Design optimization for bioMEMS studies of enzyme-controlled metabolic pathways

Abstract

Biological microelectromechanical systems (bioMEMS) provide an attractive approach to understanding and modifying enzymatic pathways by separating and interrogating individual reaction steps at localized sites in a microfluidic network. We have previously shown that electrodeposited chitosan enables immobilization of an enzyme at a specific site while maintaining its catalytic activity. While promising as a methodology to replicate metabolic pathways and search for inhibitors as drug candidates, these investigations also revealed unintended (or parasitic) effects, including products generated by the enzyme either (1) in the homogeneous phase (in the liquid), or (2) nonspecifically bound to microchannel surfaces. Here we report on bioMEMS designs which significantly suppress these parasitic effects. To reduce homogeneous reactions we have developed a new packaging and assembly strategy which eliminates fluid reservoirs that are commonly used for fluidic interconnects with external tubing. To suppress reactions by nonspecifically bound enzyme on microchannel walls we have implemented a cross-flow microfluidic network design so that enzyme flow for assembly and substrate/product for reaction share only the region where the enzyme is immobilized at the intended reaction site. Our results show that the signal-to-background ratio of sequential enzymatic reactions increases from 0.72 to 1.28 by eliminating the packaging reservoirs, and increases to 2.43 by separating the flow direction of enzymatic reaction from that of enzyme assembly step. These techniques can be easily applied to versatile microfluidic devices to minimize parasitic reactions in sequential biochemical reactions.

In situ generation of pH gradients in microfluidic devices for biofabrication of freestanding, semi-permeable chitosan membranes

“…By utilizing the pH-dependent solubility of chitosan combined with electrical signals to control local pH at electrodes, our research groups have exploited the stimuli-responsive directed assembly of chitosan at electrode surfaces in microchips 33,36,[44][45][46][47] and bioMEMS devices. [36][37][38]48 This paper reports the in situ microfabrication of freestanding, semi-permeable chitosan membranes by pH gradients across aperture openings in microfluidic networks (as shown in Scheme 1(b)). By utilizing the unique pH-dependent solubility of chitosan, we demonstrate that hydrophilic permeable biopolymer membranes can be formed in microfluidic networks by pH gradients generated at the converging interface between a slightly acidic chitosan solution and a slightly basic solution.…”

“…Permeability tests further confirmed the pore size of the membranes to be a few nanometres, similar to the size of proteins (antibodies). Building on the use of chitosan as a soft interconnect for biological components 32,33,[35][36][37]46,[48][49][50] and the broad applications of membrane functionalities in microsystems, [1][2][3][4][5][6][7][11][12][13][14][15][16] we believe that the facile, rapid in situ biofabrication of freestanding chitosan membranes in microfluidics can be applied to many biochemical, bioanalytical and biosensing applications.…”

Biofabrication: programmable assembly of polysaccharide hydrogels in microfluidics as biocompatible scaffolds

“…For example, we have previously engineered enzymes from a bacterial signaling pathway to facilitate their assembly onto these hydrogel matrices and to enable the localized synthesis of the signaling molecule on-chip. 59,60 By replicating the synthesis pathway for signaling molecules (e.g. autoinducer-2) central to bacterial quorum sensing, the resulting system may be used as a testbed for evaluating drug candidates that would operate by inhibiting the enzyme(s), presenting a new approach to antimicrobial therapy.…”

Chitosan: an integrative biomaterial for lab-on-a-chip devices

“…Luo et al continued the work involving enzyme assembly with chitosan by improving the microfluidic packaging in order to optimize the signal-to-noise ratio of the enzymatic conversion. 144 The device is improved by using an alignment scheme for the fluidic interconnects which reduces the dead volume created by reservoirs at the inputs and outputs. In addition, a cross channel design over the sensor site allows the user to separate the path of the enzyme flow from that of the substrate.…”