Iron-based Single Atoms Nanozymes For Fluorescent And Electrochemical Dual-platform Detection Of Glucose

Diabetes mellitus is a metabolic disease characterized by elevated blood glucose concentration,which can be mainly divided into type 1 diabetes,type2 diabetes,other special types of diabetes and gestational diabetes,affecting millions of people around the world.In both type 1 and type 2 diabetes,genetic and environmental factors can lead to decreased beta cell function or even loss,which can lead to hyperglycemia.Long-term hyperglycemia can lead to organ failure and tissue damage,leading to multiple complications such as blindness,cardiovascular disease,and kidney failure.In patients with typical symptoms,measurement of blood sugar is sufficient to diagnose diabetes,so monitoring the patient’s blood sugar levels and prompt medical control are critical for people with diabetes.In addition,glucose concentration is an important parameter for the health indicators of diabetic patients,and stabilizing it at an appropriate level can control the occurrence and development of certain complications.Therefore,accurate and timely monitoring of blood glucose concentration is very important for the prevention and control of diabetes.In recent years,nanozyme has been considered as a highly promising alternative to natural enzymes with unique advantages such as low cost,high stability,easy mass production and storage.Nanozymes have been widely used in environmental protection,biosensing,antibacterial and so on.The components of the currently developed nanozymes mainly include metal oxides,carbon materials and precious metals.In recent years,single-atom nanomaterials have attracted extensive interest from researchers due to their high atomic utilization and excellent catalytic performance.Single-atom nanozymes refer to catalysts in the form of fixed,stable,isolated single atoms on supports,which have a high degree of enzyme-like catalytic activity.The representative system in the field of single-atom catalysis is that the transition metal atoms are dispersed on the nitrogen-doped carbon substrate,such as atomic-scale Fe anchored on the nitrogen-doped carbon substrate,and Fe-N_xas catalytic active center has a structure similar to that of natural peroxidase.Therefore,Fe-N-C also shows peroxidase-like catalytic activity and has been widely used in the detection of hydrogen peroxide（H₂O₂）and glucose.However,due to the strong migration and aggregation tendency of active single metal atoms in the preparation or subsequent applications,which can easily lead to the degradation or even deactivation of the catalytic performance of SANs,supporting monodisperse atoms on suitable supports is an ideal solution to enhance the activity of SANs.Based on the above research background,in this dissertation,Fe single atoms in 3D porous N-doped carbon aerogels（NCAG/Fe）nanozyme was constructed.The NCAG/Fe nanozymes use multilayer three-dimensional nitrogen-doped porous carbon aerogels as a support carrier,which increases the iron loading and enhances the stability of SANs by virtue of its large surface area and abundant mass transfer channels.It was used for dual-platform quantitative detection of glucose on fluorescent biosensing and electrochemical biosensing platforms,respectively.The specific work content is as follows:Part 1:Preparation of NCAG/Fe Nanozyme and Its Use in Fluorescence Detection of GlucoseIn this chapter,we successfully prepared and characterized the NCAG/Fe nanozyme,and used it in a fluorescence sensing platform for the highly sensitive detection of H₂O₂ and glucose,and finally successfully applied it to the analysis of clinical samples.NCAG/Fe nanozymes were prepared by repeatedly freezing and thawing silica（Si O₂）nanoparticles and Fe（PM）₃²⁺complexes at-20°C to obtain gelatin-zinc hydrogels,followed by pyrolysis and acid etching to obtain NCAG/Fe.We use scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,scanning transmission electron microscopy（STEM）,X-ray photoelectron spectroscopy（XPS）,X-ray diffractometer（XRD）and elemental mapping analysis to characterize the prepared NCAG/Fe,and the results all proved the successful preparation of NCAG/Fe single-atom nanozyme.To investigate its peroxidase-like activity,we then demonstrated its feasibility to catalyze H₂O₂ using fluorescence spectroscopy.Using