title: Non-fluorescent quantification of single mRNA with transient absorption microscopy.
abstract: 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.
authors.affiliation: Bindley Bioscience Center and Birck Nanotechnology Center, Agriculture & Biological Engineering, Purdue University, West Lafayette, IN 47907, USA. josephi@purdue.edu and Nanoscience and Nanoengineering, South Dakota School of Mines and Technology, Rapid City, SD 57701, USA. Jing.Liu@sdsmt.edu and Biochemical Spatio-Temporal NetWork Resource (BioSNTR), State of South Dakota, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Bindley Bioscience Center and Birck Nanotechnology Center, Agriculture & Biological Engineering, Purdue University, West Lafayette, IN 47907, USA. josephi@purdue.edu.
authors.forename: Joseph M K
authors.initials: JM
authors.lastname: Irudayaraj
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics; D006046:Gold; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D061986:MCF-7 Cells; D053768:Metal Nanoparticles; D008853:Microscopy; D012333:RNA, Messenger / Q000302:isolation & purification*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*
chemical_list: D006046:Gold; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human