Detection Of Gene Mutation Using DNA-gold Nanoparticle Probes And Gene Chips

The DNA detection method was established based on gold nanoparticles immobilized with DNA probes and protein molecules.The DNA detection methods will be more beneficial for diagnostic of cancer,infectious diseases or other diseases.Gold nanoparticles were prepared through sodium citrate reduction of chlorouric acid.Three kinds of gold nanoparticle probes(AuNPs) were prepared through immobilization with thiolated-oligonucleotides and protein molecules(alkaline phosphatase,AP) respectively.The quality of the particles was monitored with UV-vis spectroscopy and transmission electron microscopy(TEM).The coverage of the oligonucleotide probes on the surface of the AuNPs was determined based on the fluorescencet.These three kindS of AuNP probes were DNA labeled AuNP probes,DNA and AP labeled AuNP probes,and Cy5-DNA labeled AuNP probes,respectively.Through hybridization,three kinds of sandwich structures(gene chips- target DNA- AuNP probes) were formed with three kinds of AuNP probes.Then,silver staining, membrane transfer assay and fluorescence detection were used to amplify the signals on the gene chips.The amount of target DNA was determined by detecting the different signals of the AuNPs.The synthesized gold nanoparticles had uniform,good morphology and dispersed evenly.There were 82～83 DNA probes immobilized onto the surface of gold nnaoparticles.With DNA-labeled AuNPs probes and silver staining methods,dow to 0.1 nmol/L target DNA could be detected.Dow to 1 pmol/L target DNA could be detected by measuring the fluorescent signals of the AuNPs on the gene chips with Cy5-DNA AuNP probes.Gold nanoparticles were modified with detecting probes and signal probes.The ratio of them is 1:5.The different proportions of detecting probes and signal probes could affect the detecting signals significantly.0.01μmol/L target DNA could be detected by measuring the signals on the nylon membrane with DNA and AP labeled AuNPs probes.AP catalyzed substrate solution(BCIP/NBT) into colorful products,which deposite on the nylon membranes.The nucleic acid detection results showed excellent specificity and high sensitivity. The AuNPs could be beneficial to hybridization reaction and amplify the detecting signals.By marking the different signal markers on the AuNPs,the signals could be observed easily.These three methods would be applied widely for ultrasensitive detection of target DNA and gene mutation.