Microplate differential calorimetric biosensor for ascorbic acid analysis in food and pharmaceuticals

In this paper we report on the development of a label-free low-volume (12.5 microL), high-throughput microplate calorimetric biosensor for fast ascorbic acid quantification in food and pharmaceutical products. The sensor is based on microplate differential calorimetry (MiDiCal) technology in which the heat generation, due to the exothermic reaction between ascorbic acid and ascorbate oxidase, is differentially monitored between two neighboring wells of an IC-built wafer. A severe discrepancy is found between expected and observed sensor readings. To investigate the underlying mechanisms of these findings a mathematical model, taking into account the biochemical reactions and diffusion properties of oxygen, ascorbic acid, and ascorbate oxidase, is developed. This model shows that oxygen depletion in the microliter reaction volumes, immediately after injection of sample (ascorbic acid) into the well, causes the enzymatic reaction to slow down. Calibration experiments show that the sensor's signal is linearly correlated to the area under the output versus time profile for the ascorbic acid concentration range from 2.4 to 350 mM with a limit of detection of 0.8 mM. Validation experiments on fruit juice samples, food supplements, and a pain reliever supplemented with ascorbic acid reveal that the designed method correlates well with HPLC reference measurements. The main advantages of the presented biosensor are the low analysis cost due to the low amounts of enzyme and reagents required and the possibility to integrate the device in fully automated laboratory analysis systems for high-throughput screening and analysis.