Nanozymes in Pesticide Residues and Veterinary Drugs: Comparison
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Food safety issues caused by foodborne pathogens, chemical pollutants, and heavy metals have aroused widespread concern because they are closely related to human health. Nanozyme-based biosensors have excellent characteristics such as high sensitivity, selectivity, and cost-effectiveness and have been used to detect the risk factors in foods. 

  • nanozyme
  • pesticide residues
  • veterinary drugs

1. Introduction

Agriculture and food production are directly related to the survival and development of mankind. Food safety remains one of the most crucial issues globally because food could be contaminated at all stages of production, packaging, storage, transportation, and value-added processing, giving rise to outbreaks of foodborne diseases [1]. The World Health Organization (WHO) pointed out that an estimated 600 million—almost 1 in 10 people in the world—fall ill after eating contaminated food and 420,000 die every year, resulting in the loss of 33 million healthy life years (DALYs). Food hazards have a variety of factors, such as plant, animal, and microbial metabolites, soil and water pollution hazards from the environment, and purposefully added illegal additives, generated during food packing and processing [2][3]. The revisewarch was organized by the category of food risk factors, including pathogenic microorganisms, toxins, heavy metals, pesticide residues, veterinary drugs, and others [4]. At present, the frequently used detection immunoassays, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), gas chromatography/mass spectrometry (GC-MS), liquid chromatography/mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA), have the advantages of outstanding specificity and accuracy [5]. However, they require tedious pretreatment steps, expensive instruments, specialized technical personnel, and a long testing cycle, which are inappropriate for point-of-care testing (POCT) [6]. In such contexts, with the rapid development and deepening understanding of nanotechnology, nanomaterial-based biosensors to detect food contamination and food adulteration have revolutionized the global food industry [7][8][9]. Nanozymes are artificial nanomaterials with intrinsic enzyme-like properties, which are distinct from “nano-enzymes” with natural enzymes or catalytic ligands immobilized on nanomaterials. On the whole, natural enzymes are readily digested by proteases and lose their enzymatic activity after exposure to extreme pH and high temperatures, which considerably impedes their practical application [10]. Nanozymes are structurally stable and capable of catalyzing reactions not only under mild physiological conditions but also retaining enzymatic activity in extreme environments. For instance, peroxidase substrates could be catalyzed by iron-based nanozymes at extreme pH (1–12) and temperature (−20–80 °C). The term “nano-zyme” was coined by Pasquato et al. in 2004 to employ triazacyclonane-functionalized gold nanoparticles as catalysts for transphosphorylation reactions [11]. In 2007, Yan et al. reported that Fe3O4 nanoparticles possessed the inherent catalytic activity of horseradish peroxidase (HRP), which could catalyze the conversion of substrates as natural peroxidase under mild physiological conditions [12]. To date, the catalytic performance of nanozymes have been extended from the initial single oxidoreductase (peroxidase) to the current four categories, including oxidoreductase, hydrolase, lyase and isomerase [13][14]. Dozens of inorganic nanomaterials have been found to hold different catalytic activities, for instance, cerium dioxide nanoparticles and ferromagnetic nanoparticles with peroxidase activity, gold nanoparticles with oxidase activity and cadmium sulfide and cadmium selenide nanoparticles with nitrate reductase activity [15][16]. The most outstanding feature of nanoparticles is their superior catalytic activity, low cost, high stability, and controllable and adjustable enzyme activity, which is unmatched by other simulated enzymes [17]. In addition, nanozymes can be size-controlled and surface-modified utilizing sophisticated nanotechnology to modulate their enzymatic activities, which is thought to be an inorganic material with unique physicochemical properties [18]. The discovery of nanozymes breaks the previous notion that inorganic nanomaterials are inert and reveals that they also hold catalytic activities similar to enzymes. Extensive experiments have confirmed that nanozymes are intended to be applied as an alternative to enzymes in the life sciences and the food industry (Table 1) [19][20]. However, the scant biometric events, inadequate water solubility, rational batch design, and catalytic mechanisms of synthetic enzymes based on nanomaterials and the lower catalytic efficiency of some nanozymes compared to natural enzymes are still the prime hindrances confining their applications [21].
