Bindley Bioscience Center and Birck Nanotechnology Center, Agriculture & Biological Engineering, Purdue University, West Lafayette, IN 47907, USA.
Nanoscale. 2016 Nov 24;8(46):19242-19248. doi: 10.1039/c6nr04433f.
Single molecule detection is confounded by the background signals from the biological environment, such as autofluorescence, Rayleigh scattering, or turbidity in cells and tissues. In this article, we report on the utilization of gold nanoparticles (AuNPs) as an orthogonal probe for non-fluorescence detection of single molecules with a transient absorption microscopy (TAM). The developed system and concepts were validated by quantitative evaluation of human epidermal receptor 2 (Her2) mRNA in cancer cells and tissues at single copy sensitivity. Results from TAM suggest that the average number of Her2 copies in SK-BR-3 and MCF-7 breast cancer cells is 203.19 ± 80.48, and 11.29 ± 4.47, respectively. Furthermore, TAM offers excellent signal-to-noise ratio in detecting mRNA in clinical tissues, indicating a significantly higher expression of Her2 genes in breast cancer tissues than that of normal tissues. Our single cell quantification TAM strategy was validated with a fluorescence in situ hybridization approach. Our demonstration shows that TAM has the potential to provide a new dimension in biomarker quantification at single molecule sensitivity in turbid biological environments providing a strong basis for clinical monitoring.
单分子检测受到生物环境背景信号的干扰，如 autofluorescence、Rayleigh scattering 或细胞和组织中的浑浊度。在本文中，我们报告了利用金纳米粒子 (AuNPs) 作为瞬态吸收显微镜 (TAM) 中非荧光单分子检测的正交探针。通过在单细胞灵敏度下定量评估癌细胞和组织中的人类表皮受体 2 (Her2) mRNA，对开发的系统和概念进行了验证。TAM 的结果表明，SK-BR-3 和 MCF-7 乳腺癌细胞中 Her2 拷贝的平均数量分别为 203.19 ± 80.48 和 11.29 ± 4.47。此外，TAM 在检测临床组织中的 mRNA 时具有出色的信噪比，表明乳腺癌组织中 Her2 基因的表达明显高于正常组织。我们的单细胞定量 TAM 策略通过荧光原位杂交方法进行了验证。我们的研究表明，TAM 有可能在浑浊的生物环境中单分子灵敏度下提供生物标志物定量的新维度，为临床监测提供了坚实的基础。
