**1. Introduction**

Food safety is an assurance for access to healthy and safe food for sustaining life and good health. To ensure food safety, food hygiene must be undertaken in order to preserve the nutritional value of food and protect it from microbial attack from production to consumption. This food safety must ensure the nutritional requirement of the public and, at the same time, it must not expose them to any foodborne illness. Currently, malnutrition and foodborne disease are the major food-related concerns in global population. To avoid foodborne disease, timely detection of pathogens in the food responsible for toxin production and disease is necessary. Food may contain microbes in the form of bacteria, fungus, protozoa, or viruses that are responsible for causing hundreds of diseases from mild through to severe. The United States reported an outbreak of foodborne infections, particularly bacterial infections associated with fresh farm produce in multiple states from 2010 to 2017 [1]. Likewise, a retrospective study was performed to mark the status of foodborne diseases (involving enteric bacteria) in South Africa, from 2013 to 2017 where the presence of *Salmonella* species, *Escherichia coli*, *Bacillus cereus, Listeria monocytogenes*, and *Clostridium perfringens* were reported in food samples [2].

Foodborne diseases are consequences of harmful toxins or other chemical substances produced by naturally occurring microbes in the food material which, upon entering the host body, lead to digestive-system dysfunction [3]. The escalation in foodborne diseases and associated mortality is a result of the prevalence of harmful pathogens in food due to the evolution in agricultural practices, food production and storage methods, under-cooked animal products, ready-to-eat mixes, and, most importantly, globalization of the food

**Citation:** Kakkar, S.; Gupta, P.; Kumar, N.; Kant, K. Progress in Fluorescence Biosensing and Food Safety towards Point-of-Detection (PoD) System. *Biosensors* **2023**, *13*, 249. https://doi.org/10.3390/ bios13020249

Received: 20 December 2022 Revised: 26 January 2023 Accepted: 6 February 2023 Published: 9 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

trade [4]. In pursuance of safe food supply and reduced incidence of foodborne diseases, an early, quick, and accurate detection of pathogens in food items is required [5]. A number of conventional methods for detection of foodborne pathogens are available. These are based on culturing microbes on differential media, biochemical characterization, sequencing, and characterization through HPLC, MS, PCR, etc. [6]. However, these methods are expensive, time-consuming, and unwieldy, thus restricting their use in point-of-care (PoC) applications [7]. In the food industry, rapid detection of microbes, even at very low numbers in food samples (both raw and processed), is of utmost importance in order to ensure the food quality and safety [8,9]. With the advancement in point-of-care detection methods, researchers have been now able to offer ASSURED (affordable, sensitive, specific, userfriendly, rapid and robust, equipment-free, and deliverable) technologies to the users [10]. The signals in PoC applications are usually fluorescence-, colorimetric-, or electrochemicalbased and are simple and easy to interpret/read [11]. Nevertheless, PoC applications have evolved greatly, with advancements still continuing to address challenges in translation of methods from laboratory- into industrial-application detection systems. Some of the key challenges which need attention are sensitivity, multiplexing, quantification, and multi-functionality.

Indeed, rapid and sensitive detection methods have evolved greatly, and are still evolving, making them highly sensitive, compact, and reusable with almost no detection time. In the present review, we have summarized the fluorescence biosensing basics of a variety of fluorescence-sensing methods. The review describes fluorescence biosensing materials stretching from nano to molecular to protein-based biomolecules. Further, the different materials used for integrating fluorescence-biosensing and fabrication-detection systems are also been described. The various sequential aspects and approaches of fluorescent-based biosensors are summarized under the schematic presentation in Figure 1. The figure is a detailed flow-chart for detection of food microbes/toxins/ions with the help of bioreceptors such as DNA/proteins generated against these food analytes conjugated with fluorescent active bioprobes viz. nanoparticles/graphene/quantum dots, etc. The fluorescent signal output thus generated can be in the form of FCS, FRET, or FILM; each of these components is discussed in the following sections of the review.

**Figure 1.** The schematic diagram presents the fundamental components for designing a fluorescence biosensing platform for food sensing. The food analytes (microbes, pesticides, adulterants, pollutants) are detected by using the specific bioreceptors (proteins, enzymes, cells, DNA) generated against various toxins/pesticides/adulterants, etc. These bioreceptors are coupled with bioprobes (nanoparticles, CNT, graphenes, quantum dots, etc.) that are fluorescently active to generate a fluorescent signal (MEF, FERT) response.

