*3.4. Proteins/Enzymes*

Several protein-based assays have employed studies of protein modification/interaction, kinase activity, time-bound fluorescent assays, detection of toxins/adulterants, identification of viral antigens/pathogens, etc. These have incorporated fluorescent dyes viz. Cy5, BEBO (cyanide dye), lanthanides (e.g., Eu3+, Sm3+, Tb3+ and Dy3+), SYBR green, NanoOrange, and RiboGreen that have been utilized in fluorescent biomolecular assays such as bimolecular fluorescence complementation (BiFC), lanthanide fluorescent immunoassay, fluorescent-dye-based assay, chemifluorescent enzyme-linked immunosorbent assay (ELISA), real-time immuno-PCR, immuno-detection, and sandwich fluoroimmunoassay [79]. Green fluorescent protein (GFP) or yellow fluorescent protein (YFP) have quite often been used as reporter conjugates/markers in the detection of pathogens for food sensing, helping in enumerating/tracking of bacterial cells. For complex sample preparation, fluorescent proteins with their longer wavelengths avoid the limitation associated with fluorescent dyes. Along with GFP and YFP, R-phycoerythrin (PE) isolated from red algae is also used as stable fluorescent protein [80]. A study has reported a novel TurboGFP expression vector for labeling of Yersinia species *Y. enterocolitica* biova*r* 1A, biovar 2, biovar 4, and *Y. pseudotuberculosis.* After being transformed with the vector, these bacteria expressed fluorescence of bright green color that could be seen with the naked eye [81]. Similarly, a fiber-optic toxicity biosensor incorporating GFP label modification of *Escherichia coli* was designed for detection of hazardous heavy metals such as Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I), and Fe(III) and their toxicity in the samples [82]. Apart from proteins, certain enzyme-based sensors utilizing peroxidase (HRP), glucose oxidase, lactase, urease, alkaline phosphatase, etc., integrate fluorescent properties of coenzymes that absorb light or substrates for catalytic reactions play a crucial role in sensing of food, toxins, pathogens, etc. Enzymes, being different moieties than generalized bioreceptors, are not directly involved in detection of analytes but they amplify the signal by catalyzing certain reactions. Likewise the fact that they only need a substrates in order to work, but are not affected by the working medium/environment, makes them outstanding as potential substitutes for sensing and food-monitoring applications.
