This work presents a novel development that exploits the concept of in-situ gas-separation together with a specific enzymatic colorimetric detection to produce a portable biosensor called "Blood Alcohol Micro-pad" for direct quantitation of ethanol in whole blood. The thin square device (25 mm × 25 mm × 1.8 mm) comprises two layers of patterned filter paper held together with a double-sided mounting tape with an 8-mm circular hole (the headspace). In operation, the reagent is deposited on one layer and covered with sticky tape. Then 8 μL of a blood sample is dispensed onto the opposite layer and covered with sticky tape. Diffusion of ethanol across the 1.6 mm narrow headspace permits selective detection of ethanol by the enzymatic reagents deposited on the opposite layer. This reagent zone contains alcohol oxidase, horseradish peroxidase and 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, as the chromogenic reagent. The color intensity, measured from the recorded digital image, resulting from the enzymatic assay of ethanol, correlates with the concentration of blood alcohol. The results obtained with spiked mice and sheep blood samples, using an external calibration in the range of 1-120 mg dL-1ethanol, gave recoveries of 93.2-104.4% (n = 12). The "Blood Alcohol Micro-pad" gave good precision with %RSD <1 (50 mg dL-1 ethanol, n = 10) and limit of quantification (10SD of intercept/slope) of 11.56 mg dL-1. The method was successfully validated against a headspace gas chromatography-mass spectrometric method. It has good potential for development as a simple and convenient blood alcohol sensor for on-site testing.
Keywords: ABTS; Blood alcohol; Colorimetric biosensor; Paper-based analytical device; Whole blood; in-situ headspace separation.
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