Recent advances in the biomolecules mediated synthesis of nanoclusters for food safety analysis

The development of nanoclusters based on incorporating biomolecules like proteins, lipids, enzymes, DNA, surfactants, and chemical stabilizers creates a stable and high fluorescence bio-sensors promising future due to their high sensitivity, high level of detection and better selectivity. This review addresses a comprehensive and systematic overview of the recent development in synthesizing metal nanocluster by various strategized synthesis techniques. Significantly, the application of nanometal clusters for the detection of various food contaminants such as microorganisms, antibodies, drugs, pesticides, metal contaminants, amino acids, and other food flavors have been discussed briefly concerning the detection techniques, sensitivity, selectivity, and lower limit of detection. The review further gives a brief account on the future prospects in the synthesis of novel metal nanocluster-based biosensors, and their advantages, shortcomings, and potential perspectives toward their application in the field of food safety analysis.


Introduction
In recent years, concerns related to food safety issues are on the raise due to the contamination of food stuffs with microbes, metal ions, pesticides and organic chemicals. The consumption of unsafe food causes 200 different types of diseases that affect infants to elders, from diarrhea to cancers. Almost 1 in 10 people are falling ill after eating the contaminated food each year, resulting in 420 000 deaths with the loss of 33 million people healthy life years in an estimate of at least 600 million population. According to a report, 40% Abbreviations: MNCs, Metal nanoclusters; NPs, Nanoparticles; SPR, Surface Plasmon resonance; NCs, Nanoclusters; AAS, Atomic absorption spectrometry; ICP-MS, Inductively coupled Plasma mass spectrometry; SRXRS, synchroton radiation X-ray spectrometry; EPR, electron paramagnetic resonance; GSH, Glutathione; QYs, Quantum yields; PMAA, Poly methacrylic acid; PEG, Polyethyelene glycol; THPC, Tetrakis-(hydroxymethyl) phosphonium chloride; CDs, Carbon dots; CUNCs, Copper nanoclusters; CEW, Chicken egg white; OVA, Oval albumin; FRET, Fluorescence resonance energy transfer; BSA, Bovine Serum Albumin.
of children under five years of age are suffering from food-borne related disease out of these an estimated 125 000 deaths were reported every year by World Health Organization, 2020. Most of these infections are caused by microbes such as Salmonella, E. coli, Listeria, Vibrio, Viruses, Parasites and chemicals like naturally occurring toxins, persistent organic pollutants and heavy metals [1]. Therefore, it is necessary to find a cost effective and real time sensing strategy to accurately detect trace levels of various toxic chemicals, metal cations, anions, drugs, amino acids and pesticides in various environmental, biological and food samples. These contaminants are easily identified by a variety of detection strategies including GC-MS, HPLC and other biological assays.
Fluorescent metal nanoclusters (MNCs) are emerging fluorophores have attracted immense interest from researchers because of their excellent features such as biocompatibility, photostability, sub-nanometer size, distinctive luminous capabilities and ease of synthesis (Fig. 1). MNCs are the missing link between metal atoms (which have different optical properties) and nanoparticles (NPs) (which have plasmons) and exhibit molecule-like behavior. Metals' electrical and optical characteristics are strongly influenced by their size, especially in the nanoscale range. The conduction band in bulk metal has no energy gap separating it from the valence band. Therefore, electrons do not encounter a barrier and can travel freely. The mean free path of electron determines the scattering of electrons. When the size of the metal NPs is comparable to or smaller than the electron mean free path, the electron's motion is constrained by the size of the NPs, and interaction is expected to occur mainly with the surface, resulting in surface plasmon resonance (SPR) band [2]. In metal NCs, the size of metals is further reduced around 1 nm or less, down to a few atoms, and the continuous band structure is broken into discrete energy levels. Nanoclusters (NCs) are non conductive and plasmonic. Interaction with light still exists, but it occurs through electronic transitions between energy levels, similar to organic dye molecules, resulting in light absorption and emission. As fluorescent probes in fluorescent biosensing and bio-imaging, they have the same optical property as quantum dots and fluorophores. Fluorescent MNCs are crucial for recognition of metal atoms from NPs via optical properties and Plasmon effects [2] (Fig. 2). These optical properties lead to a variety of fluorescent probes that have been used in biological imaging, bio-sensing of metal ions, insecticides, and other applications [3].
