Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Abstract

Genetically encoded nanosensor 3.2 mM High throughput glucose assay Immobilization on 96-well microtiter plates FRET a b s t r a c t Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu-3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports.The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50-90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0-24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

"Production, Purification and Immobilization of Lipase from Wild-Type and Mutant Strains of Pseudomonas Aeruginosa Grown in Culture Media with Olive and Used Cooking Oils"

“…Ten cycles were performed in 10 days and each cycle of the immobilized lipase was run for 45 min. The residual enzyme activity was expressed as the percentage of the activity at several cycles compared with the first cycle.Covalent Immobilization of Lipase with Glutaraldehydeon Microtiter Plates: 96 and 24-well microtiter plates were used as supports for lipase immobilization as described previously (Charneca, et al[19]). As far as 96-well microtiter plates are concerned, 0, 10, 20, 40, 60, 80 and 100 μL of purified lipase (0,2 U/ mg protein) from mutant strain 3B of P. Aeruginosa were added to the wells and the solution allowed to dry at room temperature.…”