Fluorescence-based biosensors

Optical biosensors based on fluorescence detection often use the combination of a fluorescent bioreceptor associated with an optical transducer. Fluorescent biosensors may also be obtained by immobilizing whole cells on the surface of a sensor layer. This bioactive layer is usually placed in front of the tip of an optical fibers bundle to generate a fluorescent signal. The optical fibers are required to send the excitation radiation to the fluorescent bioelement and convey the fluorescence radiation up to a fluo-rimeter. In order to improve the simpHcity and reliability of fluorescence-based biosensors, optically translucent supports are used because their optical properties enable detection of fluorescence emitted by the algal cells. 

A common fluorescence technique used for biosensing is the sandwich assay. In this experiment, the analyte is selectively bound to a surface by a targeting molecule (like an antibody), which has been immobihzed covalently on the surface of a well or other cell. By labehng the analyte molecule with a fluorescent tag, its surface concentration may be measured via highly sensitive fluorescence spectroscopy. 

Fluorescence spectroscopy has been widely applied in analytical chemistry. It is a sensitive technique that can detect very low concentrations of analyte because of the instrumental principles involved. At low analyte concentrations, fluorescence emission intensity is directly proportional to the concentration. Fluorescent materials and green fluorescent protein have been extensively used in the construction of the fluorescent biosensor. 

Type 1 Forster (or fluorescent) resonance energy transfer (FRET)-based biosensors Type 2 Bimolecular fluorescence complementation (BiFC)-based biosensors Type 3 Single FP-based biosensors 

In this FRET-based biosensor (a) one of the FPs is linked to the MRE and the other is linked to the analyte protein. When the sensory protein domain binds with the substrate, the donor and acceptor FPs are brought together, thus increasing the acceptor fluorescence intensity while reducing the donor fluorescence intensity. This strategy is commonly used to tag protein—protein interactions in live cells. 

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In a second experiment, Cy5-labelled antiBSA antibodies were immobilised on a silanised glass slide precoated with metallic nanoislands using a polydimethylsiloxane (PDMS) flow-cell. The antibody solution was left for 1 hour to attach and then the cell was flushed with deionised water. The slide was then dried with N2. For this experiment, a portion of the slide was not coated with metallic nanoislands, in order to act as a reference. Figure 20 shows the image recorded using the fluorescence laser scanner mentioned previously. The enhancement in fluorescence emission between those areas with and without nanoislands (B and A, respectively) is again evident. For both chips, an enhancement factor of approximately 8 was recorded. There is considerable interest in the elucidation and exploitation of plasmonic effects for fluorescence-based biosensors and other applications. 

Biomolecules Analysis Introduction Fluorescence-based Biosensors... 

Our work has focussed on two key areas which underpin the eventual exploitation of plasmonic enhancement features in fluorescence-based biosensors ... 

Fluorescence and chemiluminescence sensors are considered the most sensitive in the class of optical sensors. They have a higher selectivity because the chemiluminescent and fluorescent reactions take place in certain medium conditions, and only a limited group of ions and molecules can be involved. When the selectivity of this type of sensor is not sufficient, the quality of analysis can be improved by using a biochemical reaction such as an enzymatic reaction (chemiluminescence- or fluorescence-based biosensors) or an immunoreaction (chemiluminescence- or fluorescence-based immunosensors). By using these types of optical sensors the chemical analysis becomes most sensitive and selective.265... 

Fig. 2 Changes in the calibration of a fluorescence-based biosensor for detection of estrone due to the non-specific interaction of biorecognition elements with residual matrix components [9]...

In addition to the development of absorbance-based fiber-optic biosensors, we have recently demonstrated the feasibility of fluorescence-based biosensors (9, 10). Our initial work in this area has focused on the development of fiber-optic biosensors based on the fluorometric detection of reduced nicotinamide adenine dinucleotide (NADH). Here, a dehydrogenase enzyme supplies the biocatalytic activity, and either the generation or consumption of NADH is oionitored. 

Patra, D. (2010). Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly Biosensors and Bioelectronics 25, 1149-1154. 

Viveros L, Paliwal S, McCrae D, Wild J, Simonian A (2006) A fluorescence-based biosensor for the detection of organophosphate pesticides and chemical warfare agents. Sens Actuators B 115 150-157... 

The first FRET-based biosensors employing fluorescent proteins were developed over 10 years ago. These protease sensors consisted of a BFP donor fused to a GFP acceptor by a protease-sensitive linker [44, 119]. BFP and GFP have well separated emission spectra, resulting in little fluorescence bleed-through (Figs. 5.5A and 5.6A). This facilitates data analysis for FRET ratio imaging... 

Lubbers D.W., Fluorescence Based Chemical Sensors, in Advances in Biosensors, Vol. 

While planar optical sensors exist in various forms, the focus of this chapter has been on planar waveguide-based platforms that employ evanescent wave effects as the basis for sensing. The advantages of evanescent wave interrogation of thin film optical sensors have been discussed for both optical absorption and fluorescence-based sensors. These include the ability to increase device sensitivity without adversely affecting response time in the case of absorption-based platforms and the surface-specific excitation of fluorescence for optical biosensors, the latter being made possible by the tuneable nature of the evanescent field penetration depth. 

As opposed to conventional analytical techniques, optical sensors and biosensors, particularly those employing absorption and fluorescence-based sensing materials potentially allow for measurement through transparent or semi-transparent materials in a non-destructive fashion4, 5> 9 10. Chemical sensor technology has developed rapidly over the past years and a number of systems for food applications have been introduced and evaluated with foods. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

None of the involved species are fluorescent. Therefore, for fluorescence signaling of citrate recognition, carboxyfluorescein is first added to the medium because binding to the receptor in the absence of citrate is possible and causes deprotonation of carboxyfluorescein, which results in high fluorescence. Citrate is then added, and because it has a better affinity for the receptor than carboxyfluorescein, it replaces the latter, which emits less fluorescence in the bulk solvent as a result of protonation. Note that this molecular sensor operates in a similar fashion to antibody-based biosensors in immunoassays. It was succes-fully tested on a variety of soft drinks. 

Russsell RJ, Pishko MV, Gefrides CC, McShane MJ, Cote GL. A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a polyiethylene glycol) hydrogel. Analytical Chemistry 1999, 71, 3126-3132. 

Pickup JC, Hussain F, Evans ND, Rolinski OJ, Birch DJS. Fluorescence-based glucose sensors. Biosensors Bioelectronics 2005, 20, 2555-2565. 

Scognamiglio V, Staiano M, Rossi M, D Auria S. Protein-based biosensors for diabetic patients. Journal of Fluorescence 2004, 14, 491-498. 

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