*3.1. Nanomaterials*

**Metallic nanoparticles** acquire quantum mechanical effects such as photoluminescence and the photobleaching resistance of gold nanoparticles encourages the development of in vivo fluorescence biosensors with less toxicity [17]. Gold nanoparticles are excellent FRET-quenchers due to their surface plasmon in visible range, which causes strong absorption and scattering with huge extinction coefficients [18]. A study has reported the gold-nanoparticle-based combined fluorometric and spectrophotometric biosensing of biogenic amines in poultry meat samples. The excitation and emission of histamine conjugated with gold nanoparticle was measured and showed 50 times enhanced fluorescence compared to histamine alone [19]. Silver nanoparticles are great substrates for metal-enhanced fluorescence (MEF) as they contribute towards enhanced fluorescence

signal intensity lowering the detection limit of bioassays. These particles are also known to be great acceptors in FRET, and they even promote the efficacy of the assays as FRET pair enhancers. A recent work published by Kato et al. demonstrated a one-pot method for stable coating of silver nanoparticles with a thiolated polymer to form polymeric shells that behaved as an excellent quencher. Great potential for increase in fluorescent plasmonics was observed along with efficient masking of fluorescence quenching with polymer-coated silver nanoparticles [20].

**Carbon nanotubes (CNTs),** which have a unique arrangement of sp2 hybridized carbon atoms that form a π-conjugated network, have been explored in depth in developing fluorescent biosensing assays. The ability of CNTs to quench the fluorescence of organic dyes or quantum dots in the NIR region is combined with photoluminescence properties through dynamic energy transfer [21]. Chen et al., reported on the development of an acetylcholine-based, cost-effective, and sensitive electrochemical sensor to detect pesticides in food samples. The assay used MWCNTs that increased surface area for effective electrochemical polymerization, yet maintained the enzymatic activity, exhibiting a stable response towards multiple real samples such as carbonated drinks, milk, orange juice, and beer [22].

**Quantum dots (QDs)**, also known as semiconductor crystalline materials, are novel fluorescence materials with quantum confinement effect, good photostability, and effective biocompatibility, and they possess composition-based emission tunability [23]. With large Stokes shift and flexible fluorescence, their applications include biosensing, biomedicine, and optoelectronics [24]. QDs possess superior attributes of broader excitation with narrow emission spectra, longer time of fluorescence, and 100 times higher molar extinction coefficient than conventional organic dyes [25]. All these exceptional properties have led to the development of highly efficient and stable optical biosensing systems enabled via QD-based FRET systems. QDs directly enable the sensing phenomenon by enhancing or quenching via direct adsorption/chelation/interaction of specific conjugated bioreceptors or metal ions [26]. Many studies have reported the applicability of QDs and their conjugated derivatives in developing fluorescence-based platforms for pathogen sensing and food safety [27–29].

**Graphene**-based nanomaterials are graphene sheet, graphene oxide (GO), and a reduced form of graphene-oxide nanosheet (rGO). Graphene and its derivatives possess outstanding ability in quenching fluorescent dyes so they are used as potential energy acceptors in designing fluorescent sensors. They are often combined and conjugated with fluorophores such as QDs and UCNPs in the form of FRET pairs [30]. Various aspects of biomedical applications such as chemi-sensors, electrochemical sensors, and fluorescent biosensors serving either as quenchers or fluorophores have been explored [31]. A study reported a conjugated system of QD–aptamer–GO for detecting β-lactoglobulin in food samples [32]. Other nanomaterials, such as metal organic frameworks, up-conversion nanoparticles, silica nanoparticles, and phosphors, also contribute to the development of point-of-care fluorescent biosensing technologies for food safety. Various food analytes and the detection limit for these analytes are summarized in the Table 1. In conclusion, all these nanomaterials, with their advanced properties have resulted in the development of efficient fluorescent biosensors for food safety. Table 1 provides a comparative analysis of the bioreceptors employed for detection and their LoD. Although major nanomaterials exhibiting fluorescent properties have been discussed in this review, high-end nanohybrids incorporating conjugated nanomaterials, magnetic nanoparticles, and co-embedded manipulations that are easily fabricated have been reported to be upcoming substitutes. Moreover, depending upon the fluorescent phenomenon being used, such as quenching/masking or fluorescence enhancement involved in food sensing, the particular nanomaterial is selected for its respective application providing improved sensitivity compared to traditional dyes.


