Preparation Of Graphene Oxide Quantum Dots Nanocomposites And Their Application In Biochemical Analysis

Graphene oxide quantum dots?GOQDs?are carbon-based nanomaterials with a layered structure,displaying unique properties,such as excellent optical characteristics,good dispersibility,good biocompatibility,Preparation process eco-friendly,low toxicity,and easy modifiability together with possessing a large number of oxygen-containing functional groups.In addition,they have been widely used in bioimaging,light-emitting,and environmental fields.However,to the best of our knowledge,GOQDs modified as a fluorescent probe,used in biological detection at a very early stage.In this dissertation,the selectivity of GOQDs as fluorescent probes has been improved by coupling them to highly selective biomacromolecules?aptamers,single-stranded DNA binding proteins,and single-stranded DNAs?.Based on the diverse changes in the fluorescent intensity of the probes after the addition of the corresponding quencher and target,detection techniques for the antibiotics,sulfamethazine?SMZ?and kanamycin?KAN?,as well as the sequence-specific detection for the Genetically Modified Organism Marker Sequence?NOSt?gene,have been achieved.The main research contents are as follows:?1?This study developed a novel homogeneous and signal“off–on”graphene oxide quantum dots?GOQDs?and aptamer-based fluorescence switch.This switch was synthesized by connecting aptamer-labeled GOQDs?GOQDs@apt?as signal source with graphene oxide?GO?as quencher,and it was employed for detecting sulfamethazine?SMZ?in milk samples.The detection mechanism is based on:When SMZ is absence to the system,GOQDs@apt is adsorbed on the surface of GO and quench the fluorescence of GOQDs@apt due to the strong?-?interaction between the aptamer and GO?turning off?.In the presence of SMZ,aptamers bind to SMZ with high specificity and are folded,lose the hydrogen bonds that can bind to GO,fluorescence was recovered by release of GOQDs@apt from GO?turning on?.Moreover,the degree of fluorescence recovery is significantly positively correlated with the amount of SMZ added,Consequently,this process was utilized for the detection of SMZ through optical responses.Under optimal conditions,fluorescence intensity increased linearly with increasing concentrations of SMZ from 0.008 ng mL^-11 to 60 ng mL^-1.Limit of detection of fluorescence aptamer switch measured 0.005 ng mL^-1 for SMZ detection,further suggesting good selectivity and specificity of the assay to SMZ.This method is reliable and was successfully applied in detection of SMZ in practical samples.?2?We have successfully designed a novel biosensor based on fluorescence resonance energy transfer?FRET?for ultrasensitive detection of NOS terminator gene sequences?NOSt?.This biosensor was synthesized by connecting single-stranded capture DNA?sDNA?-labeled graphene oxide quantum dots?GOQDs??QDs-sDNA?as fluorescent probe and energy donors,and the quencher graphene oxide?GO?as an energy acceptor.The detection principle based on hybridization combinations can occur between QDs-sDNA and complementary target DNA;moreover,QDs-sDNA can bind to GO with significantly higher affinity than QDs-dsDNA.In the absence of complementary target DNA,QDs-sDNA was absorbed onto the surface of GO,and the fluorescence of QDs-sDNA was quenched due to fluorescent resonant energy transfer.In the presence of a complementary target DNA,its hybridization with QDs-sDNA formed QDs-dsDNA,which cannot be adsorbed to the GO surface and this leads to reduced quenching.By comparing the fluorescence intensity of QDs-sDNA and QDs-dsDNA in the presence of GO,we can achieve target DNA detection.Thus,rapid,simple,sensitive,efficient,and eco-friendly detection of NOSt was realized.This biosensor had a detection limit of 0.008 nM and a linear range of 0.05–50 nM.Moreover,this sensor can selectivity detect target DNA compared with random and single-base-mismatched sequences,and was successfully applied to the determination target DNA sequences in biological fluids directly.?3?We coupled single-stranded DNA-binding protein?SSB?with graphene oxide quantum dots?GOQDs?to form QDs–SSB,as a new type of fluorescent probe.Considering the superiority of FRET?fluorescence resonance energy transfer?and the urgency of realizing kanamycin?KAN?sensitive detection,we combined this fluorescent probe with the quencher BHQ₁coupled aptamer?Apt-BHQ₁?,and applied the FRET principle to achieve ultra-trace detection of KAN,and successfully applied to real milk sample detection.The mechanism of detection is based on:SSB has a strong affinity for aptamer?apt?in free state but not in collapsed state;The aptamer is in a free state when not bound to the target and is folded when combined with the target.In the absence of KAN,the distance between QDs-SSB and Apt-BHQ1 is shortened due to the strong affinity between the aptamer and the SSB,and the fluorescence of the QDs–SSB was quenched by Apt-BHQ₁ via FRET.However,when the KAN was added to the QDs–SSB/apt-BHQ₁ system,the combination of aptamer with KAN caused the aptamer to assume a folded state and lose its ability to bind with SSB,thereby reducing FRET and the fluorescence of QDs–SSB recovery.Moreover,the degree of fluorescence recovery was significantly correlated with the amount of KAN added.In this way,the detection of the target is achieved.The detection limit of this aptasensor was low to 6pg/mL with a linear range of 0.01–90 ng/mL.Moreover,the method had high selectivity,stability,anti-interference,accuracy and repeatability.In summary,three simple,efficient,economical,and environmentally friendly quantum dots-based biosensors with high specificity were developed through the surface modification of the GOQDs.Moreover,a sensitive method for the detection of antibiotics?sulfamethazine,kanamycin?,and biological macromolecules?single-stranded DNA?has been achieved.This work extends the application of GOQDs to the field of fluorescent probes and detection of biological molecules.It also demonstrates the potential for the coupling of biological macromolecules?SSB and DNA?and GOQDs,providing new directions for the detection of specific target molecules in actual samples as well as the design of novel FRET biosensors.Further,the present protocol has significant application value for biochemical and analytical chemistry research.