Synthesis Of Gold And Silver Nanomaterials And Their Use For MiRNA Detection

This thesis presents an in situ method for miRNA detection based on silver nanoclusters(AgNCs)and an enzymatic photo-induced electron transfer strategy and a colorimetric analysis method based on magnetic beads of DNA hydrogels encapsulated with Au NPs for miRNA detection.The fluorescence properties and structures of AgNCs were characterized using fluorescence spectrophotometry(FL)and transmission electron microscopy(TEM),and the functionalized DNA hydrogels were investigated by electrochemical square wave voltammetry(SWV)and ultraviolet absorption spectroscopy(UV).The specific studies in this thesis are as follows:(1)In situ detection of miRNAs based on AgNCs and enzymatic photo-induced electron transfer strategy:This paper present a method that uses photoinduced electron transfer(PET)for the analysis of micro RNA(miRNA)in clinical serum samples and complicated cell samples using smartphone.The maximum excitation and emission wavelengths of AgNCs were found to be at?ex=518 nm and?em=585 nm,respectively.Despite the advantages of the PET strategy for biosensing,the sensitivity of the PET-based strategy needs to be further improved.Here,we developed an enzymatic multiplex PET strategy to accomplish this task.A 3?-phosphorylated DNA template silver nanoclusters(AgNCs)were first synthesised as a fluorescent signal and hybridised to the target miRNA.The probe is then cleaved by a double-stranded specific nuclease to form a 3?-hydroxylated product.In the presence of d GTP,extra-long poly-G extension by terminal deoxynucleotidyl transferase(Td T)leads to the formation of a G-quadruplex/hemin structure in the presence of K~+and chlorohemoglobin(hemin).In this way,PET can occur between the AgNCs and the produced G-quadruplex/hemin unit,leading to a fluorescence burst of AgNCs.Notably,the fluorescence images can be captured by smartphones and converted into digital information,forming a direct quantitative determination of miRNA.Thus,the strategy in this paper yielded satisfactory results for miRNA detection with a limit of detection of 1.43 p M.Furthermore,this is superior to other reported PET-based sensors where Td T-powered ultra-long G-quadruplex/hemin units act as multiple electron acceptors to promote sensitivity for miRNA monitoring.By adjusting the complementary sequence of the probe,the detection of different miRNA can be achieved.These features not only broaden the scope of practical applications of PET-based strategies,but also provide new insights into nucleic acid detection.(2)A bead-based colorimetric analysis of DNA hydrogel-encapsulated Au NPs for miRNA detection:In this paper,we developed a colorimetric analysis strategy for miRNA detection by deepening the colour of ABTS with DNA hydrogel-encapsulated Au NPs.Nanoparticles are electrostatically adsorbed onto the negatively charged bio-DNA.miRNAs possessing complementary sequences are complementarily paired with the bio-DNA to form a bio-DNA-Au NPs-miRNA double-stranded structure.Subsequently,nucleic acid exonuclease III(Exo III)specifically cleaves the duplex,releasing Au NPs and streptavidin magnetic beads.Because the peroxidase activity of Au NPs,catalyzed by H2O2 and Ag~+,can catalyze the ABTS substrate to deepen its colour,and the catalytic performance of DNA hydrogel-encapsulated Au NPs is better,the miRNA can be detected rapidly by colorimetric method.this method has short detection time and high sensitivity,which provides a new idea for miRNA detection.