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Sensors & Transducers Journal, Vol. 113, Issue 2, February 2010, pp. 1-17

Fluorescence Resonance Energy Transfer (FRET) is a nonradiative energy transfer process from excited state donor molecule to an acceptor molecule, when appreciable overlap exists between the emission spectrum of the donor and the absorption spectrum of the acceptor. This radiation-less transfer of energy, when the excited state fluorophore and the second chromophore lie within a range of approximately 10 nm, provides vivid structural information about the donor-acceptor pair. This is a quantum mechanical process that does not require a collision and does not involve production of heat. When energy transfer occurs, the acceptor molecule quenches the donor molecule fluorescence, and if the acceptor is itself a fluorochrome, increased or sensitized fluorescence emission is observed [35, 36, 38]. Fig. 5 shows the underlying principle of FRET. The information obtained by this method is unique because the surrounding solvent shell of a fluorophore does not affect the FRET measurements.

Donor

Acceptor

molecule

molecule

Wavelength (λ)

Fig. 5. Fluorescence resonance energy transfer.

The Surface Enhanced Raman Spectroscopy or Surface Enhanced Raman Scattering (SERS) is a surface sensitive technique that results in the enhancement of Raman scattering by molecules adsorbed on rough metal surfaces. The vibrational modes of the adsorbates on the roughened surface are sometimes observed to have about one million times the intensity that would be predicted by comparison with their Raman spectra in the gaseous phase [39].

Fluorescent measurement techniques are commonly used for the detection of biomolecules. In fluorescence spectroscopy, fixed or living cells or single stranded DNA probes are often labeled with fluorescent tags or fluorophores, each specific to a particular intercellular component, which absorps light at one wavelength (excitation), followed by a subsequent emission of secondary fluorescence at a longer wavelength. The excitation and emission wavelengths are usually separated from each other by tens to hundreds of nanometers.

Cellular components are labeled with specific fluorophores to identify their localization within fixed and living parameters. In microarray systems, the target molecules are labeled, which is a process of covalently binding a molecule or particle to the target DNA strand, for generating transducer signal. This approach takes care of the issue related to safety and disposal associated with radioactive markers and allows the researchers to study several experimental parameters simultaneously with multiplex samples. In the case of multiple probes, different dyes are attached to different probes which can be simultaneously detected at different wavelengths using optical filters. After hybridization, the fluorescent signals from a DNA chip are studied using specific instruments. However, this method of labeling with fluorophores is not possible everywhere [64] because the optical labels are costly and unreliable and also the optical scanners are expensive and the procedure of extracting information from the data is complicated [70]. Researchers are trying to work it out with label free techniques or reagent-less optical biosensors where the target sample can be detected in a heterogeneous solution without adding anything but the sample [96].

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