Researchers have paid close attention to the unique physical, chemical, electrical, and optical properties of NCs. Nowadays, NCs are synthesized by various methods in different sizes with fluorescence properties that can be tuned from the UV to the near range -IR region. NCs are capped with biomolecules such as proteins, oligonucleotides, enzymes, and peptides because they have high luminescence quantum yields, nontoxic, tunable luminescence features and biocompatible nature. The advantages and the development of biosensing and imaging applications are based on the unique features of optical probes [5,6]. The synthesis of fluorescent NCs and their application for the development of sensor platforms are the subject of extensive research activities.
Food, biomedical, forensic, and environmental materials are contaminated by several chemical and biological agents. Fundamental biology, chemistry, and materials science are required to develop advanced technologies with sensitive, low-cost sensors [ [7,8]]. However, the technology possesses several challenges requiring sophisticated instrumentation, modification of existing techniques, and complex sample handling. Therefore, the detection of toxic chemicals and biomolecules in a simple, label-free and cost-effective manner using quantitative and qualitative methods has become more important. Fluorescent MNCs have gained attention for rapid, selective and sensitive detection of various toxic chemicals, biomolecules and the environmental samples. Many existing fluorescent probes are mainly based on organic fluorophores, quantum dots and fluorescent proteins [8,9]. The various organic fluorophores differ in their chemical structure spectral properties, and susceptibility to photobleaching, which limits their potential applications in the respective field. Quantum dots appear larger in physical, photostable, which may liberate hindrance in time of binding, which may compromise their use for in vivo applications and toxic nature. MNCs appear in ultra-small size, low toxicity, possess good biocompatibility, low cost with excellent photostability properties compared to over other organic dye molecules and semiconductor quantum dots [8,10]. The origin of the fluorescence, stability, binding capacity of the various functional groups, and biocompatibility properties in NCs are related to the ligand or templates. Biomolecules like proteins, thiols, peptides, enzymes, lipids, DNA, Polymers and dendrimers are used as a stabilizer to prepare noble MNCs with several advantages. For example, the emergence of fluorescent nanomaterials, especially fluorescent MNCs, has had a profound influence on optoelectronic devices, bio-marker, and biosensors. In fact, MNCs showed great promise for a number of applications, including electronics, chemistry, biology, pharmaceuticals, and biosensing. MNCs-based biosensors found applications in a variety of domains, including healthcare and food safety, offering quick and low-cost analysis of a variety of target molecules. There are number of review articles on the synthesis, properties and versatile applications of MNCs [11,12].
Various strategies have been used to detect cations and anions at trace levels, including atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), electron paramagnetic resonance (EPR), and synchrotron radiation X-ray spectrometry (SRXRS) [13]. However, these detection methods are high cost, time consuming, need sophisticated instruments, and sample pretreatment of samples. In recent years, in order to overcome this issue, fluorometric detection techniques have been touted as a potential alternative for the detection of various analytes with high specificity, high sensitivity, and reproducibility. Moreover, in fluorometric detection, the monitoring of biological samples in real-time reduces the risk of contamination and time consumption. Further, semiconductor quantum dots and organic dyes are used for sensing of toxic chemicals and bioimaging applications. However, they possess many limitations, such as less photostability and high toxic nature. Compared to quantum dots, MNCs have distinct advantages and are widely used for sensing and bioimaging applications. For instance, luminescence sensors, electrochemical sensors, optical biosensors, electrochemical biosensors, and photoelectrochemical biosensors are examples of MNCs-based sensors. To the best of our knowledge, no systematic study of MNCs-based fluorescence sensing applications for food quality analysis has been published yet. Although several sensing methodologies utilizing MNCs have been developed, only a few studies have focused on the detection of hazardous compounds and physiologically essential molecules for food safety applications. This review article provides an overview of recent advances in the synthesis and photophysical properties of MNCs and their application in food safety analysis.

