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.
In this paper we present a multilayer device based on a-Si:H/a-SiC:H that operates as photodetector and optical filter. The use of such device in protein detection applications is pertinent in Fluorescence Resonance Energy Transfer (FRET) measurements that demand the detection of visible fluorescent signals located at specific wavelengths bands. This device was designed to operate in the visible range with a selective sensitivity dependent on the applied electrical bias. Several nanosensors were tested with a commercial spectrophotometer to judge the performance of the FRET signals using glucose solutions of different concentrations. Two nanosensors (FLIPglu-90μM and FLIPglu-600μM) were tested with a commercial spectrofluorimeter to judge the performance of the FRET signals by using glucose solutions of different concentrations. These measurements were carried out by using these nanosensors both in the free form and immobilized form on inner epidermis of onion bulb scale. The proposed device was used to demonstrate the possibility of FRET signals detection, using visible signals of similar wavelength and intensity. The device sensitivity was tuned to enhance the wavelength band of interest using adequate electrical biasing.
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