Nanozymes in Pesticide Residues and Veterinary Drugs: Comparison
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食源性病原体、化学污染物、重金属等引起的食品安全问题，与人体健康息息相关，引起广泛关注。基于纳米酶的生物传感器具有高灵敏度、选择性和成本效益等优异特性，已被用于检测食品中的危险因素。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. 简介

农业和粮食生产直接关系到人类的生存和发展。食品安全仍然是全球最关键的问题之一，因为食品可能在生产，包装，储存，运输和增值加工的所有阶段受到污染，从而导致食源性疾病的爆发[1]。世界卫生组织（

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）指出，估计有) pointed out that an estimated 6亿人（几乎每10人中就有1人）在食用受污染的食物后生病，每年有42万人死亡，导致3300万健康生命年（DALYs）的损失。食品危害有多种因素，如植物、动物和微生物代谢产物，来自环境的土壤和水污染危害，以及食品包装和加工过程中故意添加的非法添加剂[2，3]。
目前常用的检测免疫分析，如高效液相色谱（00 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 review 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）、气相色谱（), gas chromatography (GC）、质谱（MS）、气相色谱/质谱（GC-MS）、液相色谱/质谱（LC-MS）和酶联免疫吸附测定（ELISA），具有突出的特异性和准确性[5]), 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].然而，它们需要繁琐的预处理步骤、昂贵的仪器、专业的技术人员和较长的检测周期，这些不适合即时检测[6]。在这种情况下，随着纳米技术的快速发展和对纳米技术的深入了解，基于纳米材料的生物传感器检测食品污染和食品掺假已经彻底改变了全球食品工业[7，8，9]。
纳米酶是具有内在酶样特性的人造纳米材料，与固定在纳米材料上的天然酶或催化配体的 Nanozymes are artificial nanomaterials with intrinsic enzyme-like properties, which are distinct from “纳米酶nano-enzymes”不同。总体而言，天然酶很容易被蛋白酶消化，在暴露于极端pH和高温后失去酶活性，这极大地阻碍了其实际应用[10]。纳米酶结构稳定，不仅能够在温和的生理条件下催化反应，而且能够在极端环境中保持酶活性。例如，过氧化物酶底物可以在极端 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）和温度（-20-80°C）下由铁基纳米酶催化。术语“纳米酶”是由–12) and temperature (−20–80 °C). The term “nano-zyme” was coined by Pasquato等人于2004年创造的，用于使用三氮杂环烷官能化的金纳米颗粒作为转磷酸化反应的催化剂[11]。 et al. in 2004 to employ triazacyclonane-functionalized gold nanoparticles as catalysts for transphosphorylation reactions [11]. In 2007年，, Yan等人报道了 et al. reported that Fe3O4纳米颗粒具有辣根过氧化物酶（ nanoparticles possessed the inherent catalytic activity of horseradish peroxidase (HRP）固有的催化活性，在温和的生理条件下可以催化底物转化为天然过氧化物酶[12]。
迄今为止，纳米酶的催化性能已从最初的单一氧化还原酶（过氧化物酶）扩展到目前的四类，包括氧化还原酶、水解酶、裂解酶和异构酶[13，14]。已经发现数十种无机纳米材料具有不同的催化活性，例如，具有过氧化物酶活性的二氧化铈纳米颗粒和铁磁性纳米颗粒，具有氧化酶活性的金纳米颗粒以及具有硝酸盐还原酶活性的硫化镉和硒化镉纳米颗粒[15，16]。纳米粒子最突出的特点是催化活性优越，成本低，稳定性高，酶活性可控可调，这是其他模拟酶无法比拟的[17]。此外，纳米酶可以利用复杂的纳米技术进行尺寸控制和表面改性，以调节其酶活性，这被认为是具有独特物理化学性质的无机材料[18]。纳米酶的发现打破了以前无机纳米材料是惰性的观念，并揭示了它们也具有类似于酶的催化活性。大量实验证实，纳米酶有望作为酶的替代品应用于生命科学和食品工业（表), 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]。然而，基于纳米材料的合成酶的生物特征事件少、水溶性不足、批次设计合理、催化机理多，以及一些纳米酶与天然酶相比催化效率较低，仍然是限制其应用的主要障碍[21]。) [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].
表Table 1.基于纳米酶的生物传感器，用于检测食品污染物。
Nanozyme-based biosensors for the detection of food contaminants.
分析物Analytes 生物传感器Biosensors 纳米酶Nanozymes LODs 食物基质Food matrix 裁判。Ref.
病原微生物Pathogenic microorganism          
大肠杆菌 O157：H7 （大肠杆菌Escherichia coli O157:H7 O157：H7）(E. coli O157:H7) 色度Colorimetric 铂涂层磁性纳米颗粒簇（铂Platinum-coated magnetic nanoparticle clusters (Pt/跨国公司）MNCs) 10 立方英尺CFU/毫升mL 牛奶milk [86][22]
