Triglyceride Assay by Amperometric Microbial Biosensor: Sample Hydrolysis and Kinetic Approach

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Abstract
A triglyceride assay based on triglyceride hydrolysis and glycerol detection was developed. Non-specific lipase isolated from Candida rugosa and intact Gluconobacter oxydans cells, containing membrane-bound glycerol dehydrogenase, were used to develop a biosensor. Two approaches were investigated: analysis of pre-hydrolysed samples and a kinetic approach. The sensor prepared from G. oxydans cells exhibited sensitive and fast response to glycerol: detection limit 20 μM (S/N=3), linear range up to 2 mM and response time 84 s (90% of steady-state). The triglyceride assay of pre-hydrolysed samples was based on a 20 min hydrolysis and determination of released glycerol by the biosensor. A calibration curve linear up to 12 mM was obtained for triolein samples. The kinetic approach was based on simultaneous glyceride hydrolysis and glycerol detection. Analysis time of 10 min, linear range up to 30 mM, and estimated detection limit of 50 μM were achieved using the kinetic approach. The kinetic triglyceride assay is not influenced by free glycerol present in a sample. Storage stability, expressed as a half life (50% of the initial response), was 7 days when trehalose was used as a stabiliser.