Assad Ossama N, Di Fiori Nicolas, Squires Allison H, Meller Amit
Department of Biomedical Engineering, Boston University , Boston, Massachusetts 02215 United States.
Nano Lett. 2015 Jan 14;15(1):745-52. doi: 10.1021/nl504459c. Epub 2014 Dec 22.
Optical sensing of solid-state nanopores is a relatively new approach that can enable high-throughput, multicolor readout from a collection of nanopores. It is therefore highly attractive for applications such as nanopore-based DNA sequencing and genotyping using DNA barcodes. However, to date optical readout has been plagued by the need to achieve sufficiently high signal-to-noise ratio (SNR) for single fluorophore sensing, while still maintaining millisecond resolution. One of the main factors degrading the optical SNR in solid-state nanopores is the high photoluminescence (PL) background emanating from the silicon nitride (SiNx) membrane in which pores are commonly fabricated. Focusing on the optical properties of SiNx nanopores we show that the local membrane PL intensity is substantially reduced, and its spectrum is shifted toward shorter wavelengths with increasing e-beam dose. This phenomenon, which is correlated with a marked photocurrent enhancement in these nanopores, is utilized to perform for the first time single molecule fluorescence detection using both green and red laser excitations. Specifically, the reduction in PL and the concurrent measurement of the nanopore photocurrent enhancement allow us to maximize the background suppression and to detect a dual color, five-unit DNA barcode with high SNR levels.
固态纳米孔的光学传感是一种相对较新的方法，能够实现对纳米孔集合的高通量、多色读出。因此，它对于基于纳米孔的DNA测序和使用DNA条形码进行基因分型等应用极具吸引力。然而，迄今为止，光学读出一直受到单荧光团传感需要实现足够高的信噪比（SNR）的困扰，同时还要保持毫秒级分辨率。固态纳米孔中光学SNR下降的主要因素之一是通常制造有孔的氮化硅（SiNx）膜发出的高光致发光（PL）背景。聚焦于SiNx纳米孔的光学特性，我们表明，随着电子束剂量的增加，局部膜PL强度显著降低，其光谱向更短波长移动。这种现象与这些纳米孔中显著的光电流增强相关，首次被用于使用绿色和红色激光激发进行单分子荧光检测。具体而言，PL的降低以及纳米孔光电流增强的同时测量使我们能够最大限度地抑制背景，并以高SNR水平检测双色五单元DNA条形码。
