In this work we demonstrate a novel microfluidic based platform to investigate the performance of 3D out-of-plane microspike array based glucose and lactate biosensors. The microspike array was bonded with a glass slide and modified with glucose oxidase or lactate oxidase using covalent coupling chemistry. An epoxy-polyurethane based membrane was used to extend the linear working range (from 0 to 25 mM of substrate) of these biosensors. Both lactate and glucose sensors performed well in the clinically relevant substrate concentration range. Glucose microspikes were further investigated with respect to the effects of substrate transfer by incorporation into a microfluidic system. Data from the microfluidic system revealed that the sensor response is mainly dependent on enzyme kinetics rather than membrane permeability to glucose. The robustness of the sensors was demonstrated by its consistency in performance extending over 48 h.

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A permselective membrane is a critical component that defines the linear detection limits, the sensitivity, and thus the ultimate efficacy of an enzymatic biosensor. Although membranes like epoxy-polyurethane (epoxy-PU) and Nafion are widely used and provide the desired glucose detection limits of 2 to 30 mM, both the within batch and batch-to-batch variability of sensors that use these materials is a concern. The hypothesis for this study was that a crosslinked hydrogel would have a sufficiently uniform porosity and hydrophilicity to address the variability in sensor sensitivity. The hydrogel was prepared by crosslinking di-hydroxyethyl methacrylate, hydroxyethyl methacrylate and N-vinyl pyrrolidone with 2.5 mol% ethylene glycol dimethacrylate using water soluble initiators -ammonium persulfate and sodium metabisulfite under a nitrogen atmosphere. The hydrogel was applied to the sensor by dip coating during polymerisation. Electrochemical measurements revealed that the response characteristics of sensors coated with this membrane are highly consistent. Scanning electrochemical microscopy (SECM) was used to spatially resolve glucose diffusion through the membrane by measuring the consequent H 2 O 2 release and compared with an epoxy-PU membrane. Hydrogen peroxide measurements using SECM revealed that the epoxy-PU membranes had uneven lateral diffusion profiles compared to the uniform profile of the hydrogel membranes. The uneven diffusion profiles of epoxy-PU membranes are attributed to a fabrication method that results in uneven membrane properties, while the uniform diffusion profiles of the hydrogel membranes are primarily dictated by their uniform pore size.

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The concept of a biosensor is well established and the idea of integrating a molecular recognition layer with a base sensor, such that analyte binding or reaction at the former results in a measureable change (in current or voltage) in the latter has seen many ingenious embodiments. Despite this and the huge amount of research worldwide in biosensors, as outlined in Chap. 2, many challenges still remain in building reliable, long-lived biosensors, especially in the hostile environment of the human body. The enormous potential for in vivo sensing of pathophysiological molecules over time and space has led to many attempts to achieve this and the rewards, both in terms of clinical benefit and improved understanding, cannot be underestimated. As new tools for producing biosensors become available, they are rapidly recruited. In recent years, developments in two areas of science and engineering have provided new opportunities to look again at how biosensors are built and deployed. These developments were not driven by the needs of those building and using biosensors but by much broader scientific and technological trends, which nonetheless have found ready applicability in this area.One of the trends is an increasing knowledge of the structural factors that determine function in biological macromolecules and the other is the appreciation that the properties of materials with a characteristic length scale from 1 to 100 nm are not those expected from dividing macroscopic materials into smaller pieces nor those expected from adding atoms or molecules together. The first of these trends is sometimes referred to as biomolecular engineering and the second as nanotechnology.There are many drivers for the use of molecular engineering and nanotechnology in the design and application of biosensors and the past decade has seen these tools

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