Synthesis of metal nanoclusters
The structure, size, and surface properties of NCs depend upon the concentration of templates or ligands, reducing agents, metal ions and temperature. Proteins, peptides, polymers, and chemicals act as capping agents to produce highly stable fluorescent nanocluster. The origin of the fluorescence, the sensitivity of NCs with different spectral regions depends on the stabilizer. NCs are mainly synthesized based upon photoreduction, chemical reduction and templates (Fig. 3). The Ag + , Au 3+ and Cu 2+ metal ions are reduced in the presence of the stabilizers there by resulting in the formation of fluorescent NCs. These processes proved that proteins itself acted as effective reducing and capping agents and did not require any additional reducing agents. Another method for creating NCs is to employ small molecules such as thiol compounds in an alkaline solution, as well as template-free techniques and the electrochemical methods for etching bigger NPS (2-4 nm). The chemical reduction method was the most commonly used technique to obtain MNCs. For instance, AuNCs are generally synthesized by reducing Au 3+ with capping and reducing agents [12]. Thiol compounds are used as a capping agent to minimize the complicated bonding character of Au-S through Au atoms/ions. Reducing agents are typically used to synthesize NCs in the presence of thiol compounds such as tetrakis-(hydroxymethyl), phosphonium chloride (THPC), and sodium borohydride, (NaBH 4 ). It was previously reported that when NaBH 4 is utilized as a reducing agent in the presence of glutathione (GSH), glutathione-stabilized AuNCs (GSH-AuNCs) are formed from Au 3+ [14]. Different sizes of GSH− AuNCs emit at the emission wavelengths after separation and purification with 0.1 percentages of quantum yields (QYs) (Fig. 4). Stabilized AuNCs are made by utilizing  different thiols such as polyethylene glycol attached lipoic acid, phenylethyl thiolate, tiopronin, and thiolate cyclodextrin by altering an identical technique. When the molar ratio of thiol to Au ions is increased, the size and QY of thiol-stabilized AuNCs usually decrease. However, the majority of the thiol-stabilized AuNCs produced had low QYs. On the other hand, GSH is used as a reducing agent in the production of NCs. GSH acted as a potent capping agent and a weak reducing agent which leads to inadequate reduction of Au 3+ to Au 2+ to produce the Au NC core, which was stabilized with thiolate-Au + complexes. Fluorescent Au @ Au + -thiolate core-shell NCs were synthesized with low thiol Au (1.5:1) by the thiolate-based complex's generated Au cores at controlled aggregation with the QY of 15% [15]. In other hand, synthesized CuNCs from CuCl 2 by a simple chemical reduction method using N 2 H 4 H 2 O as a reducing agent and BSA as a stabilizer. The light-yellow color indicated the formation of CuNCs.

Template-based synthesis
Template-based synthesis is an effective way to synthesis MNCs. Many templates are used to synthesize MNCs such as polymer, thiols, molecular sieves, peptides and proteins, DNA oligonucleotides, dendrimers, polyelectrolytes etc. The core size and size distribution of the MNCs can be controlled by the template's method [17].

Polymers
Many MNCs are synthesized by the polymer-based template method. For instances, polymer with carboxylic acid and alcoholic groups is used as a stabilizing agent. By varying the polymer to metal ratio, MNCs shape, size and fluorescent properties are tuned easily. Researchers reported the preparation of Ag NCs by utilizing Poly methacrylic acid (PMAA) as a template by reducing the AgNO 3 precursors under ultraviolet irradiation. It has been reported that when 60 mg/mL AgNO 3 and 10 mg/mL PMAA solution were exposed to UVA for 60 min, the solution turned pink color which indicates the formation of AgNCs [18].
Photoreduction methods are used in the preparation of MNCs by preventing hazardous inorganic reducing agents [17]. The MNCs size and QYs are mainly dependent on the nature of polymers used in the synthesis and can be varied using the molar ratio of polymer and metal ions. PVP was used as a template for the preparation of CuNCs by a facile chemical method 0.2 g PVP, 300 μL L-ascorbic acid, 100 μL of CuCl 2, and 10 mL of double distilled water added into a 100 mL of Carousel 6 Plus solution which was then kept under 90 • C for 21 h to the yield PVP capped CuNCs [19]. [20] Aparna et al., synthesized CuNCs using polyethyleneimine (PEI) as a capping agent or stabilizing agent. CuSO 4 and ascorbic acid were mixed with 0.094 g mL − 1 PEI solution to form a colloidal suspension. This reaction mixture was exposed to microwave irradiation for 20s to obtain CuNCs-PEI [21]. Hu et al., used poly (sodium-p-styrene sulfonate) (PSS) as a template for silver nanocluster synthesis. In a typical synthesis they used 17 mg of AgNO 3 , 14.9 mg of D-penicillamine and 50 mL of double distilled water mixed with 150 mg of PSS, and the complexes were exposed to UV-lamp of 300W for 25 min to obtain the PSS-DPA-AgNCs (D-Penicillamine stabilized argentum nanoclusters) ( Table 3) [22]. Lewis et al., used 5 mL of polyethylene glycol (PEG) with 50 mL of 1 mM HAuCl 3 and 4 mL of trisodium citrate into the solution. A color change from black to red indicated the formation of AuNCs-PEG.

