Maximizing Signal Level

Since the "native" fluorescence of biomolecules is relatively weak in most cases, it is a common practice to label the biomolecules with fluorescent probes, which can be covalently and site-specifically attached to biomolecules [16,17]. To maximize signal using fluorescence-based techniques, fluorophores with high quantum yields and favorable photophysical properties such as large absorption cross sections and high photostability need to be used. Some commonly used classes of fluorescent probes used in biological labeling include rhodamines, cyanines, oxazines, etc. [7,18]. More recently, a new class of single-molecule fluorophores has been found among molecules originally optimized for nonlinear optical properties [19]. Alternatively, variants of green fluorescent protein (GFP) can be fused onto proteins as fluorescent tags, which have been successfully used to monitor motor proteins [20,21]. In principle, any protein can be fluorescently labeled by constructing cDNAs of desired proteins fused to the genes of GFPs and expressing them in living cells. However, dramatic blinking of GFP is a problem that needs to be further addressed [22]. Strong fluorescing semiconductor quantum dots have also been proposed as biological fluorescent labels. Advantages offered by quantum dots include narrow emission lines and resistance to photobleaching. However, their application has been limited by poor attachment of the quantum dots to biomolecules, which has been the subject of intense ongoing research [23]. Biocompatible quantum dots have been developed by encapsulating quantum dots in an organic disguise that prevents them from coming into direct contact with the aqueous biological environment.

For Raman-based techniques, efficient and reproducible substrates for surface enhancement need to be prepared to increase the cross section for Raman scattering. Different schemes have been developed to produce highly uniform and reproducible localized surface plasmon resonance (LSPR) nanostructures from several different materials [24-27]. Silver nanostructures are the most common media and provide the largest enhancements. Gold is another material frequently used in various surface-enhanced Raman scattering (SERS) applications because of their large enhancement, biocompatibility, chemical robustness, and established functionalization chemistry. The production schemes include vapor deposition through self-assembled monolayer masks [25], electrochemical etching procedures [26], templated self-assembly of colloidal crystals [24,27,28], and annealing of vapor-plated metal islands [29].

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