Paper-fluidic electrochemical biosensing platform with enzyme paper and enzymeless electrodes

A miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform was developed and the effects of fluidic behaviors in paper substrate on electrochemical sensing were systemically investigated. The biosensor is composed of an enzyme-immobilized pure cellulose paper pad, an enzymeless screen printed electrode (SPE) modified with platinum nanoparticles (PtNPs), and a pair of clamped acrylonitrile butadiene styrene (ABS) plastic holders to provide good alignment for stable signal sensing. The wicking rate of liquid sample in paper was predicted, using a two-dimensional Fickian-diffusion model, to be 1.0 x 10(-2) cm(2)/s, and was verified experimentally. Dip-coating was used to prepare the enzyme-modified paper pad (EPP), which is amenable for mass manufacturing. The EPP retained excellent hydrophilicity and mechanical properties, with even slightly improved tensile strength and break strain. No significant difference in voltammetric behaviors was observed between measurements made in bulk buffer solution and with different sample volumes applied to EPP beyond its saturation wicking volume. Glucose oxidase (GO(x)), an enzyme specific for glucose (Glc) substrate, was used as a model enzyme and its enzymatic reaction product H2O2 was detected by the enzymeless PtNPs-SPE in the presence of ambient electron mediator O-2. Consequently, Glc was detected with its concentration linearly depending on H(2)O(2)oxidation current with sensitivity of 10.5 mu A mM(-1) cm(-2) and detection limit of 9.3 mu M(at S/N = 3). The biosensor can be quickly regenerated with memory effects removed by buffer additions for continuous real-time detection of multiple samples in one run for point-of-care purposes. This integrated platform is also inexpensive since the EPP is easily stored, and enzymeless PtNPs-SPEs can be used multiple times with different EPPs. The green and facile preparation in bulk, excellent mechanical strength, well-maintained enzyme activity, disposability, and good reproducibility and stability make our paper-fluidic biosensor platform suitable for various real-time electrochemical bioassays without any external power for mixing, especially in resource-limited conditions. (C) 2014 Elsevier B. V. All rights reserved.