Thiols
The thiols-based template contains a small stabilizer molecule which provides a strong interaction for synthesizing MNCs. Various thiols have been used to synthesize MNCs like glutathione, 3-mercaptopropionic acid, thiolate-cyclodextrin and tiopronin [35]. X. Liu and coworkers prepared CuNCs by using 2-mercapto-5-n-polypyrimidine (MPP) as a protecting thiol group and using NaBH 4 as a reducing agent. The chemical reduction method is the most commonly used technique to obtain MNCs. For example, the most typical process for the production of AuNCs is the reduction of Au + from Au 3+ to obtain Au 0 . Thiol groups, as capping agents, are known to bind strongly to gold surfaces due to sulphur bonding. To prepare NCs, thiol reducing agents such sodium borohydride (NaBH 4 ) and tetrakis-(hydroxymethyl) phosphonium chloride (THPC) are commonly utilized. It has been reported in the presence of reducing agent NaBH 4; glutathione (GSH) stabilized AuNCs (GSH− AuNCs) have been prepared from Au 3+ ions. GSH− AuNCs with <0.1% of quantum yields (QYs) emit at emission wavelength obtained in various sizes after the purification and separation process. AuNCs are stabilized using many other thiols such as phenylethyl thiolate; polyethylene glycol appended lipoic acid, thiolate cyclodextrin, and tiopronin following the same methods of preparation. The concentration of molar ratio of thiol increases with Au ions, resulting in smaller size of Au ions with lower QY. The prepared thiol stabilized AuNCs have consistently low QYs. GSH, which works as a capping and reducing agent, is commonly used for fluorescent NCs. AuNC core was produced under neutral conditions by the semi-reduction of Au 3+ , which was stabilized by monolayer thiolate-Au + complexes. GSH works as a weak reducing agent with a QY of 15% at fluorescent Au@Au +thiolate core-shell NCs, which are synthesized by controlled aggregation of thiolate and Au + complex, at lower molar concentrations of thiol-Au (1.5:1). Glutathione was used to prepare CuNCs by the following method: 50 mM of CuCl 2 was mixed with 0.21 mM of glutathione under the stirring condition to form copper thiolate complexes. Then the colloidal suspension was centrifuged and dialyzed to obtain CuNCs with a quantum yield of 8.6% [36]. [37] Gayen et al., used 10 mM of HAuCl 4 and 0.11 M of MPA (Mercaptopropionic acid) and heated at 50 • C for 2 min. Then the solution is illuminated under a UV trans illuminator at 305 nm; on excitation, a bright luminescence was observed. MPA-AuNCs were synthesized using MPA and NaOH [38]. [39] Nath et al., synthesized AuNCs using 2-mercapto-4-methyl-5-thiazoleacetic acid (MMT). In a typical synthesis, 2.5 mg of MMT, 0.1 M HAuCl 4 , 10 mL of citrate and 1.0 mL of NaOH were mixed at room temperature and stirred for 8 h to obtain MMT-AuNCs.

Dendrimers
Dendrimers are used to prepare small MNCs. A low yield of MNCs was obtained from this method [40]. Zheng et al. prepared stabilized AuNCs by dendrimers with a high yield of 42%. By varying the concentration of (poly (amidoamine) dendrimers) PAMAM/Au, produced AuNCs with ranging emission colors from UV to near-infrared region.

