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

• Published in: Sensors and actuators. B, Chemical Chemical, 2015-12, Vol.221, p.236-241
• Main Authors: Charneca, Ana, Karmali, Amin, Vieira, Manuela
• Format: Article
• Language: English
• Subjects: Assaying; Binding; Fluorescence; Fluorescent indicator proteins; FRET; Fretting; Genetically encoded nanosensor 3.2 mM; Glucose; High throughput glucose assay; Immobilization on 96-well microtiter plates; Monitoring; Nanostructure; Onion membranes; Proteins
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2015.06.037

•Immobilization of whole-cells and cell-free extract containing FRET nanosensors.•Genetically encoded nanosensors immobilized on onion membrane and 96-well plates.•Novel and cheap high throughput method for glucose assay by using FRET analysis.•Assay method does not involve a chemical conversion of glucose+O2 into products.•Immobilized novel FRET nanosensors exhibit high storage and operational stabilities. 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- 3.2mM. 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 0–24mM with a limit of detection of 0.11mM 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.