The natural world is an elaborate mixture teeming with a plethora of compounds. On a macroscopic scale, the structural organization of these compounds is ubiquitous; however, to the unaided observer, their role in biological and chemical systems can be easily overlooked. Typically, scientists employ analytic assays to better visualize, quantify, and assess the activity of a target species, or analyte. The allure of these assays arises from the ability to provide relevant physical and biomedical information at varying levels (Misiuk, 2010). Recent developments with analytical instruments such as fluorescence-based sensors have yielded promise in a number of scientific applications, ranging from quantitative quality assurance to efficient health monitoring.
To better understand the utility of fluorescent sensors, it is first important to consider relevant underlying physical processes. Fluorescence is an electrical phenomenon that occurs when some of the internal energy of a molecule is lost as its electrons transition from a higher to lower energy state (Figure 1) (Lakowicz, 2006; Valeur and Berberan-Santos, 2013). In other words, when a fluorophore absorbs energy, such as in the form of light at a particular wavelength, it will re-emit that energy as light of a longer wavelength over a certain interval of time (Lakowicz, 2006). Generally, fluorescence is characterized by radiative processes of electron demotion, while a similar phenomenon known as quenching results from non-radiative processes, which decrease the fluorescence intensity of a molecule (Lakowicz, 2006). Fluorescence quenching forms the basis of fluorescence spectroscopy, where physical parameters such as the Stoke’s shift, fluorescence intensity with and without a quencher, and ultimately the concentration of the analyte can be measured (Lakowicz, 2006).
In practice, the effectiveness of a fluorescent sensor mainly depends on the stability and brightness of the fluorophore (Carter, Young, and Palmer, 2014). Additionally, an important consideration is the sample matrix and setting of the analyte; in biological systems, photodamage and photobleaching incurred by the excitation and emission wavelengths may hinder detection (Carter, Young, and Palmer, 2014; Ma et al., 2014). To mitigate these factors, the design of a fluorescent sensor should account for the reactive nature of the analyte. For example, fluorescent sensors for metal ions consist of a metal chelating moiety and a fluorophore (Carter, Young, and Palmer, 2014). This allows for a high degree of selectivity and accurate measurements since only specific metal ions will chelate and quench the fluorescence signal (Carter, Young, and Palmer, 2014). These metal ion fluorescence sensors have been used to test for environmental contaminants like mercury, as well as in living systems to gauge the presence of important cofactors, such as iron (Carter, Young, and Palmer, 2014; Ma et al., 2014). In addition, they exhibit especially high spatial and temporal resolution, giving them an advantage over some conventional optical sensors (Wu et al., 2017).
Fluorescent biosensors also have widespread applications in assessing food safety. In particular, they have been developed to detect mycotoxins, which are fungal secondary metabolites responsible for food poisoning (Sharma et al., 2018). Since mycotoxins are not inherently fluorescent, special fluorescent aptamers, which are small oligonucleotides that bind with high affinity and specificity to a variety of target molecules, or antibodies are used as probes to label the analyte (Hong, Li and Li, 2012; Sharma et al., 2018). As such, the simplest biosensors require a biological component and a signal-generating component that converts the biochemical interaction into a physical signal (Hong, Li and Li, 2012). For example, ochratoxin A (OTA) can be quantified in coffee, wine, corn, and other matrices via a fluorescence resonance energy transfer (FRET) immunoassay system (Sharma et al., 2018). In this case, there is an observable change in the fluorescence signal when the anti-OTA antibody complexes with OTA, which can then be amplified with a physiochemical transducer (Figure 2) (Hong, Li and Li, 2012; Sharma et al., 2018). The output of these methods, essentially a calibration curve with a specified linearity and limit of detection, can then be used to construct safe ranges and quantify toxin levels in a vast array of samples (Sharma et al., 2018).
Overall, fluorescence-based sensors operate on the well-known physical principles of fluorescence. Depending on the compound of interest, these sensors can be adapted to target specific metal ions or biomolecular agents with excellent sensitivity and resolution. While continued research will improve its capabilities, fluorescent sensors are versatile analytical tools that offer a breadth of uses, directly impacting human health and safety.
References
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