][3][4] In open microchannels having dimensions on the order of 100 µm, mass transfer is dominated by laminar flow, and thus mixing is driven principally by diffusion. 5,6 However, the diffusional time scale is often too long for technological applications such as synthesis and bio/chemical sensing. This problem has most often been addressed by using either slow flow rates or long channels, neither of which is optimal. We have discovered that interstices between microbeads packed within channels provide a simple means for reducing the effective thickness of fluid laminae, thereby greatly increasing the mixing rate for fluids. Moreover, the microbeads themselves have a high surface area relative to that of the channel walls; thus, they also act as a convenient support for heterogeneous catalysts. 7 Herein we demonstrate both of these functions.The principles of laminar shear flow, extensional flow, and distributive mixing are relevant for efficient mixing under conditions of laminar flow. 8 Laminar shear flow and extensional flow lead to an increase in interfacial area of the fluid elements and a sufficient reduction in the thickness of the fluid laminae that molecular diffusion alone results in rapid mixing. Distributive mixing physically splits fluid streams into smaller segments and redistributes them in such a way that the striation thickness is significantly reduced. Several approaches for mixing confluent liquid streams have recently been reported. For example, Stroock et al. 9 presented a general strategy for creating transverse flow in microchannels that can be used to induce chaotic stirring at low Reynolds number (Re, which is proportional to the ratio of the inertial and viscous forces). Johnson et al. 10 fabricated a series of slanted wells within a polycarbonate sheet to induce rapid lateral transport of two confluent streams. He et al. 11 described mixing of liquids transported by electroosmotic flow in a microfabricated device having multiple intersecting channels of varying length and a bimodal width distribution. This problem has also been studied using numerical simulations. 12 A schematic diagram of the first microfluidic system used in this study is shown in Figure 1A. The device was fabricated from poly(dimethylsiloxane) (PDMS) using standard photolithographic and repl...