Table 1. Nanozyme-based biosensors for the detection of food contaminants.
Analytes Biosensors Nanozymes LODs Food matrix Ref.
Pathogenic microorganism          
Escherichia coli O157:H7 (E. coli O157:H7) Colorimetric Platinum-coated magnetic nanoparticle clusters (Pt/MNCs) 10 CFU/mL milk [22]
E. coli O157:H7 Colorimetric Hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers) 4.1 CFU/mL milk [23]
Salmonella Enteritidis Colorimetric Fe-MOF nanoparticles 34 CFU/mL milk [24]
Salmonella typhimurium Colorimetric Prussian blue nanoparticles (PBNPs) 6 × 103 CFU/mL powdered milk [25]
Salmonella enterica serovar typhimurium Colorimetric ZnFe2O4-reduced graphene oxide nanostructures 11 CFU/mL milk [26]
Listeria monocytogenes (L. monocytogenes) Colorimetric AgNCs 10 CFU/mL pork [27]
Toxins          
Aflatoxin B1 (AFB1) Colorimetric Mesoporous SiO2/Au-Pt (m-SAP) 0.005 ng/mL peanut [28]
AFB1 Colorimetric Porphyrin NanoMOFs (NanoPCN-223(Fe)) 0.003 ng/mL milk [29]
AFB1 and Salmonella Enteritidis Colorimetric/Fluorescent Pt@PCN-224-HRP-initiator DNA (PP-HRP-iDNA) 6.5 × 10−4 ng/mL and 4 CFU/mL for AFB1 and Salmonella Enteritidis respectively rice and milk [30]
Ochratoxin A (OTA) Colorimetric Co(OH)2 nanocages 2.6 × 10−4 ng/mL corn [31]
Saxitoxin (STX) Colorimetric AuNPs 4.246 × 10−4 nM shellfish [32]
Pesticide residues          
Diazinon Fluorescent Fe3O4 nanoparticles@ZIF-8 (Fe3O4 NPs@ZIF-8) 0.2 nM water and fruit juices [33]
Acetamiprid Colorimetric Gold nanoparticles (GNPs) 0.1 ng/mL - [34]
Methyl-paraoxon Colorimetric/Fluorescent nanoceria 420 nM Semen nelumbinis, Semen Armeniacae Amarum, Rhizoma Dioscoreae [35]
Paraoxon Fluorescent Carbon quantum dots (CQDs) 0.05 ng/mL tap and river water [36]
Paraoxon Fluorescent MnO2 Nanosheet-Carbon Dots 0.015 ng/mL tap water, river water, rice, and cabbage [37]
Paraoxon, Parathion, Fenitrothion and Diazinon Colorimetric AuNPs 0.13 ng/mL, 0.37 ng/mL, 0.42 ng/mL and 0.20 ng/mL for paraoxon, parathion, fenitrothion and diazinon, respectively water [38]
Glyphosate Colorimetric/Fluorescence/Photothermal N-CDs/FMOF-Zr 13.1 ng/mL, 1.5 ng/mL and 11.5 ng/mL for colorimetric, fluorescence and photothermal respectively rice, millet, and soybeans [39]
Veterinary drugs          
Tetracycline (TC) Colorimetric AuNCs 46 nM drugs and milk [40]
Kanamycin Colorimetric Gold nanoparticles (GNPs) 1.49 nM - [41]
Enrofloxacin Chemiluminescence Co(OH)2 nanosheets 4.1 × 10−5 ng/mL shrimp, chicken, and duck meat [42]
Norfloxacin (NOR) Colorimetric 1-methyl-D-tryptophan-capped gold nanoclusters (1-Me-D-Trp@AuNCs) 200 nM drugs [43]
Sulfaquinoxaline (SQX) Chemiluminescence Cu(II)-anchored unzipped covalent triazine framework (UnZ-CCTF) 7.6 × 10−4 nM milk [44]
Chloramphenicol (CAP) Electrochemiluminescence Ultrathin PtNi 2.6 × 10−7 nM pig urine, river water, and milk [45]
Heavy metals          
Hg2+ Colorimetric Pt NP 16.9 nM, 26 nM and 47.3 nM for MilliQ water, tap water and ground waters, respectively MilliQ water, tap water, and ground waters [46]
Hg2+ and MeHg Fluorescent Copper oxide-based nanocomposites 3.0 nM and 3.3 nM for Hg2+ and MeHg, respectively tap water, river water, seawater, and dogfish muscle [47]
Ag2+ Colorimetric Chitosan-PtNPs (Ch-PtNPs) 4 nM tap and lake water [48]
Ag2+ Colorimetric Pt nanoparticles 7.8 × 10−3 nM river water [49]
Pb2+ Colorimetric Tungsten disulfide (WS2) nanosheets 4 ng/mL tap water, soil, wheat, and fish serum [50]
Pb2+ Colorimetric Au@Pt nanoparticles 3.0 nM lake water [51]
Pb2+ and Hg2+ Fluorescent Metal-deposited bismuth oxyiodide (BiOI) nanonetworks nanomolar quantities tap water, river water, lake water, and sea water [52]
Others          
Sulfide Colorimetric GMP-Cu nanozyme with laccase activity 670 nM baking soda, rock sugar, konjac flour, and xylitol [53]
Nitrite Colorimetric/Electrochemical Histidine(His)@AuNCs/rGO 2 nM and 700 nM for Colorimetric and Electrochemical respectively sausage [54]
Nitrite Colorimetric Hollow MnFeO particles 200 nM sausage, pickles, and salted eggs [55]
Salbutamol Colorimetric AgNPs 2.614 × 10−4 ng/mL tap water and artificial urine

