Department of Applied Chemistry, School of Engineering, The University of Tokyo, Hongo, Bunkyo, Japan.
Anal Chem. 2011 May 1;83(9):3352-7. doi: 10.1021/ac103185j. Epub 2011 Apr 4.
The rolling circle amplification (RCA) is a versatile DNA amplification method in which a DNA molecule is amplified using a single DNA primer, allowing the product to be counted as a single dot. Circular templates for RCA can arise from padlock probes in highly specific DNA target-mediated ligation reactions. However, improvement of detection efficiency represents an important challenge. In homogeneous assays, the detection efficiency is generally only under 0.1%, mainly because the sample volume is too large compared with the detection volume. Here, we used microchannel surfaces in a glass microchip for DNA detection in small volume samples. First, DNA patterning on glass surfaces in microchannels was demonstrated using chemical surface patterning by UV light. By using a photochemical reaction, we realized DNA patterning in a closed space. Second, RCA was demonstrated using dilutions of target molecules, and a calibration curve was obtained. The highest detection efficiency was 22.5% by virtue of the reduced sample volumes from several hundred microliters to 5.0 nL. Accordingly, a countable number of DNA molecules was successfully detected. This method is suitable for analysis of very small volume samples such as single cells, especially by using extended-nanochannels with dimensions of 10-1000 nm.
滚环扩增（RCA）是一种通用的 DNA 扩增方法，它使用单个 DNA 引物扩增 DNA 分子，使产物可以被计为一个单个点。RCA 的环状模板可以来自于高度特异性 DNA 靶介导连接反应中的发夹探针。然而，提高检测效率是一个重要的挑战。在均相测定中，检测效率通常低于 0.1%，主要是因为与检测体积相比，样品体积太大。在这里，我们在玻璃微芯片中的微通道表面上用于小体积样品的 DNA 检测。首先，通过紫外线的化学表面图案化证明了微通道玻璃表面上的 DNA 图案化。通过光化学反应，我们在封闭空间中实现了 DNA 图案化。其次，通过目标分子的稀释来证明 RCA，并获得校准曲线。通过将样品体积从几百微升到 5.0 nL 减少，最高检测效率达到 22.5%。因此，成功地检测到了可计数数量的 DNA 分子。这种方法适用于非常小体积样品的分析，例如单细胞，特别是通过使用尺寸为 10-1000 nm 的扩展纳米通道。
