Signal Amplification Based On Magnetic Nanobeads For Ultrasensitive DNA Detection And Its Application In Measure Ment Of Gene Expression Level

DNA is the genetic material and one of the indispensable biological macromolecules in the phenomenon of life. Detecting mutant genes associated with disease has significant meaning for gene screening, early diagnosis and treatment. Therefore, DNA detection methods become more and more important. This thesis developed several novel methods of DNA detection with high sensitivity and selectivity using magnetic beads as the carrier to amplify signal. This technique was successfully applied to gene expression in the cell. The content as follows:In chapter one, we reviewed DNA detection methods with and without labels, and signal amplification techniques including amplification of taget DNA and detected substances briefly.In chapter two, we reported ultrasensitive ECL methods for determination of mRNA in cells using streptavidin-coated magnetic nanobeads (SA-MBs) as the carrier of Ru(bpy)32+-NHS to amplify signal. In this chapter, we used ECL spectrometry to measure gene expression level in cells by using an optical multi-channel analyzer. Using this method, not only the limit of detection for DNA determination was as low as1.2×10-15mol/L, but also the ECL spectrum of Ru(bpy)32+-NHS on the surface of the SA-MBs was obtained. Additionally, we used carbon nanotubes (CNTs) to wrap the Ru(bpy)32+-loaded MBs on the electrode. In this case, the ECL of Ru(bpy)32+was greatly increased. Using this method, DNA of3x10-6mol/L could be detected and mRNA in cells could be quantified.In chapter three, a new signal amplification strategy based on DNA hybridization-dehybridization reaction on the surface of MBs for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MBs modified with probe DNA (DNAP-MBs) were bound to t-DNA (with a ratio of1:1) captured to a substrate. The DNAp-MBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MBs were then hybridized with other Cy5-DNAd to initiate the next hybridization-dehybridization round. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was8.5×10-15mol/L for11cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2(RPLP2) mRNA in human breast cancer cells.In chapter four, a novel multiplexed optical coding technique for single-molecule detection (SMD) of DNAs was developed using300-nm-diameter MBs. The encoded MBs were fabricated by hybridizing three kinds of coding DNAs labeled with different dyes (Cy5, FAM and AMCA), respectively, at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated MBs. Different colors from the encoded MBs could be obtained by overlapping three single-primary-color fluorescence images of the MBs corresponding to emission of Cy5(red), FAM (green) and AMCA (blue). Intraccllular multi-gene expression analysis demonstrated the DNA coding technique for SMD.