2. Pesticide Residues

One of the major factors impacting food quality is contamination from pesticide residues [56]. The majority of pesticides are neurotoxic and carcinogenic to individuals, and their residues migrate through the food chain and environment and even persist in nature for more than 15 years [57]. To analyze pesticide residues in agricultural products and the environment at trace or even ultra-trace levels, the sensitive detection of pesticide residues primarily makes use of high-performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography/mass spectrometry (GC-MS) and HPLC-MS/MS [58]. However, these methods are not appropriate for POCT, while the specific nanozyme structure holds a synergistic effect to raise the sensitivity of detected targets [59]. Pesticides can be broadly divided into four categories based on the chemical property of their active ingredients: organochlorines, organophosphates, carbamates and pyrethroids [60].
Applying a synthetic Fe-N/C SAzyme that directly oxidized TMB to produce blue oxidized product 3,3′,5,5′-tetramethylbenzidine diamine (oxTMB), Ge et al. established a novel, extremely sensitive malathion colorimetric platform. L-ascorbic acid-2-phosphate (AA2P), a substrate of acid phosphatase (ACP), could be hydrolyzed to AA, inhibiting the oxidization reaction of TM and resulting in noticeable blue color fading. With the addition of malathion, AA synthesis was reduced and ACP activity was hampered, restoring the catalytic activity of the single-atom nanozyme [61]. Iron-based metal-organic gel (MOGs) nanosheet hybrids with AuNPs immobilization (AuNPs/MOGs (Fe)) were fabricated to detect organophosphorus (OPs), which displayed excellent chemiluminescence (CL) properties. The considerable enhancement of CL was blamed for the modification of AuNPs on the MOGs (Fe) nanosheet, which synergistically increased the CL reaction by speeding the formation of OH•, O2•− and 1O2 [62].
The removal of peroxidase-like activity and color interference is crucial for colorimetric analysis of the nanozyme. The GeO2 nanozyme was found that it only possessed peroxidase-like activity but no oxidase-like capability, which rendered the related detection system free from O2 disturbance. In addition, the white GeO2 nanozyme removed its color interference. Accordingly, a colorimetric sensing platform for ultra-trace detection of OPs pesticides with paraoxon as a representative model was proposed. In the absence of paraoxon, the active acetylcholinesterase (AChE) degraded the GeO2 nanozyme and lost its peroxidase function by hydrolyzing acetylthiocholine (ATCh) to thiocholine (TCh). In the presence of paraoxon, AChE was irreversibly inactivated and TCh production was inhibited [63].
While most investigations have been devoted to further optimizing fluorescent probes or assays to enhance the sensitivity for OPs, Liang et al. conducted groundbreaking work on creating yeast-surface-displayed acetylcholinesterase (AChE) mutants (E69Y and E69Y/F330L) from the perspective of modifying the sensitivity of AChE for OPs. Using electronegative fluorescent gold nanoclusters (AuNCs) combined with AChE mutants, an ultra-trace fluorescence assay for OPs with the LOD of 3.3 × 10−14 M was established, indicating that the E69Y and F330L mutations had the potential to significantly improve the sensitivity of the nanozyme to OPs [64].

