**4. Integration of Fluorescence Biosensing for Microbe Detection**

Over recent decades, development of fluorescence-based detection of pathogenic microbes has accelerated, with the development of direct and rapid point-of-care testing techniques that maintain proper safety assessments. Fluorescence biosensing has the well-established advantages of immediate response time, highly sensitive detection, easy labelling of fluorophore with functional groups, localized fluorescence signals that provides visible output using multicolor dyes, and multiplexed detection assays [83]. For decades culture-based methodology was the gold-standard. It offers low-cost, equipment-free, and easy-operational detection assays. However, its time constraint compromises rapid and on-site detection. Then, PCR (polymerase chain reaction) and LAMP (loop-mediated isothermal amplification) assays were developed, offering high sensitivity and rapid bacterial detection. However, several bottlenecks related to expensive instrumentation, falsepositive results, and the need for trained manpower also restricted their applicability for point-of-care microbial detection systems. Moreover, immunological techniques, such as ELISA, that are increasingly recommended for pathogen detection due to their sensitive antigen–antibody interaction, also have shortcomings of cross-reactivity, longer durations for result processing, and complex sample processing [84]. Therefore, to avoid the limitations of the aforementioned methodologies, high-performance novel fluorescence-based biosensing techniques were introduced. These are sensitive up to an ultralow level microbial concentrations and satisfy the high demand for food safety. Here, we will focus upon

these fluorescence-based bioassays comprising microarray/biochip assays, microfluidics assays, paper-based hand-held devices, and lateral-flow devices.

### *4.1. Microarrays*

Fluorescence-based microarrays comprise a microtiter plate, a glass slide onto which the sample protein is bound in an array, and fluorescently labelled probe molecules which are added to deliver chemiluminescence or a colorimetric signal readout. The fluorescencelabelled probe interacts with the immobilized protein samples releasing a fluorescent signal that is further scanned by laser for detection. The biochemical activity of proteinsensing is generally studied using three types of array—analytical, functional, and reversephase protein microarrays—that are consolidated for pathogen-sensing, ensuring food safety. Studies have shown that the bead/suspension array technique provides detection of bacterial/plant toxins, mycotoxins, and pesticides in food using microsphere beads conjugated with biomolecules such as DNA oligonucleotides/proteins labeled with fluorescent dye. The DNA microarray technique comprises immobilization of cDNA probes on a solid matrix onto which PCR-amplified fluorescent-labeled DNA molecules are hybridized. Their interaction generates a signal, allowing detection of known probes on the microarray. DNA-microarray-technology applications have been extended to a great extent for detection of food pathogens. Fluorophores that are generally incorporated for labeling of probes are Cyanine5/Alexa Fluor 647 (excitation at 650 nm/emission at 668 nm), Cyanine3/Alexa Fluor 555 (excitation/emission values at 550/568 nm), and bacterial-species-specific antibody-labeled and biotinylated DNA/RNA aptamers in combination with fluorescence-labeled streptavidin [85]. An in situ generated biochip was designed for detection of food pathogens present on freshly cut vegetables and fruits. Specific sequences of Vibrio *parahemolyticus*, *Escherichia coli* O157:H7, *Salmonella typhimurium*, *Staphylococcus aureus*, and *Listeria monocytogenes* were identified using tilling array probes in a hybridization array. The assay produced strong amplification signals with detection limit of 3log CFU/gm on freshly cut lettuce and cantaloupe in 24 h time detection [86]. Another work studying the amplification of foodborne-pathogen sensing on microarray comprised Cy5-dye-labeled double biotin DNA linkage and detection antibody as Cy5–Ab complex. Simultaneous detection of *Salmonella* and *E. coli* was achieved as visual screening followed by fluorescence-based quantification. A detection limit of 103 CFU/mL and 9 CFU/mL in buffer and real food was achieved via visual screening and quantification of fluorescence intensity [87].