#### **2. Fundamental Aspects of Fluorescence Biosensing**

Among the variety of available sensing options, fluorescence biosensors are the most promising due to their high sensitivity and selectivity which extends their usefulness in biosensors for clinical and environmental monitoring. When a substance absorbs light of higher energy/shorter wavelength and emits low-wavelength light which is a very-short lived (10−<sup>9</sup> to 10−<sup>8</sup> s), this light is called fluorescence [12]. Fluorescence-based detection in biosensors is beneficial for aspects such as sensitivity, signal detection limits, and accuracy.

Developments in nanotechnology have also revolutionized the field of fluorescence biosensing and improved the specificity and sensitivity of the analyte to nano-levels. An example of this is fluorescence-based detection using a cleavable hairpin beacon coupled with LAMP (loop-mediated isothermal amplification) to probe the presence of the *Borrelia burgdorferi* recA gene where the system showed a sensitivity of detecting nearly 100 copies of the gene in 25 min [13]. This sensitivity is many folds higher than that of traditional organic dyes and other fluorescent probes. Among the variety of nanomaterials, quantum dots and carbon nanotubes/carbon dots have gained special attention due to better compatibility, higher surface-to-volume ratio, better chemical and thermal stability, and faster detection. The carbon-nanoparticle-based fluorescence detection of ferrocyanide ion in food samples such as salted foods (radish, cucumber, cabbage) was achieved with a detection limit of as low as 3 ng/mL [14]. Another efficient and sensitive quantum dot (QD)-based fluorescent system to probe the presence of thiram in food samples was reported. The QD consisted of mesoporous silica loaded with a gold nanocluster with the LoD of 0.19 ng/mL [15]. All these features favor its application in point-of-detection (PoD) devices which have maximum demand in diagnostics where sensitivity, specificity, and user-friendly quick response are needed for analyte detection. To utilize these fluorescent labels in biosensing applications, the fluorescence measuring/sensing/estimating phenomenon also need to be understood, and this is elaborated in the following section.

#### **3. Fluorescence Biosensing Materials**

With the advancements in the field of nanobiotechnology, fluorescence-based detection methodologies have replaced conventional organic dyes with nanomaterials as detection labels due to their superior optical properties viz. a wide range of excitation and emission wavelengths and brighter fluorescence with better photostability [16]. Figure 2 summarizes a wide range of nanomaterials that are used for fluorescent based point-of-care biosensing of food analytes such as varied nanoparticles, graphene derivatives, metal organic frameworks, carbon-based nanomaterials, etc. Moreover, traditional fluorescence biosensors employing organic dyes do not offer low detection limits, hence compromising the sensitivity of the assay due to limited quantum yields and low receptor binding ratio of dyes. The potential biocompatibility of fluorescent nanomaterials owing to their physico-chemical properties enhances the performance of biosensors, delivering low-cost and portable point-of-care fluorescence sensing of food contamination. Additionally, these fluorescent nanomaterials will impart a solid support system for biosensing conjugated with multiple probes with high labeling ratio yielding high sensitivity [17]. Nanomaterials as fluorescent packets are advantageous in having tunable optical properties with greater quantum yield. Hence, considering the applications of fluorescent nanomaterials in food sensing, we will discuss the major advances and improvements of various nanomaterials that are currently being used for designing fluorescence biosensors. The applications of different nanomaterials and the enhancement of their limits of detection in the system are summarized in Table 1. A list of recent studies of metal nanomaterials and carbonbased nanomaterials along with some other nanomaterials is featured in the table with comparison between their limits of detection for analyzing a wide range of food analytes.

**Figure 2.** Variety of nanomaterials and their surface modifications used in biosensing of food toxins/pathogens like metallic nanoparticles such as AgNPs (Silver nanoparticles), AuNPs (Gold nanoparticles), carbon nanomaterials viz. QDs (Quantum Dots), GO/rGO (Graphene oxide), carbon nanotubes and other MoFs (Metal organic frameworks), Silica nanoparticles, Microspheres, Phosphors, etc. materials.