3. Veterinary Drugs

Veterinary drugs are the substances applied to prevent, treat and diagnose animal diseases or purposefully regulate animal physiological functions and effectively help farmers solve the problems of livestock threatened by epidemics in breeding production, including antibiotics, antiparasitic and antifungal drugs, hormones, growth promoters, etc. [65]. In the ongoing expansion of the livestock industry today, the employment of veterinary drugs has been crucial in the production of animal-derived food [66]. Veterinary drug residues are the substances of prototype drugs and their metabolites as well as related impurities that accumulate or remain in the organisms or products of livestock and poultry (such as eggs, milk, meat, etc.) following drug application [67].
Li et al. established the Fe-gallic acid (GA) nanozymes (FGN), an artificial multi-iron peroxidase with monoclonal antibody recognition activity and high catalytic performance inspired by polyphenol–protein interactions. Afterward, clenbuterol (CLL) in pork and poultry was determined applying the nanozyme-mediated dual colorimetric immunochromatographic in conjunction with smartphones, with a detection limit of 0.172 ng mL−1 [68]. Applying aggregation-induced (AI)-electrochemiluminescence (ECL)-containing covalent organic framework materials (COF-AI-ECL) as the signal element and Co3O4 nanozyme as the signal amplification component, a CAP molecularly imprinted sensor was established. Co3O4 catalytically amplified the ECL signal of COF-AI-ECL, which was effectively quenched by CAP; thus, the ECL signal was controlled by the elution and adsorption of CAP by molecularly imprinted polymer (MIP) [69]. Kanamycin (Kana) was detected on polyaniline-nanowire-functionalized reduced-graphene-oxide (PANI/rGO) framework by catalyzing H2O2 to generate oxygen using platinum nanozymes on hairpin DNA probes. The principle of signal amplification was to produce a sizable amount of Pt nanoparticles through the coupling of catalytic hairpin assembly (CHA) reaction and strand-displacement amplification (SDA) reaction [70].
Multisignal, enhanced ultrasensitive detection of veterinary drugs is considered an advanced academic research achievement. Utilizing planar VS2/AuNPs nanocomposites as the electrode sensing platform, streptavidin-functionalized CoFe2O4 nanozyme, and methylene-blue-labeled hairpin DNA (MB-hDNA) as signal-amplifying components, an electrochemical aptamer sensor for kanamycin (Kana) proportional detection was developed. The VS2/AuNPs nanocomposites were combined with hDNA complementarily hybridized with biotinylated Kana-aptamers, and the CoFe2O4 nanozyme immobilized on the aptamer sensor possessed excellent peroxidase-like catalytic activity. In the presence of Kana, aptamer biorecognition resulted in a decrease in nanozyme accumulation and an increase in the response of MB [71].

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