大肠杆菌E. coli O157：:H7 色度Colorimetric 血红素Hemin-刀豆球蛋白A杂交纳米花（HCH纳米花）concanavalin A hybrid nanoflowers (HCH nanoflowers) 4.1 焦氧单位CFU/毫升mL 牛奶milk [87][23]
肠炎沙门氏菌Salmonella Enteritidis 色度Colorimetric 铁Fe-MOF纳米颗粒 nanoparticles 34 立方英尺CFU/毫升mL 牛奶milk [88][24]
鼠伤寒沙门氏菌Salmonella typhimurium 色度Colorimetric 普鲁士蓝纳米粒子Prussian blue nanoparticles (PBNPs) 6 × 103立方英尺 CFU/毫升mL 奶粉powdered milk [89][25]
鼠伤寒沙门氏菌Salmonella enterica serovar typhimurium 色度Colorimetric 铁锌ZnFe2O4-还原氧化石墨烯纳米结构reduced graphene oxide nanostructures 11 立方英尺CFU/毫升mL 牛奶milk [90][26]
单核细胞增生李斯特菌（单核细胞增生李斯特菌）Listeria monocytogenes (L. monocytogenes) 色度Colorimetric 农业AgNCs 10 立方英尺CFU/毫升mL 猪肉pork [91][27]
毒素Toxins          
黄曲霉毒素Aflatoxin B1 （AFB1）(AFB1) 色度Colorimetric 介孔碳化硅Mesoporous SiO2/Au-Pt （(m-SAP）) 0.005 纳克ng/毫升mL 花生peanut [92][28]
空军基地AFB1 色度Colorimetric 卟啉纳米Porphyrin NanoMOFs（纳米 (NanoPCN-223（Fe））(Fe)) 0.003 纳克ng/毫升mL 牛奶milk [93][29]
AFB1和肠炎沙门氏菌 and Salmonella Enteritidis 比色Colorimetric/荧光Fluorescent Pt@PCN-224-HRP引发剂-initiator DNA （(PP-HRP-iDNA）) 6.5 × 10−4分别针对 ng/mL and 4 CFU/mL for AFB1 和肠炎沙门氏菌的and Salmonella Eng/mL 和 4 CFU/mLteritidis respectively 大米和牛奶rice and milk [94][30]
赭曲霉毒素Ochratoxin A （OTA）(OTA) 色度Colorimetric 钴（俄勒冈）Co(OH)2纳米笼 nanocages 2.6 × 10−4纳克 ng/毫升mL 玉米corn [95][31]
虎链淀粉Saxitoxin （STX）(STX) 色度Colorimetric 金AuNPs 4.246 × 10−4纳米 nM 贝类shellfish [96][32]
农药残留Pesticide residues          
二嗪农Diazinon 荧光Fluorescent 铁Fe3O4 nanoparticles@ZIF-8 （铁(Fe3O4 NPs@ZIF-8）) 0.2 纳米nM 水和果汁water and fruit juices [97][33]
啶虫脒Acetamiprid 色度Colorimetric 金纳米颗粒（国产总值）Gold nanoparticles (GNPs) 0.1 纳克ng/毫升mL - [98][34]
甲基对氧磷Methyl-paraoxon 比色Colorimetric/荧光Fluorescent 纳米二氧化铈nanoceria 420 纳米nM 精液，亚美尼亚精液，蓣蓣科Semen nelumbinis, Semen Armeniacae Amarum, Rhizoma Dioscoreae [99][35]
对氧磷Paraoxon 荧光Fluorescent 碳量子点Carbon quantum dots （CQD）(CQDs) 0.05 纳克ng/毫升mL 自来水和河水tap and river water [100][36]
对氧磷Paraoxon 荧光Fluorescent 氧化锰MnO2纳米片 Nanosheet-碳点Carbon Dots 0.015 纳克ng/毫升mL 自来水、河水、大米和卷心菜tap water, river water, rice, and cabbage [101][37]
对氧磷、对硫磷、非硝硫磷和二嗪酮Paraoxon, Parathion, Fenitrothion and Diazinon 色度Colorimetric 金AuNPs 对氧磷、对硫磷、非硝硫磷和二嗪酮分别为 0.13 纳克/毫升、0.37 纳克/毫升、0.42 纳克/毫升和ng/mL, 0.37 ng/mL, 0.42 ng/mL and 0.20 纳克/毫升ng/mL for paraoxon, parathion, fenitrothion and diazinon, respectively 水water [102][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 [103][39]
Veterinary drugs          
Tetracycline (TC) Colorimetric AuNCs 46 nM drugs and milk [104][40]
Kanamycin Colorimetric Gold nanoparticles (GNPs) 1.49 nM - [105][41]
Enrofloxacin Chemiluminescence Co(OH)2 nanosheets 4.1 × 10−5 ng/mL shrimp, chicken, and duck meat [106][42]
