Research On Detection Method Of Bio-enzyme Activity Via Novel Carbon Dots-based Fluorescence Sensors

Bio-enzymes are biocatalysts produced by cells to promote cellular metabolism and energy conversion.Bio-enzymes do not normally leach out of cells,however,changes in enzyme activity in tissues and body fluids occur when the body is diseased and are used as a reference for early diagnosis of disease.Therefore,the sensitive and accurate determination of bio-enzymes in complex biological samples is important for the early diagnosis and precise treatment of diseases.The commonly used methods for the determination of bio-enzymes are mainly colorimetric,electrochemical,high-performance liquid chromatographic and fluorimetric methods.Among them,fluorimetric methods have the advantages of easy operation,fast detection speed,good selectivity and high sensitivity.In addition,fluorescence imaging technology can perform multiple fluorescence observation simultaneously without harming biological samples,and can analyze cell stereo images clearly.The construction of fluorescence sensing methods requires the selection of signal units that are responsive to the target as analytical probes.Carbon dots as a new zero-dimensional nanomaterial exhibit good properties,such as excellent fluorescence performance,high chemical stability,easy preparation,and good biocompatibility.Based on the above-mentioned background,we prepared a variety of new carbon dots with excellent performance and used"off-on"detection method,ratiometric fluorescence technique and host-guest identification to improve the selectivity and sensitivity of carbon dots,and used smartphones and gel reagents to improve the efficiency of detection.Based on this,we have achieved simple,highly sensitive and selective detections of various bio-enzymes in complex biological samples.The main research contents are as follows:1.A sensitive and effective screening strategy forα-glucosidase and its inhibitors was established based on carbon dots and V₂O₅ nanoribbons.Boron-sulfur-nitrogen co-doped carbon dots（BSN-CQDs）were prepared by microwave hydrothermal method using o-phenylenediamine,boric acid and thiourea as raw materials.The prepared BSN-CQDs exhibited yellow fluorescence under 410 nm excitation with a quantum yield of 15.3%.V₂O₅nanoribbons can quench the fluorescence of BSN-CQDs through inner filter effect.However,α-glucosidase catalyzed the generation of ascorbic acid from the substrate L-ascorbyl-2-glucoside could convert the V₂O₅ nanoribbons to V⁴⁺,thus achieving fluorescence recovery.In addition,as anα-glucosidase inhibitor（anti-diabetic drug）,acarbose decreasedα-glucosidase activity,which in turn reduced ascorbic acid production,resulting in impaired fluorescence recovery of the system.The linear range of the established fluorescence sensing system forα-glucosidase was 0.01-5 U/m L,and the detection limit was 0.003 U/m L.In addition,the detection limit of acarbose was 0.02μM.The sensing system has been successfully applied toα-glucosidase detection in serum,cell imaging and anti-diabetic drug screening.2.The detection efficiency was enhanced based on the study in chapter 2 by using smartphones to further improve the accuracy and visualization of the assay.A method for ratiometric fluorescence detection of acid phosphatase activity was established based on La³⁺ligand self-assembled carbon dot nanohybrids.A clever combination of La³⁺-mediated aggregation-induced emission（AIE）,reversible competition between adenosine triphosphate（ATP）and La³⁺,and acid phosphatase-induced dephosphorylation was established.At 350 nm excitation wavelength,the carbon dots nanohybrids exhibited blue and red fluorescence.Upon the addition of La³⁺,the blue fluorescence intensity was reduced by static quenching and the red fluorescence intensity was enhanced by AIE.Interestingly,the interaction between ATP and La³⁺was stronger relative to that between the carboxyl group and La³⁺,thus inhibiting the La³⁺-induced self-assembly process of the nanohybridization.Acid phosphatase catalyzed the targeted hydrolysis of ATP,which dissociated the La³⁺-ATP complex.F₆₁₀/F₄₅₂ correlated well with acid phosphatase concentration in the range of 0.1-10 U/L with a detection limit of 0.024U/L.The established sensing system exhibited multicolor fluorescence changes,which were combined with smartphones for visual quantitative analysis of acid phosphatase.The established method was applied to the detection of acid phosphatase in serum and cell imaging,and satisfactory results have been obtained.3.Based on the visualization study in chapter 3,hydrogels were introduced to improve the portability for immediate on-site detection.A sensitive method for the detection ofβ-glucuronidase was established using functionalized carbon dots（β-CD-SiCDs）as fluorescent probes.The functionalized carbon dotsβ-CD-SiCDs were found to be obtained through in situ autopolymerization by mixing the solutions of methyldopa,mono-6-ethylenediamine-β-cyclodextrin and N-（β-aminoethyl）-γ-aminopropyltrimethoxysilane at room temperature.