Here we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the “branch point effect”, a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of Glucose Oxidase (GOx) and coupled it to a second, non-signaling reaction catalyzed by the higher affinity enzyme Hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter’s response. Above an arbitrarily selected “threshold” substrate concentration, the non-signaling HK enzyme saturates leading to a “sudden” activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

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The fabrication, evaluation and attractive performance of multiwall carbon nanotube(MWCNT)/polysulfone biocomposite membrane modified thick-film screen-printed electrochemical biosensors are reported. The fabricated carbon nanotube/polysulfone (CNT/PS) strips combine the attractive advantages of carbon nanotube materials, polysulfone matrix and disposable screen-printed electrodes. Such thick-film carbon nanotubes/polysulfone sensors have a well defined performance, are mechanically stable, and exhibit high electrochemical activity. Furthermore, biocompatibility of CNT/PS composite allows easy incorporation of biological functional moiety of horseradish peroxidase by phase inversion technique. The comparison of graphite with MWCNT as conductor material is described in this paper. The proposed H(2)O(2) biosensor exhibited a linear range (applied potential, -0.2 V) from 0.02 to 0.5 mM and a K(M)(app) of 0.71 mM.

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The aim of this study was the fabrication and characterization of biomembranes by the phase inversion (PI) method followed by their subsequent casting onto screen-printed electrodes (SPE) for biomedical applications. The combination of multiwalled carbon nanotubes (MWCNT) as a transducer with polysulfone (PSf) polymer enables easy incorporation of biological moieties (hormones or antibodies), providing a 3D composite with high electrochemical response to corresponding analytes. Antibody/MWCNT/PSf biosensors were characterized by confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM), and electrochemical methods. For biomedical purposes, human chorionic gonadotropin (hCG) hormone was tested by competitive immunoassay. The detection limit was determined to be 14.6 mIU/mL with a linear range up to 600 mIU/mL. We concluded that the easy and fast incorporation of biomolecules by the PI method, as well as their stability and distribution throughout the 3D polysulfone composite, are testament to the utility for the versatile fabrication of biosensors for clinical diagnosis.