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

2015-12-31Journal article Open access
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dc.contributor.authorCharneca, Ana
dc.contributor.authorKarmali, Amin
dc.contributor.authorVieira, Manuela
dc.date.accessioned2016-03-11T15:20:04Z
dc.date.available2016-03-11T15:20:04Z
dc.date.issued2015-12-31
dc.description.abstractGlucose 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.pt_PT
dc.identifier.citationCHARNECA, Ana; KARMALI, Amin; VIEIRA, Manuela; - Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins. Sensors and Actuators B-Chemical. ISSN. 0925-4005. Vol. 221, (2015), pp. 236-241pt_PT
dc.identifier.doi10.1016/j.snb.2015.06.037pt_PT
dc.identifier.issn0925-4005
dc.identifier.urihttp://hdl.handle.net/10400.21/5837
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevier Science SApt_PT
dc.relation.publisherversionhttp://www.sciencedirect.com/science/article/pii/S0925400515008126pt_PT
dc.subjectFluorescent indicator proteinspt_PT
dc.subjectOnion membranespt_PT
dc.subjectGenetically encoded nanosensor 3.2 mMpt_PT
dc.subjectHigh throughput glucose assaypt_PT
dc.subjectImmobilization on 96-well microtiter platespt_PT
dc.subjectFRETpt_PT
dc.titleNon-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteinspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage241pt_PT
oaire.citation.startPage236pt_PT
oaire.citation.volume221pt_PT
person.familyNameKarmali
person.familyNameVieira
person.givenNameAmin
person.givenNameManuela
person.identifier10792
person.identifier.ciencia-id9516-E25E-BB8E
person.identifier.orcid0000-0003-0419-401X
person.identifier.orcid0000-0002-1150-9895
person.identifier.ridE-4787-2011
person.identifier.ridV-7860-2017
person.identifier.scopus-author-id6603778216
person.identifier.scopus-author-id7202140173
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublication23975b4b-3234-4139-9675-89ea6f9a2332
relation.isAuthorOfPublicationb77c6785-9cfb-4cd4-90b5-c2b816bd7d11
relation.isAuthorOfPublication.latestForDiscovery23975b4b-3234-4139-9675-89ea6f9a2332

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