Bui Minh-Phuong Ngoc, Baek Taek Jin, Seong Gi Hun
Department of Applied Chemistry, Hanyang University, Ansan, South Korea.
Anal Bioanal Chem. 2007 Jul;388(5-6):1185-90. doi: 10.1007/s00216-007-1354-4. Epub 2007 May 30.
The potential ability of atomic force microscopy (AFM) as a quantitative bioanalysis tool is demonstrated by using gold nanoparticles as a size enhancer in a DNA hybridization reaction. Two sets of probe DNA were functionalized on gold nanoparticles and sandwich hybridization occurred between two probe DNAs and target DNA, resulting in aggregation of the nanoparticles. At high concentrations of target DNA in the range from 100 nM to 10 microM, the aggregation of gold nanoparticles was determined by monitoring the color change with UV-vis spectroscopy. The absorption spectra broadened after the exposure of DNA-gold nanoparticles to target DNA and a new absorption band at wavelengths >600 nm was observed. However, no differences were observed in the absorption spectra of the gold nanoparticles at low concentrations of target DNA (10 pM to 10 nM) due to insufficient aggregation. AFM was used as a biosensing tool over this range of target DNA concentrations in order to monitor the aggregation of gold nanoparticles and to quantify the concentration of target DNA. Based on the AFM images, we successfully evaluated particle number and size at low concentrations of target DNA. The calibration curve obtained when mean particle aggregate diameter was plotted against concentration of target DNA showed good linearity over the range 10 pM to 10 nM, the working range for quantitative target DNA analysis. This AFM-based DNA detection technique was three orders of magnitude more sensitive than a DNA detection method based on UV-vis spectroscopy.
通过在DNA杂交反应中使用金纳米颗粒作为尺寸增强剂，证明了原子力显微镜（AFM）作为定量生物分析工具的潜在能力。两组探针DNA在金纳米颗粒上进行了功能化，并且在两个探针DNA与靶DNA之间发生了夹心杂交，导致纳米颗粒聚集。在100 nM至10 μM范围内的高浓度靶DNA下，通过用紫外可见光谱监测颜色变化来确定金纳米颗粒的聚集情况。DNA-金纳米颗粒与靶DNA接触后，吸收光谱变宽，并且在波长>600 nm处观察到一个新的吸收带。然而，在低浓度靶DNA（10 pM至10 nM）下，由于聚集不足，金纳米颗粒的吸收光谱没有观察到差异。在该靶DNA浓度范围内，AFM被用作生物传感工具，以监测金纳米颗粒的聚集并定量靶DNA的浓度。基于AFM图像，我们成功地评估了低浓度靶DNA下的颗粒数量和尺寸。当平均颗粒聚集体直径与靶DNA浓度作图时获得的校准曲线在10 pM至10 nM范围内显示出良好的线性，这是定量靶DNA分析的工作范围。这种基于AFM的DNA检测技术比基于紫外可见光谱的DNA检测方法灵敏三个数量级。