Peptides and proteins
Biological macromolecules like peptides and proteins were also used as a template for the synthesis of MNCs. The macromolecules provided high binding sites to reduce metal precursors, which offered small MNCs. Several proteins such as human serum protein, insulin, lysozymes proteins, Ovalbumin etc., were used as templates. Some enzymes were capable of catalytic activity towards the production of MNCs [41]. Bhamore et al., synthesized AuNCs using amylase solution. They used 17 mg of HAuCl 4 , 0.25 g of amylase and 2 mL of NaOH to obtain amylase capped-AuNCs. CuNCs are prepared using bovine serum albumin (BSA) through a simple one-pot method [42]. CuSO 4 (20 mM) was added to BSA solution (5 mL @ 15 mg/mL) with the addition of NaOH, the pH was maintained at 12 and the reaction mixture was incubated at 55 • C for 8 h to obtain fluorescent CuNCs. CuNCs from CuCl 2 were synthesized by a simple chemical reduction method using hydrazine as a reducing agent and BSA as a stabilizer agent. The light-yellow color indicated the formation of CuNCs -carbon dots/CuNCs nanohybrid (CDs/CuNCs). CuNCs were made using a simple chemical reduction process with hydrazine as a reducing agent and BSA as a stabilizer agent, whereas carbon dots were made using a hydrothermal method [15]. Kalaiyarasan et al., first used chicken egg white (CEW) as a template for the preparation of AuNCs with the size of 2 nm and also generated a red fluorescence at 720 nm with excitation at 535 nm [43]. In a similar way Guo et al. [44], used CEW for the synthesis of AuNCs. Similarly  [48]. To this, 0.5 mL of NaOH was added and incubated at 37 • C for 10 h to obtain AuNCs-BSA. Serum protein BSA was used to prepare AuNCs by a facile chemical reduction method, 12.5 mL of HAuCl 4 , 1.25 mL of NaOH, 12.5 BSA, and 1.25 mL of MPA were mixed and incubated at 4 • C for 1hr to get BSA/MPA-AuNCs [49]. To make AuNCs, Cheng and his colleagues used soybean protein: 5 mL HAuCl4 and 5 mL soybean protein were combined with 1 mL NaOH which resulted in light yellow to pale brown colour indicated the creation of SP-AuNCs [50].

Chemical etching
Chemical etching offers a straight forward approach to prepare NCs from metallic NPs. For example, the ligand-induced etching method produces excess ligands used to prepare AuNCs from the Au NPs with 2-4 nm core size. THPC acts as a capping and reducing agent in preparing AuNCs using thiol ligands such as 11-mercaptoundecanoic acid (11-MUA) as an etching agent under alkaline conditions. 11-MUA causes a significant etching ability on the surface of Au atoms. At a high pH (>12.0), it creates strong coordination to form stable 11-MUA-Au complexes on the Au surface in each core of the shell, resulting in fluorescent capped AuNCs with a QY of 3.1% [52]. Different thiol compounds can be used to alter the size and optical characteristics of AuNCs. Alkane thiols act as ligands in the production of alkane thiol-bound AuNCs with varied chain lengths and emission wavelengths ranging from 501 to 613 nm QYs ranging from 0.0062 to 3.1%. The emission properties of 11-MUAAuNCs are tuned from 524 to 456 nm by changing the molar ratio of Ag + /Au3 + from 0 to 1.6. The variable fluorescent wavelength, long lifespan (>200 ns), and considerable Stokes shift are all features of these fluorescent AuNCs. (>100 nm) [52]. Sun et al., used 3:1 HNO 3 : HCl, 1% trisodium citrate and 0.075% NaBH 4 for the chemical etching of HAuCl 4 solution to obtain AuNCs of size 13 nm [53]. Mecker et al., developed AuNCs with sodium citrate and HAuCl 4 under reflux for 15 min, the golden color solution turns into red wine color indicating the formation of AuNCs. Blue emission is seen in the prepared AuNCs, with QYs of 3.8%, 14.3%, and 20.1%, respectively [54].

Template-free method
The template-free methods were a simple, facile and effortless method for the production of MNCs. There is no extra substance, minimal post processing using the strong ionic or acid-alkali condition to obtained pure MNCs in this method [55][56][57].

Inverse micelle synthesis method
The inverse micelle synthesis technique was a liquid phase synthesis method. A droplet-like inverse micelle was used for this synthesis. It is an inexpensive, facile and simple technique. In this process, the cluster size was controlled by regulating the concentration of metal salt precursors and surfactant concentration [58][59][60].

Electrochemical synthesis method
The electrochemical synthesis method was a promising route for the preparation of MNCs. Here, the shape and size can be altered using the electrochemical parameters. In electrochemical synthesis anode acts as the metal source. By anodic dissolution or reduction at the cathodic surface, the MNCs were formed and balanced using surfactants in the electrolyte solution [35]. under electrochemical synthesis using Cu anode, CuNCs are made using Cu as an anode, Pt as a cathode and 0.1 M of tetrabutyl ammonium nitrate as an electrolyte solution and Ag/AgCl as a reference electrode.

