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Fluorescent Protein-Based Biosensors
What are biosensors?

A biosensor, in simple terms, is a biological component that can sense an analyte. It contains three parts; a biological element that detects the change, a transducer element that measures the signal of the sensing element and a signal processor that displays the result.

The sensing elements include enzymes, antibodies, microorganisms, biological tissues, nucleic acid etc. and the transducer can be electrochemical, optical, acoustic, colourimetric etc.

Fluorescent Protein-Based Biosensors

In fluorescent protein-based biosensors the sensing element consists of one or more fluorescent proteins (FPs) linked to one or more polypeptide chains. The polypeptide chain acts as the molecular recognitions element (MRE) that undergoes conformational changes upon binding with the analyte, thus producing a change in fluorescence properties.
Generally, FP-based biosensors can be described under three types based on their structure.

Type I : Förster (or Fluorescent) Resonance Energy Transfer (FRET) based biosensors
Type II : Bimolecular Fluorescence Complementation (BiFC) based biosensors
Type III : Single FP based biosensors

FRET based biosensors

FRET describes the energy transfer between two chromophores. A donor chromophore, in a higher energy state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of the energy transfer is determined by the distance and orientation between the donor and acceptor proteins. Generally, FRET efficiency, measured by a fluorescence emission spectrum, is used to determine the proximity of the two chromophores.

In FRET based biosensors, two fluorescent proteins are genetically linked either to each end of a polypeptide chain (MRE) which is sensitive to the analyte or two separate polypeptides, the MRE and the analyte protein. Upon interaction with the analyte, conformation of the sensor protein changes, thus altering the distance between two chromophores. This causes a change in the fluorescence intensities of the donor and acceptor FPs which is measured in terms of FRET efficiency. An increased FRET efficiency indicates that the two FPs are aligned together while a decrease in FRET efficiency suggests that the donor and acceptor FPs are separated.

FRET-based biosensors are widely used to detect a range of molecular events such as protein-binding interactions, protein conformational changes, enzyme activities (e.g. proteolysis, phosphorylation, dephosphorylation, and GTPase activities), and concentration of biomolecules.

Some common examples of FRET-based biosensor designs are illustrated below.
[Image: fretbiosensorsfigure1.jpg]

(a). Here, one of the FP 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. That is, according to this example, the fluorescence hue of the specimen changes from cyan to yellow. This strategy is commonly used to tag protein-protein interactions in live cells.

(b). In this type of biosensors, the donor FP and the acceptor FP are fixed to the opposite ends of the MRE. When the analyte binds to the MRE the conformation of the sensor protein changes thus placing the donor and acceptor FPs side by side. This increases the FRET efficiency. This is usually used for the detection of glucose, maltose, glutamate and cyclic nucleotides.

©. Two FPs are attached to each end of a complex unit composed of a sensory domain linked to its binding substrate. When the sensory domain is stimulated by the analyte, it binds to the substrate protein inducing a large overall conformational change that changes the FRET signal. Ca2+ biosensor that employ calmodulin as the substrate and calmodulin-binding peptide as the MRE is a classic example for this. When the calcium ion concentration is high, calmodulin binds to the MRE, bringing ther FRET pair closes and produces a FRET signal. When the calcium ion concentration drops below a certain level, calmodulin dissociates from the peptide, decreasing FRET.

(d) This model of biosensors is used to detect proteolytic activity. MRE, a substrate for the protease of interest, is cleaved by the enzyme thus detaching the FRET pair and this turns the FRET signal off.

Check out this link for an interactive flash tutorial on FRET-Based biosensors.

Bimolecular Fluorescence Complementation (BiFC)-based biosensors

In this type of biosensors, the FP which is split up and MRE is linked to one portion while the analyte protein is linked to the other portion. When the two proteins interact, the two fragments fuse together, refolding properly into its 3-D structure and produce a fluorescence signal. . BiFC biosensors are commonly used to detect protein-protein interactions in cells. It is even possible to combine pieces of different fluorescent proteins together thus producing chimeral FPs with a variety of fluorescent shades enabling the simultaneous study of multiple protein interactions in the same cell.

[Image: image2.jpg]

Single FP based biosensors

A single fluorescent protein coupled with a MRE makes up single FP based biosensors. The MRE can be either exogenous or endogenous. Analyte binding to the MRE causes conformational changes of the fluorescent protein consequently altering its fluorescent properties. This strategy is useful in pH sensitive biosensors, Zn2+ biosensors etc.

[Image: b907749a-f5.gif]


However, recent research advancements have resulted in biosensor designs that combine two or more of the above strategies.

A growing field

Owing to the comparative advantages such as ease of manufacture using standard molecular biology techniques, ability to noninvasively observe biological process in the live cells, high sensitivity, high selectivity, the progress in the development of the genetically encode fluorescent protein-based biosensors has been revolutionary.
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Green fluorescent protein and its principles have thrown their light over the wide spectrum of biological causes. Today, with the help of color palette which is comprised of fluorescent proteins in various spectrum allow researchers to paint living cells according to their own desires.

Besides, sophisticated bio sensors have been engineered with regard to containing single or double or multiple fluorescent proteins. These include spatiotemporally FRET-based bio sensors. They unveil molecular methodologies involved in underlying physiological processes.

It is also true that these molecular have immensely contributed for basic researches so far. It is also necessary to mention that their functionality could be used in applied sciences is yet to investigate.
Genetically produced bio sensors has the tendency to allow noninvasive imaging of certain bio recognition or biochemical processes with the preservation of temporal and sub-cellular spatial information. Apart from this, aequorea green fluorescent protein and their engineered variants are very important and critical components of genetically produced bio sensors because they provide read-out of bio-recognition chain of events even during investigations.

The family of fluorescent protein based bio sensors includes the diverse range of arrays of various designs that can be utilized for different photo-physical characteristics of fluorescent proteins.
Notwithstanding, these fluorescent bio sensors can be used for studying molecular chain of events from single cells to whole organism. They are unique in the context that they have the capability to target both organelles and tissues. They can also determine different molecules events such as protein binding interactions, phosphorylation, proteolysis, and De-phosphorylations. There is option of designing them with four different and common patterns.

These patterns includes intra-molecular interactions, intermolecular interactions, multiple FPs or proteo-lytic cleavage. Binding interactions between proteins are essential for signal transduction and catalytic activation. For instance, many protein pathways are activated by the association of membrane receptors, which activate enzymes for propagation of signals.
Sasa Milosevic
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