o-phenylenediamine（OPD）as the substrate,when H₂O₂and NCAG/Fe were present in the reaction system,we observed a clear fluorescence signal,proving the peroxidase-like catalytic activity of NCAG/Fe.The highly sensitive detection of H₂O₂ can be achieved by measuring the fluorescence signal.Since glucose can specifically generate H₂O₂ under the catalysis of glucose oxidase（GOx）,this method can be further applied to the detection of glucose.The NCAG/Fe nanozyme-based fluorescence assay has a linear range of 0.02 m M to 1 m M for the detection of glucose with a detection limit of approximately 3.1μM.In addition,the sensing method has good selectivity,and we selected several interfering substances（fructose,maltose,sucrose,glycine,and common inorganic ions）to test whether they would cause non-specific interference.The results showed that only glucose produced a significant fluorescence response,and no significant fluorescence signal was detected even with a higher concentration of the interferer（3 m M）.The good selectivity of NCAG/Fe in distinguishing glucose from potential interferents may be attributed to the high selectivity of GOx.Then we use the NCAG/Fe nanozyme-based fluorescence method to detect glucose in serum,simulated sweat and saliva,and compared with commercial blood glucose meters.The results show that the NCAG/Fe-based fluorescence method can be accurate detect glucose in serum,simulated sweat and saliva with small relative errors.These results demonstrate the potential and practical value of fluorescence assays using NCAG/Fe nanozymes to detect blood glucose in clinical samples.Part II:NCAG/Fe Nanozyme-Based Electrochemical Sensor for Glucose DetectionThe detection accuracy of the single platform is easily disturbed by external factors,such as non-standard test procedures,different environments,and even different operators.To address this challenge,in this chapter we apply NCAG/Fe nanozymes to an electrochemical platform and construct an electrochemical glucose sensor based on NCAG/Fe nanozymes.We first modified the nanocomposite on the surface of glassy carbon electrode,and analyzed the feasibility of glucose detection by chronoamperometry.The experimental results show that the addition of glucose does not change the current signal in the presence of a single glassy carbon electrode.When the glassy carbon electrode was modified with NCAG without iron atom doping,the addition of glucose only produced a small current step,while the NCAG/Fe nanozyme-modified electrode produced a significant current signal change after the addition of glucose.This indicates that the Fe N₄ site of the NCAG/Fe nanozyme is highly active during the electrocatalytic oxidation of glucose.Then,we performed a sensitivity analysis,which showed that the NCAG/Fe nanozyme had a linear response range of 2-2000μM for glucose detection,and its detection limit was as low as 0.5μM.To investigate the selectivity of NCAG/Fe nanozymes,we modified NCAG/Fe nanozymes on electrodes using various interfering substances（dopamine,L-AA,NH₄Cl,L-glutamic acid,lactose,uric acid and L-histidine）,to test whether they produce nonspecific signals.Compared with the glucose-responsive signal,the signal changes generated by the interfering components were negligible,indicating the excellent selectivity of the NCAG/Fe nanozyme for glucose detection on the electrochemical platform.In addition,we also studied the stability of the electrochemical sensor based on NCAG/Fe nanozyme,and the results showed that the electrochemical signal of the NCAG/Fe nanozyme-modified electrode to 100μM glucose did not decrease significantly within one week,proving that NCAG/Fe nanozyme-modified electrodes have excellent stability.Based on the previous work,we finally used the NCAG/Fe nanozyme-based electrochemical sensor for glucose detection in clinical serum,simulated sweat and saliva samples.The electrochemical sensor based on the NCAG/Fe nanozyme is comparable to the detection results of commercial blood glucose meters,which proves that the nanozyme-based sensor can accurately detect glucose in serum,simulated sweat and saliva samples.The above results demonstrate that NCAG/Fe can be used as a reliable,low-cost,high-performance,and highly selective nanozyme for both fluorescent and electrochemical sensors,enabling dual-platform detection of glucose in real clinical samples.