Photo reduction
To eradicate the hazardous inorganic reducing materials like NaBH 4 , trisodium citrate etc. photoreduction methods have been utilized for the formation of MNCs. For example, MNCs with fluorescent characteristics, are produced by photoreduction of tridentate thioether terminated polymers such as poly (n-butyl methacrylate), poly (tetra-butyl methacrylate), and poly (methyl methacrylate). The size and yield of MNCs are determined by the nature of polymers, which can be changed by adjusting the molar concentration of polymer to metal ions [61,62].

Microbial detection
Food and drinking water were contaminated by microorganisms like viruses, bacteria and protozoa. The microbes causing food spoilage were initially screened by biosensing with good selectivity and high sensitivity. Several MNCs -based sensors have been developed for the detection of pathogens [24]. Liu et al., detected Listeria monocytogenes by monitoring the visible color change of Ag NCs from blue to red in the concentration range of 10-10 6 CFU. mL − 1 . The sensing assay exhibited a detection limit as low as 10 CFU mL − 1 . In other hand immune invertase-NCs (INCs) detected E.coli O157:H7 at concentrations ranging from 10 2 to 10 7 CFU mL − 1 , with the lowest detection at 79 CFU/mL, according to Huang et al. [63] (Fig. 5) (Table 3) [28]. Zhang et al., used DNA/Ag NCs to detect Ochratoxin's (Ap1) and Aflatoxin (Ap2) binding with mycotoxins. Since Ochratoxin (OTA) and Aflatoxin B 1 (AFB 1 ), the DNA/Ag NCs have set detection limits of 0.2 pg mL − 1 and 0.3 pg mL − 1 , respectively [33]. Zhang et al., made a biosensor to detect Salmonella species using Fe-NCs with a lower detection limit of 14 CFU/mL and recovery as ~105.0% in spiked chicken samples [32]. Subramaniyan et al., used phytoprotien functionalized Pt-NCs with size ~ 5 nm in a spherical shape. A minimum inhibitory concentration of 12.5 μM of salmonella typhi was found using Pt-NCs, and the hemolytic test showed low cytotoxicity at 100 μM [27]. Zhang et al., produced a biosensor to detect staphylococcal enterotoxin A (SEA) using DNA/Ag NCs-PPy. The SEA concentration was in a range of 0.5-1000 ng mL − 1 with the detection limit of 0.3393 ng mL − 1 . This sensor was used in milk samples with the highest recovery efficiency of 94.56% [29]. Yao et al., synthesized silver NCs and detected a food-borne pathogen, i.e., Staphylococcus aureus, using Ag NCs. The S. aureus ranges from 10 to 10 6 cfu mL − 1 , and the limit of detection of 4.9 CFU mL − 1 with the recovery of 85.6%-103.7% [64]. Chen and his coworkers made a dual recognition with aptamer and antibiotic to detect Staphylococcus aureus (SA) using Vancomycin-Au-NCs. The sensitivity of Vancomycin-AuNCs detection assay towards SA is in the range of 3x10 8 cfu. mL − 1 with the detection limit of 16 CFU mL − 1 . Tan  samples. The same group synthesized the chitosan-AuNCs to detect three different kinds of bacteria, such as S. aureus, E. coli and B. subtilis. This biosensor had more selectivity towards the S.aureus than the E.coli and B. subtilis with the detection of S.aureus as 4 × 10 2 CFU/mL. Khan et al. [26], detected the T-2 (trichothecenes mycotoxin) using aptamer functionalized Ag NCs (apt-Ag NCs) by fluorescent resonance energy transfer detection method. The T-2 range was found 0.005-500 ng mL − 1 and 0.93 pg mL − 1 in maize and wheat samples and LOD with improved recovery efficiency ranged from 89.46% to 102.08% and 90.41% to 107.75% [38]. Ariani and his coworkers detected Listeria monocytogenes by MPA-AuNCs with fluorescent technique. In a 10-μL sample, the detection of bacteria was sensed quickly with LOD of 2000 CFU (Table 1).