β-CD-SiCDs prepared fromβ-cyclodextrin emitted green fluorescence at 380 nm excitation with a quantum yield of 7.9%.4-nitrophenyl-β-D-glucuronide was introduced as a substrate forβ-glucuronidase to generate p-nitrophenol.Subsequently,p-nitrophenol self-assembled withβ-CD-SiCDs through host-guest recognition to form a non-luminescent complex,resulting in the fluorescence quenching ofβ-CD-SiCDs.The linear range ofβ-CD-SiCDs for detectingβ-glucuronidase activity was 0.5-60 U/L with a detection limit of 0.14 U/L.For immediate on-site detection,gel reagents were prepared by a simple method and the images were visualized and quantified using smartphones,avoiding the use of large instrumentation.The constructed fluorescence sensing platform has the advantages of easy operation and time saving,and has been successfully used for the detection ofβ-glucuronidase activity in serum and cell imaging.4.The carbon source selection of fluorescent probes in the above research was improved,and the carbon dots were synthesized using biomass as the carbon source,and a bio-enzyme detection system was developed to improve the utilization value of biomass.An efficient method for the detection of trypsin was established using biomass nitrogen-doped carbon dots（N-CDs）as a fluorescent probe.The N-CDs were synthesized by hydrothermal method using enzymatic lignin as the carbon source and ammonia as the solvent and nitrogen source.The prepared N-CDs with good water solubility and stable optical properties were used as label-free biosensors for the detection of trypsin.The fluorescence of N-CDs was quenched with positively charged cytochrome c（Cyt c）by electrostatic induced aggregation and static quenching.However,in the presence of trypsin,cytochrome c tended to be hydrolyzed into short peptides,which led to fluorescence recovery of the N-CDs/Cyt c complex.The linear range of the N-CDs/Cyt c complex for trypsin was 0.09-5.4 U/m L,and the limit of detection for trypsin was 0.013 U/m L.The method was applied to the detection of serum trypsin with recoveries of 95.5-102.0%.Enzymatic lignin-based biomass carbon dots can be prepared at low cost and mass production,and the developed method has been successfully used for the detection of trypsin activity in serum and cell imaging.5.Based on the study of fluorescent carbon dots prepared from lignin in chapter 5,an in-depth study was conducted to realize the development of lignin-derived carbon dots with dual functions of fluorescence and enzyme-like activity by modifying lignin,and then a ratiometric fluorescence sensing strategy was constructed to detect xanthine oxidase activity based on carbon dots.Modified lignin carbon dots（LS-CDs）were prepared by hydrothermal method using sodium lignosulfonate and hydrochloric acid,and o-phenylenediamine carbon dots（OPD-CDs）were prepared by hydrothermal method using o-phenylenediamine as raw material.The mixed solutions of carbon dots exhibited blue and yellow fluorescence at 370 nm excitation wavelength.In addition to their excellent fluorescence properties,LS-CDs also possessed peroxidase-like properties and can rapidly catalyze the generation of hydroxyl radicals from H₂O₂.Xanthine oxidase generated H₂O₂ in the process of catalyzing the production of uric acid from the substrate xanthine.H₂O₂ was then catalyzed by LS-CDs to generate hydroxyl radicals.The hydroxyl radicals oxidized the residual o-phenylenediamine groups on the surface of OPD-CDs to enhance their yellow fluorescence,accompanied by the blue fluorescence quenching of LS-CDs.The established fluorescence sensing system showed a linear range of 0.5-120 U/L for xanthine oxidase with a detection limit of 0.13 U/L.Compared with the monochromatic fluorescence-catalyzed system,the system exhibited a more pronounced color change.The sensing platform was successfully applied to the sensing and imaging of xanthine oxidase in serum and cells.In summary,different methods were used to prepare carbon dots with different properties and to improve the selectivity and sensitivity of carbon dots by using host-guest identification,"off-on"detection and ratiometric fluorescence techniques.The mechanism of interaction between enzymatic reaction products and fluorescent materials and the quenching mechanism were investigated.The visualization,accuracy and immediate detection capability of the method are enhanced by using smartphones and gel reagents.In this study,high sensitivity and high specificity sensing methods have been developed for a variety of bio-enzymes that are closely related to life activities,and the detection of serum samples and imaging of related enzymes in cells were successfully achieved.This study opens up new ideas for the development and application of novel fluorescent sensors,and provides a technical support and theoretical basis for the detection of related disease markers,which is of great significance for the diagnosis of related diseases.