Synthesis And Application Of Non-metal Doped Carbon Dots And Their Composites With Enzyme

Carbon dots(CDs)are a new type of carbon nanomaterials with sizes generally below 10 nm.Due to their many advantages such as high fluorescence quantum yield(QY),good photo-stability,anti-photobleaching,extremely low toxicity and easy functionalization,CDs have great potential in broad applications of bio-detection and sensing,bio-imaging,cancer treatment,nano-drugs and etc.Enzymes are a class of highly efficient and specific biocatalysts widely used in biochemistry.However,the poor stability and inactivation of the enzyme makes it particularly important to modify or immobilize the enzyme by using a suitable material or matrix.In this thesis,two kinds of non-metallic elements doped CDs were synthesized by hydrothermal method and microwave-assisted thermal decomposition method:fluorine and nitrogen co-doped carbon dots(F,N-CDs),phosphorus and nitrogen co-doped carbon dots(P,N-CDs).The morphology,structure,surface composition and optical properties of as-synthesized F,N-CDs and P,N-CDs were studied carefully,and then four composites of these CDs with horseradish peroxidase(HRP)and laccase(Lac)were prepared as follows:HRP-F,N-CDs,Lac-F,N-CDs,HRP-P,N-CDs and Lac-P,N-CDs.Finally,the applications of these four composites in electrochemical sensing and biocatalysis were investigated.The main research contents and results are summarized as follows:1.Construction of Lac-F,N-CDs based electrochemical biosensor for detection of catechol.The F,N-CDs with bright green fluorescence were synthesized by a one-step hydrothermal method using p-phenylenediamine and pentafluorouracil as starting materials.The morphology,structure,surface composition and optical properties of F,N-CDs were characterized by employing transmission electron microscopy(TEM),ultraviolet-visible absorption spectroscopy(UV-Vis),photoluminescence spectroscopy(PL),X-ray photoelectron spectroscopy(XPS)and other instruments.The obtained F,N-CDs have an average particle size of 1.68 nm,and the lattice fringes of graphitic carbon atoms can be observed clearly in the core area.The doping amount of N and F atoms in F,N-CDs is about 15.90%and 2.26%,respectively.The F,N-CDs exhibit bright yellowish green fluorescence under an UV light,with a fluorescence QY as high as 45.6%.The preparation of two composites(Lac-F,N-CDs and HRP-F,N-CDs)were explored.Compared with HRP,it was found that Lac was easier to form a stable complex with F,N-CDs,thus the properties of Lac-F,N-CDs complex and its application in electrochemical biosensor were studied here.A new Lac-F.N-CDs/GCE biosensor was constructed by coating Lac-F,N-CDs onto a glassy carbon electrode(GCE).The ability of Lac-F,N-CDs/GCE biosensor for detecting catechol was investigated by means of cyclic voltammetry(CV)and chronoamperometry(i-t)on an electrochemical workstation.It was obtained from the i-t result that,the detection limit of the biosensor for detecting catechol was 14 nM with a sensitivity of 219.17 ?A mM-1 cm-2.Moreover,the Lac-F,N-CDs/GCE sensor has a good cycle stability.When it cycled for 50 times,the redox peak positions on the CV curve almost kept unchanged and the peak current drop was rather small.Further,the interference of hydroquinone,dopamine and gallic acid on Lac-F,N-CDs/GCE sensor for detecting catechol was studied.In addition,the experiments of using Lac-F,N-CDs/GCE sensor to detect catechol in tap water and lake water were also performed.This work provides a new approach to construct electrochemical biosensors based on the composites of non-metal doped CDs and enzyme for the detection of catechol and its analogues.2.Preparation of low-temperature biocatalysts based on HRP-P,N-CDs and HRP-F,N-CDs.P,N-CDs were synthesized by microwave-assisted thermal decomposition method,using citric acid,sodium tripolyphosphate and ethylenediamine as starting materials.The morphology,structure,surface composition and optical properties of P,N-CDs were characterized by employing TEM,UV-Vis,PL,XPS and other methods.The results show that the P,N-CDs have an average particle size of 2.72 nm,and the lattice fringes of graphitic carbon atoms can be observed clearly in the core area.The doping amounts of N and P atoms are 13.09%and 0.21%,respectively.The P,N-CDs exhibit bright blue fluorescence under an UV light,with a fluorescence QY as high as 53.1%.Two composites of HRP-P,N-CDs and HRP-F,N-CDs were formed and characterized by using UV-Vis absorption and PL spectroscopy.It was found that HRP was easier to form a stable complex with P,N-CDs than Lac,and the PL intensity of the P,N-CDs decreased a little after combined with the HRP while the absorption peak also shifted slightly.Finally,at a low temperature such as 4 ?,the interference of the added hydrogen peroxide(H2O2)on three composites of HRP-P,N-CDs,Lac-P,N-CDs and HRP-F,N-CDs for catalyzing ABTS substrate was studied.The results showed that the amount of H2O2 has a great influence on the catalytic oxidation of ABTS by HRP-P,N-CDs and HRP-F,N-CDs.When ABTS was oxidized,the substrate solution changed from colorless to dark green,and the degree of discoloration visually could reflect the enzymatic activity and catalytic ability of HRP in the above-mentioned composites.In summary,HRP-P,N-CDs and HRP-F,N-CDs composites are expected to be stable low-temperature biocatalysts capable of maintaining high catalytic activity of HRP enzymes.