Detection Of Cancer Cell DNA Using Nanoparticle Signal Amplification Technology

DNA is the basic genetic material of an organism.It has the function of storing,reproducing and transferring genetic information.It plays a very important role in every stages of life activities of organisms.Specific DNA is closely related to cell carcinogenesis.The detection of such DNA can detect early cancer cells at the genetic level,so has great significance for the diagnosis of cancer and the development of new anticancer drugs.The existing DNA detection technology mostly amplifies the target DNA to enhance the signal intensity.However,in many cases,the target DNA cannot be directly amplified and its concentration is too low to be directly detectable,so it is necessary to develop the technique of amplifying the signal of the DNA detector.Fluorescence in situ hybridization(FISH)technology can detect tissues,cells and nucleic acids in chromosomes in morphology,and display the location of the target nucleic acids.According to the mutation genes of cancer cells,DNA probes designed with specific hybridization can effectively detect cancer cells.Nanoparticles have many advantages such as volume effect,quantum size effect and surface effect,which can amplify the signal by modifying specific antibodies.In this paper,DNA,a biological macromolecule,was taken as the research object.By combining nanoparticle signal amplification technology with fluorescence in situ hybridization technology,a new high sensitive and selective detection method based on signal amplification of detectors was developed,which could be used to detect DNA of cancer cells.Surface carboxylated magnetic nanoparticles(MNP_S)were prepared by chemical coprecipitation using iron salts and fulvic acid,and carboxylated MNP_S was covalently coupled with anti-digoxigenin antibody(Anti DIG)by amino coupling.The UV spectra of MNPS coupled with Anti DIG under different experimental conditions were measured by UV spectrophotometer,and the optimal coupling conditions were derived from the UV spectra.The MNP_S-Anti DIG was applied to FISH to detect DNA signals in SMCC-7721cancer cells by labeling DNA probes.A control experiment was designed to investigate its sensitivity,accuracy and specificity.The results showed that carboxylated MNP_S were successfully prepared.In a 37?water bath,when the volume ratios of carboxylated MNPs(51.7?g/mL),Anti DIG(1.0mg/mL)and Carbondiimide/N-Hydroxysuccinimide(0.4mol/L/0.1 mol/L)were 1:2:2,the coupling rate of MNP_S-Anti DIG was the highest.Its average diameter is about 20 nm.The particles are nearly spherical and uniformly dispersed without agglomeration.Successfully labeled digoxigenin on SC6-BAC-MBD4 probe by random primer method,and used this probe to perform fluorescence in situ hybridization based on MNP_S-Anti DIG,successfully detected the fluorescence signal of SMCC-7721 cells and detected normal liver.The cells did not show a fluorescent signal,and the unlabeled digoxigenin SC6-BAC-MBD4 probe was used to detect the absence of fluorescent signal production in liver cancer cells.The fluorescence intensity of fluorescence in situ hybridization based on MNP_S-Anti DIG detected by fluorescence spectrometer is much higher than that of traditional fluorescence in situ hybridization.The experimental results demonstrated that fluorescence in situ hybridization based on nanoparticles-could detect unamplified low-concentration DNA,amplify the signal of the DNA detection objects,and detect cancer cells due to the specificity of the probe.