270 Use of Polylysine as a Carrier for Signal Amplification in DNA Detection

Thursday, November 5, 2009: 12:20 PM
Rio Grande (Camino Real Hotel)
Hong Qian , Chemistry, North Carolina State University, Raleigh, NC
The positive charged polylysine (PLL) forms stable complex with negative charged DNA (DNA/PLL). Modification of amino groups on the side chain of polylysine with desired functional groups such as polymerization initiators or fluorophores provides an easy way to bring multiple functional groups to each DNA/PLL complex. Further signal amplification is achieved via polymerization which renders direct visualization of the presence of DNA by naked eye, or signal can be directly detected via fluorescence instrument with extraordinary sensitivity. The use of positively charged PLL as a carrier for signal amplification eliminates the need for covalent modification of individual detection probes prior to DNA detection and provides a simplified label-free detection scheme, which is of importance for high throughput detection.  In polymer-based detection, AGET ATRP (activators generated by electron transfer for atom transfer radical polymerization) was adapted as the method of polymerization to realize a well controlled, quantitative detection without the needs of oxygen-free environment. The effect of PLL length, modification ratio of PLL, and the concentration of PLL were studied. It was found use of longer PLL macroinitiator yields thicker polymer film atop of target DNA molecules and the optimal modification ratio of PLL was around 55% to ensure stable complexation of DNA with PLL and bring the most of initiators to DNA/PLL complex. The maximum polymer film thickness was reached at 48 uM PLL concentration (in repeat units) despite the different modification ratio. Under optimized condition, as low as 1nM of DNA was detected. In fluorescence-based detection, DNA was detected directly after the adsorption of PLL macrofluorophores. The limit of detection was improved to 75 fM, corresponding to 104 improvement compared with polymer-based detection.
This work is partially supported by NSF (No.0644865).