Construction Of Nanozyme Colorimetric Sensors And Their Application In Environment And Food Analysis

Environmental pollution and food quality and safety issues are increasingly becoming the focus of attention.Therefore,the development of simple,portable,rapid,accurate and sensitive sensor for environmental and food analysis is essential to safeguard public health.As an emerging field,nanozyme-based colorimetric analysis has shown great application prospects in the field of instant detection owing to its cost effectiveness,easy operation,fast response,and instrument-free visualization.However,there are still some problems in the application of nanozymes in colorimetric analysis,such as low catalytic activity and selectivity,single enzyme-mimicking activity,and complicated operation and analysis procedures,which limit the detection sensitivity,application range,and on-site analysis.Therefore,in this paper,a series of functional cerium-and cobalt-based nanozymes were prepared by regulating the material composition,morphology,and surface modification,and the catalytic performance and mechanism of their enzyme mimics were systematically studied.Based on different sensing strategies,nanozyme-based colorimetric sensors in solution-and paper-based reaction system were respectively constructed for various target analytes detection in the environment and food,including antibiotics,phenolic pollutants,antioxidants and mycotoxins.The focus was on the detection sensitivity,stability,repeatability and anti-interference ability of the constructed sensors.In addition,the portable sensing platform was built by further integrating with a smartphone,and its application potential for on-site analysis was evaluated.The specific research conclusions are as follows:（1）Using CeO₂/Ce（OH）₃ as the precursor,mixed with boron nitride quantum dots（BNQDs）for hydrothermal treatment,the BNQDs/CeO₂ with a porous rod-like structure was synthesized by a one-step hydrothermal method,which exhibited excellent peroxidase-like activity.However,after the modification of aptamer,its activity was increased by nearly 2.1 times.Through the study of surface charge and the effect of different molecular functionalization on the enzyme-like activities,and combined with the analysis of catalytic kinetics,it was proved that the aptamer significantly enhanced the catalytic activity by improving the affinity of BNQDs/CeO₂to 3,3′,5,5′-tetramethylbenzidine（TMB）and H₂O₂.Whereas the catalytic enhancement was suppressed in the presence of specific targets.It was because the aptamer would desorb from the surface of BNQDs/CeO₂ due to the higher affinity to its targets.Thus,based on the strategy of aptamer-regulated nanozyme activity,a simple,rapid,highly sensitive and selective colorimetric assay for kanamycin（KAN）was established.This method showed a good linear response to KAN in the range of 0.01-100 n M with an ultra-low detection limit of 4.6 p M.Meanwhile,it exhibited a good anti-interference ability and storage stability.In addition,the spiked recoveries of KAN in pig urine,municipal sewage and milk samples were 98.94-101.22%,and the relative standard deviation（RSD）was within 3.3%,demonstrating its excellent practical application ability.This study provides theoretical support for improving the catalytic activity and target-specific recognition of nanozymes.（2）Using polydopamine-coated MnCo Prussian blue analogs（Mn Co-PBA@PDA）as the precursor,the Mn Co bimetallic oxides@carbon composite with yolk-shell nanocage structure（Mn Co@C NCs）was synthesized by one-step calcination under air atmosphere.Mn Co@C NCs exhibited multi-enzyme mimic features,including oxidase,laccase and catalase,thereinto,its oxidase-like activity was confirmed to be the result of the joint participation of multiple reactive oxygen species.With TMB as the substrate,the maximum reaction rate(V_max)of the oxidase-like activity of Mn Co@C NCs was 3-18 times higher than that of other reported nanozymes,while the V_max of laccase-like activity for 2,4-dichlorophenol（2,4-DP）showed 8-fold higher that of natural laccase,and the above reactions are both completed within 3 min.Based on the excellent oxidase-like and laccase-like activities,the simple,rapid and highly sensitive colorimetric assays for ascorbic acid and 2,4-DP were established,with the detection limits as low as 0.29μM and 0.76μM,respectively.In addition,a portable sensing platform was constructed by integrating with a smartphone,which was successfully applied to on-site and quantitative determination of total antioxidant capacity（TAC）in fruits,vegetables and commercialized beverages,and 2,4-DP in municipal sewage.This study provides a research basis for the design of highly active and multi-enzyme mimic nanozymes as well as their multifunctional applications in environmental and food analysis.（3）Similar to the preparation method mentioned above,Ni Co bimetallic oxides@carbon composite with hollow nanocage structure（Ni Co@C HCs）was synthesized,which exhibited excellent peroxidase-like activity.Its V_max for TMB was at least 5 times stronger than other reported nanozymes,allowing it to rapidly catalyze TMB oxidation and further produce colored product within 3 min,while the generated hydroxyl radicals（·OH）by catalyzing H₂O₂ decomposition played a dominant role in the TMB oxidation process.Moreover,the modified aptamer could significantly inhibit the catalytic activity of Ni Co@C HCs due to its masking effect on the active site,but the catalytic inhibition could be restored through binding to the specific target.By introducing aptamer-modified nanozyme probes into the paper pieces,the low-cost,portable,easy-to-operate,and low-reagent-consumption paper-based biochips were constructed,and further integrated with a smartphone sensing platform,enabling on-site and quantitative analysis of enrofloxacin（ENR）.The sensor exhibited high sensitivity with the liner detection range from 0.1 ng/m L to 50μg/m L,and the detection limit was as low as 0.029 ng/m L.It was successfully validated in field analysis of swine urine,livestock wastewater and milk samples.This study provides a new insights into the design of paper-based colorimetric sensing platform with simplicity,portability,low reagent consumption,and field application.（4）A polydopamine-coated CuCo Prussian blue analogues（Cu Co@PDA）was synthesized by a facile self-assembly method,which performed the multifunctional characteristics of good dispersion stability,easy biomodification,visible body color and excellent peroxidase-like activity,and its catalytic activity was proved to be derived from·OH generation and electron transfer.The probe labeled Cu Co@PDA was used for color development on aptamer-functionalized lateral flow chromatography test strip,due to the hybridization reaction with the complementary DNA,the Cu Co@PDA probe was captured on the T line to generate a colorimetric signal,and the signal was further amplied by catalyzing chromogenic reaction in situ.The visualization and dual-signal readout detection of aflatoxin B1（AFB1）was realized based on the competitive reaction.Thereinto,in the catalytic colorimetric mode,the visual detection limit（v LOD）and cut-off value were 0.1 ng/m L and 500 ng/m L,respectively.Compared with the ontological colorimetric mode,it showed higher sensitivity and wider detection range.A smartphone sensing platform was further integrated to realize the quantitative analysis of AFB1 with the detection limit of 2.2 pg/m L.The sensor showed good anti-interference ability,accuracy and precision with the spiked recoveries between 103.38-108.08%and coefficient of variation less than 9.1%,and it was successfully applied to the on-site analysis of AFB1 in peanut,corn and wheat samples.This study provides new proposals for the design of simple,portable,accurate,sensitive test strip sensing platform with instrument-free visualization.