Pesticides
Pesticides are the chemical substances that protect agricultural products from insects, pests or weeds. These chemical substances when accumulated in water, soil, air and food, leads to more severe health issues in animals and humans. Hence, adequate measures must be taken to control sense and detect the pesticides using MNCs [21]. Hu    using amylase-AuNCs by "turn off" fluorescence quenching mechanism. Detection of deltamethrin and GSH were done under fluorescent probe in the range of (0.01-5 μM) and (0.05-5 μM) with good linearity and LOD of 6 and 10 nm [83]. Sun et al., developed an electrochemical immunosensor to detect carbofuran pesticide using Au NPs and Prussian blue-multiwalled carbon nanotubes-chitosan (PB-MWCNTs-CTS) nanocomposite film. The system provided a wide linear range between 0.1 and 1 μg/mL with a low detection limit of 0.021 ng mL − 1 [45]. Yan and his colleagues developed the assays of organophosphorus pesticides (OPs) using tyrosinase-gold NCs (TYR-AuNCs) by fluorometric technique. Rapid detection of OPs by fluorescence (paraoxon as a model) with a LOD of 0.1 ng mL − 1 also provides excellent sensitivity [67]. Yan and his coworkers used AuNCs-MnO 2 for the detection of carbamate pesticide by Fluorescence resonance energy transfer (FRET) effect. A dual-output assay, via color and fluorescence, was used to detect carbaryl with LOD of 0.125 μg L − 1 [84]. The BSA-CuNCs used for the detection of paraoxon organophosphates by enzyme-free electrochemical biosensor technique. BSA-CuNCs were used as redox-active on the surface electrode with SWCNT and glassy carbon electrodes. The reduction peak current vs. paraoxon concentration was linear over the range from 50 nM to 0.5 μM and from 0.5 to 35 μM respectively, with a LOD of 12.8 nM [78]. Zhao et al., used GSH-AuNCs to sense trace amounts of thiram residues in agriculture and food samples via an aggregation-induced emission enhancement (AIEE) mechanism. The detection assay showed an LOD of 0.05 μg mL − 1 (Table 1).

Metal ions
Lin et al., prepared bi-ligand CuNCs to detect hexavalent chromium in water [86]. A wide linear range of 0.1-1000 mM with a lower LOD of 0.03 mM was obtained in chromium assay. The recoveries of samples were between 98.3 and 105.0% indicating a better repeatability. Huang et al., used CuNCs for the detection of Fe 3+ ion in water [36]. In real water sample assay, the fluorescence of CuNCs was linearly quenched upon the increasing Fe 3+ concentrations in the range of 1-100 μM, with the LOD of 0.3 μM (Fig. 6) ( Table 2). A biological sensor was developed based on AuNCs-GO (gold NCs/graphene oxide) and investigated to detect Hg 2+ in the water sample. In an actual water sample, the fluorescence peak intensity linearly decreases with increasing Hg 2+ concentration in the range of 1.0 × 10 − 5 to 5.0 × 10 − 13 M with a detection limit of 1.8 × 10 − 13 M [79].

Antibiotic drugs
The substance or compounds that could kill/inhibit the growth of microorganisms were classified into drugs and antibiotics (Fig. 7). These compounds had been widely used in animal husbandry or agriculture as a growth promoter or inhibitor for the microorganisms. However, the usages of these compounds are frequently increased; there should be a proper control towards the usage and detection of these materials towards the environmental benefit [77]. The AuNCs-immunoassay biosensor has been reported for the sensing of clenbuterol and ractopamine. The sensing assay exhibited good linear relationship over the concentrations range of clenbuterol and ractopamine were 0.06 and 0.32 μg L − 1 , respectively, with LOD of 0.003 and 0.023 μg L − 1 [87]. A CuNCs-BSA was used to identify the kojic acid by fluorescence technique. This biosensor exhibited an excellent detection of the kojic acid in the range of 0.2 μM-50 μM, with LOD of 0.07 μM [50]. Cheng and his colleagues successfully detected bismerthiazol by using soybean protein-capped AuNCs. High selectivity and sensitivity in the concentration range of 5-100 μg mL − 1 with as low as 5 μg mL − 1 of bismerthiazol [30].
Hosseini et al., prepared a biosensor using DNA aptamer-Ag NCs to detect oxytetracycline (OTC) by fluorescence technique. The DNA-AgNCs were quenched linearly in the range of 0.5 nM-100 nM with a LOD of 0.1 nM [46]. detected a series of antioxidant compounds using CEW-AuNCs by measuring the Cu (II)-induced prooxidant activity (Table 1). Borse et al. [31], used CEW-Pt NCs for the detection of carbidopa assay. The carbidopa acts as a quencher, resulting in the range of 5.0-35 μM and exhibited a linear response with the LOD of 1.71 μM.

