Fabrication Of Fluorescent Sensors Based On Carbon Dots For Enzyme And Pesticide Detection

Carbon dots（CDs）as excellent fluorescent materials possess outstanding photostability,tunable emission spectra,low toxicity and better biocompatibility,and present incomparable advantages in sensing.Enzymes,as biocatalysts,speed up chemical reactions to reach equilibrium in living organisms.The abnormal enzyme activity is closely related to many diseases,and they are regarded as biomarkers in clinical medicine.Simultaneous assay of multiple enzymes can decline false positive rate,improve diagnostic accuracy and provide guidance for prognosis of disease.CDs-based fluorescent sensors provide alternative platforms to monitor enzyme activity.Although the design of detection principles and various sensing mechanism have been fabricated to monitor enzyme activity,their explorations are still in initial stages.With the increasing concerns of food safety and environmental protection,the detection of pesticide residues has attracted more attentions.The methods for pesticide detection are based on large-scale equipment with accurate results and high reproducibility.The time consuming and multi-step operations of these methods also limit their applications in practical life.Pesticides as inhibitor can suppress the enzyme activity by combination with enzyme specifically.Enzyme-inhibition-based methods have been employed for assay pesticides with merits of facile and sensitive performance.In real life,the design of low cost,user-friendly and portable platform is significant for high-throughput detection for pesticides.Based on the fluorometry of CDs,we design sensors and construct detection platforms for enzyme activity and pesticide,and the main works as follows:（1）Bright green emissive N-CDs were synthesized by one-step hydrothermal technique with catechol and ethylenediamine.The excitation and emission wavelengths of N-CDs were 408 and 510 nm,respectively.γ-L-Glutamyl-4-nitroanilide was employed as the substrate of GGT.The absorption spectrum of GGT catalytic product（4-nitroaniline,4-NA,Abs.maximum at 381 nm）overlapped greatly with the excitation spectrum of N-CDs.4-NA acted as absorber in inner filter effect（IFE）to quench the fluorescence of N-CDs.The established fluorescent method offered good linear relationship within 2.0–10.0 U L^-1（R²,0.982）and 10.0–110.0 U L^-1（R²,0.998）with limit of detection（LOD）of 0.6 U L^-1.This method was successfully applied to investigate GGT activity in human serum samples with acceptable recoveries（99.1–105.0%）.The approach was also employed for screening GGT inhibitors from different polar extracts of Schisandra chinensis.This method has great potential as a candidate for GGT detection,GGT-related diseases diagnosis and high-throughput drug discovery.（2）Simultaneous detection of multiple enzymes is urgently needed for diagnosis,therapeutics and prognostic of related diseases in clinic.Here,a new strategy for simultaneous monitoring GGT and alkaline phosphatase（ALP）activity has been fabricated based on dual-emission CDs.Dual-emission CDs were prepared by solvothermal treatment of Actinidia chinensis,which presents two fluorescent emissions at 471 nm（blue channel）and 671 nm（red channel）.GGT and ALP activity can be detected based on IFE and static quenching effect of blue and red channels of CDs,respectively.Linear ranges were 2.5-90 U L^-1 and 5-200 U L^-1,and LODs were 0.71 U L^-1 and 1.2 U L^-1 for GGT and ALP,respectively.Dual-emission CDs can monitor GGT and ALP activity in human serum samples with satisfied recoveries（99.3%-108.6%for GGT,98.4%-105.4%for ALP）.Furthermore,the combination of CDs to sense GGT and ALP activity with OR logic gate can predict human health status.The design and application of dual-emission CDs can also be extended as promising tools to detect multianalytes using different channel signals.（3）Based on the excellent performance of CDs-based sensor for enzyme activity,a ratiometric fluorescent sensor was designed for visual organophosphorus pesticide（OP）detection according to the special inhibition of OPs for acetylcholinesterase（ACh E）.CDs with dual emissions at 450 nm and 675 nm under 365 nm excitation were synthesized by cucumber.OPs can inhibit ACh E to suppress the production of thiocholine（binding with Ag⁺）.Then,free Ag⁺oxidizes o-phenylenediamine（OPD）into yellow fluorescent 2,3-diaminophenazine（ox OPD,Em.maximum at 560 nm）,which quenches blue emission of CDs.In real sample detection,OPs rapidly diffused into surface deposited sodium alginate（SA）solution and were then extracted by an in situ formed SA hydrogel network.After that,the released OPs were visually detected using a smartphone-assisted platform.The fluorescent images presented a distinct color variation from blue to orange with a wide linear range from1.2 to 57.0μg L^-1 and low LOD of 0.50μg L^-1 for parathion-methyl,a model sample of OPs.The facileness,high sensitivity and nondestructive technology for OP measurements provided new insight into food safety evaluation.（4）In order to more convenient and practical detection for OPs,a threaded 3D microfluidic paper analytical device（3DμPAD）was fabricated and combined with red-emission CDs（RCDs）to form ratiometric fluorescent sensor for instrument-free and background-free OP detection in agricultural products.Butyrylcholinesterase（BCh E）can hydrolyze ATCh into TCh and TCh reduced Mn O₂ nanosheets to Mn²⁺.With addition of OPs,BCh E activity was irreversibly inhibited and prevented the generation of TCh and destruction of Mn O₂ nanosheets.Mn O₂ nanosheets oxidized OPD into yellow fluorescent ox OPD,which quenched the fluorescence intensity of RCDs via IFE.Threaded 3DμPAD had four layers,and each layer allowed to load and add reagents to trigger the sequential reactions in each step.The fluorescent images presented visual color variation from red to yellow with dichlorvos concentration ranging from 2.5μg L^-1 to 120μg L^-1 and limit of detection of 1.0μg L^-1.In the testing for practical samples,threaded 3DμPAD not only can eliminate background influence of colorimetric and fluorescent signal but also avoid the organic solvent influence on enzyme activity for OP detection.Threaded 3DμPAD has merits of accuracy response,simple operation and background-free for OP sensing and supplies a new alternative approach for on-site pesticide detection.（5）This study designed a portable paper lab-in-a-syringe（LIS）device integrated with smartphone sensing platform for rapid,visual determination of 2,4-dichlorophenoxyacetic acid（2,4-D）.2,4-D as an inhibitor could specifically suppress ALP activity,and reduce the production of ascorbic acid（AA）.The oxidation of AA into DHAA by Mn O₂nanosheets is less,and the production of blue-emission fluorescent DFQ is also less.Mn O₂nanosheets oxidate OPD into yellow fluorescent ox OPD to quench the fluorescence of RCDs via IFE.Based on above sensing principles,paper pads loaded reaction substances and assembled into reusable plastic filters.Then,reaction solution was introduced through a disposable syringe for on-site and visual 2,4-D determination.The linear range for 2,4-D was 10-1000μg L^-1with LOD of 5.0μg L^-1,and the color of fluorescent images exhibit obvious variation from blue-purple to red to yellow.This strategy can avoid the interference of color and fluorescent of real samples,and realize background-free,cost-effective and portable monitoring of 2,4-D,and expand application prospect in the field of pesticide analysis.