**Table 1.** List of food-analyte sensing by various types of nanomaterials (2018–2022).

## *3.2. Nucleic-Acid-Based Molecular Markers*

Fluorescent-based molecular markers such as DNA/mRNA covalently conjugated with fluorophore are used for sensing applications as they selectively bind to functional groups of target molecules [61]. Fluorescent DNA/RNA can also be generated by use of 2-aminopurine (for adenine) or isoxanthopterin (for guanine) nucleobase analogs and used as efficient molecular recognition elements (MREs) for developing target-detection systems [62]. Generally, fluorescent nucleic acids are classified based on their structures that begin with detecting SNP based on duplex formation. Another structural analysis of homoadenine and A-cluster systems demonstrated their applicability in three-way-junction (3WJ) probes for targeting miRNA. Moreover, G-quadruplexes with their G-rich sequences form fluorescent probes or detection of targets. Most important is the selectivity and specificity of a new group of fluorescent molecular beacon (MB) systems towards target sequence [63]. MBs are highly specific single-stranded DNA fluorescent probes that are dual modified at one end with fluorophore (F) and at the other end with a quencher (Q), leading to their applicability in detection systems [64]. An MB can acquire an open structural state where the quencher is away from the fluorophore, spatially restoring the fluorescence that generally happens in the presence of target and closed state where the fluorophore and quencher come into close proximity, diminishing the fluorescence. A recent study reported the detection of signature molecules of food-borne pathogens using the FRET mechanism of MBs, QDs, and nanoscale quenchers [65]. Moreover, MB-based multiplex real-time PCR studies have been reported for detection of various food pathogens [66–68]. Evolving from the inherent attribute of nucleic acid to form Watson–Crick duplex structures to detect complementary nucleic acid strands, there have been ground-breaking discoveries of generating affinity nucleic acids possessing specific binding properties [69]. Over the last decade, single-stranded DNA/RNA aptamers as a versatile class of bioreceptors, have been introduced. Their ease of synthesis and excellent biofunctionalization properties enable efficient fluorescent sensing [70,71]. The fluorophore is conjugated with an aptamer as a labeled/non-labeled moiety and target detection is determined by excitation-light interaction with the bioreceptor reflecting fluorescent intensity [72]. A recent study reported a signal-on fluorescent MB-aptamer-based sensor for rapid detection of mercury in food samples [73].

Comprehensively, as compared to the classical conventional bioreceptors for sensing applications, aptamers pave novel avenues for designing fluorescent detection strategies due to their exceptional properties that allow bioconjugation with a large variety of compounds. They offer high sensitivity for detection of target analytes enabling specific biorecognition abilities that promote potential sensing applications.

#### *3.3. Antibodies*

Fluorescent immunoassays generally use antibodies covalently linked with fluorochrome that absorbs light and emits at another wavelength as detection reagents. In point of fact, a few years ago, the novel concept of a fluorescent immunosensor, a Quenchbody, also known as a Q-body, was introduced by Ueda and colleagues. The key aspect of this technology comprises antibody-labeling of the N-terminal region/antigen-binding fragment (Fab) of an antibody with fluorescent dyes, delivering enhanced fluorescence when antibody–antigen interaction occurs [74,75]. Fluorescent dyes such as TAMARA, ATTO520, and rhodamine are conjugated with variable regions of antibodies via flexible linker peptides [76]. These fluorescent-labeled antibodies are utilized in designing lateral-flow test cards for sensing food contaminants. Alongside, smartphone integration with fluorescent detectors have been successfully used as efficient point-of-care systems for sensing pathogenic bacteria in food samples [7]. Huang et al., in 2017, reported a protein-sensing platform employing a combination of graphene-oxide sheets conjugated with antibodies that displayed quantitative quenching of fluorescent signals [77]. Another fluorescenceantibody-labeled sandwich immunoassay was reported using chitosan–cellulose nanocrystal membrane for rapid detection of *Listeria monocytogenes* in food samples [78]. Over the

recent years, the advancements in antibody-based detection techniques have increased due to immunological modifications, resulting in effective food-sensing applications.