Advantages and challenges associated with MNCs -based fluorescence sensors in food analysis
Many fluorescent nanomaterials such as the semiconducting QDs and MNCs have considerably less solubility and stability in complex food matrices and biological mediums. Researchers have used several surface modification strategies to afford biocompatibility and stability to address this shortfall. In contrast, MNCs are highly photostable and less toxic. It has been found that MNCs showed high stability and sensitivity toward target analytes in complex food matrices such as milk, fruits and meat samples. As discussed in the previous section, surface passivation of MNCs with suitable capping agents affords unique fluorescence properties. When ligand-capped MNCs are used to detect various analytes, their fluorescence behavior shows an excellent response. The sensing effectiveness of fluorescent nanomaterials is influenced by surface functionalization features such as charge and hydrophobicity, in addition to their inherent photophysical capabilities. Moreover, the interfacial interactions of nanomaterials against target analytes are predominantly controlled by surface functionalization. For example, small organic molecules with aromatic structures undergo strong interaction with the MNCs through electrostatic interaction, π-π stacking, or hydrogen bonding, thereby resulting in FRET or fluorescence quenching. Inspite of possessing significant advantages, MNCs -based fluorescence assays have some disadvantages as well.
For instance, factors such as low quantum yield and the unclear relationship between the surface chemistry and physicochemical properties have restricted the performance of MNCs fluorescence sensing assays. The up-conversion and two-photon fluorescence properties of MNCs remain unexplored till now, and it must be utilized for food-based assays. It can be concluded that there is still plenty of space available for future researches in the development of novel MNCs -based fluorescence sensing strategies toward food analysis.
The functionality as well as the accuracy of the results are both key factors to consider while preparing food samples. The pretreatments are carried out based on the food matrix and sensor properties. Liquid foods (drinking water or beverages) are generally easier to pre-treat than solid foods, because of the complexity of food substrate (meat or cereal). On the other hand, plant-based foods (fruits and vegetables) are, easier to prepare than animal-based foods (fish or chicken) [88,89]. According to these research, liquid foods can be measured directly without any treatment [33]. On the other hand, Solid meals require multiple procedures to extract analytes, including crushing, combining with solvents, and centrifugation, before the built-in sensors can be tested. For 220 unique foods, numerous innovative preparation methods have recently been investigated. For example, pre-degassing, was utilized in the preparation of beer [90]. To prevent interfering with test results, the newer procedure includes extracting the target with an organic solvent and then evaporating the solvent [91]. These pretreatment techniques are gradually improving, resulting in more efficient operations and processes. Most of the reported MNCs -based fluorescence real sample analysis follows a simple pretreatment process such as drying, grinding, centrifugation, and filtration. In recent years, various extraction techniques such as solid-phase microextraction, dispersive micro-solid phase extraction, stir bar solid-phase extraction, dispersive solid-phase extraction, magnetic solid-phase extraction etc., have been developed for effective extraction. It is to be noted that each extraction technique is unique in its functioning and extraction ability with inherent advantages and limitations. As a result, the choice of the extraction method has to be decided according to the properties of the target analytes and sample matrices.

Future and outlooks
The MNCs (M: Au, Ag, Cu etc.,) based biosensors have been reviewed to detect various elements such as microorganisms, pesticides, drugs-antibiotics, metal ions, amino acids and food additives. Many detection techniques have been discussed, such as fluorescent, colorimetric, SERS detection and some newer techniques. In most of the research, the MNCs-based biosensor shows better sensitivity, reliability, specificity and reproducibility. However, many researches still need to be done to solve issues that are crucial for MNCsbased biosensor, such as single step, simple method, minimal washing, fast results, low cost, and homogeneous assay. In MNCs based biosensor with the target analysis, the quality should be maintained qualitatively and quantitatively with binding activity. The MNCsbased biosensor cannot maintain consistent binding activity. Therefore, the standardizing detection techniques will be complex. The cross-linking principle in MNCs will help introduce NCs ' novel properties to improve and promote food safety.
In general, a simple, fast detecting, low cost-based MNCs biosensor needs to be commercialized. The complex synthesis technique and expensive reagents used for the synthesis of MNCs, need to be replaced by a simple and rapid synthesis. Still, many biomolecules in the food contaminants cannot be detected and several steps are required to detect the contaminants. Therefore, research in the field of biosensors needs to be further advanced. The MNCs based biosensor has a certain detection limit, binding activity and it can't be compromised by storage and cost in real applications. Furthermore, research on MNCs-based biosensors is needed to develop more commercial and viable products for the detection of microorganisms to protect food and the environment.

Author contribution statement
All authors listed have significantly contributed to the development and the writing of this article.

Data availability statement
Data included in article/supp. material/referenced in article.