Norfloxacin (NOR) Colorimetric 1-methyl-D-tryptophan-capped gold nanoclusters (1-Me-D-Trp@AuNCs) 200 nM drugs [107][43]
Sulfaquinoxaline (SQX) Chemiluminescence Cu(II)-anchored unzipped covalent triazine framework (UnZ-CCTF) 7.6 × 10−4 nM milk [108][44]
Chloramphenicol (CAP) Electrochemiluminescence Ultrathin PtNi 2.6 × 10−7 nM pig urine, river water, and milk [109][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 [110][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 [111][47]
Ag2+ Colorimetric Chitosan-PtNPs (Ch-PtNPs) 4 nM tap and lake water [112][48]
Ag2+ Colorimetric Pt nanoparticles 7.8 × 10−3 nM river water [113][49]
Pb2+ Colorimetric Tungsten disulfide (WS2) nanosheets 4 ng/mL tap water, soil, wheat, and fish serum [114][50]
Pb2+ Colorimetric Au@Pt nanoparticles 3.0 nM lake water [115][51]
Pb2+ and Hg2+ Fluorescent Metal-deposited bismuth oxyiodide (BiOI) nanonetworks nanomolar quantities tap water, river water, lake water, and sea water [116][52]
Others          
Sulfide Colorimetric GMP-Cu nanozyme with laccase activity 670 nM baking soda, rock sugar, konjac flour, and xylitol [117][53]
Nitrite Colorimetric/Electrochemical Histidine(His)@AuNCs/rGO 2 nM and 700 nM for Colorimetric and Electrochemical respectively sausage [118][54]
Nitrite Colorimetric Hollow MnFeO particles 200 nM sausage, pickles, and salted eggs [119][55]
Salbutamol Colorimetric AgNPs 2.614 × 10−4 ng/mL tap water and artificial urine [120]

2. Pesticide Residues

One of the major factors impacting food quality is contamination from pesticide residues [42][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 [43][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 [44][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 [45][59]. Pesticides can be broadly divided into four categories based on the chemical property of their active ingredients: organochlorines, organophosphates, carbamates and pyrethroids [46][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 [47][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 [48][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 [49][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 [50][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. [52][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 [53][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 [54][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 [55][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) [56][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 [57][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 [58][71].

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