Journal Of The American Chemical Society | September 3, 2008
Zhaohui Li, Ryan B. Hayman and David R. Walt
Journal of the American Chemical Society
DOI: 10.1021/ja8053018
Abstract: Improving the sensitivity of DNA biosensors is extremely important in clinical diagnostics, gene therapy, and a variety of other biomedical studies. In this regard, we have developed a highly sensitive single molecule DNA assay platform with a 1fM experimental detection limit using enzymatic amplification in an array of femtoliter-sized reaction wells. To validate the utility of this technology in our study, we employed a fiber optic array to create thousands of femtoliter-sized reaction wells, each specifically functionalized with oligonucleotide probes capable of capturing biotinylated target DNA. After hybridization, the fiber was incubated with streptavidin-labeled enzyme solution. The bound single enzyme molecules were confined to individual reaction vessels containing excess fluorogenic substrate and catalyzed the production of a sufficient number of fluorescent product molecules to generate a detectable signal. At low target DNA concentrations with relatively short incubation times, only a small percentage of the capture sites bind target DNA, enabling a binary readout of target concentration from the high-density fiber array. This simple binary readout-based scheme is easy to perform and exhibits a high signal-to-noise ratio in the presence of trace amounts of DNA target. Furthermore, it also should be possible to extend this technology to protein detection by modifying the reaction wells with specific capture antibodies. We expect this assay to be useful in a number of biomedical applications where accurate and highly sensitive target analysis is critical.
Related Posts
Neurology
Comparative Spatial Analyses of the Tumor Immune Landscape in Mouse Models of Glioblastoma Using the PhenoCode Discovery Mouse Neuro Panel and Customized Markers
Poster Background Developing effective therapies for glioblastoma (GBM) relies on robust preclinical animal models that enable detailed investigation of tumor biology, discovery of therapeutic targets, and evaluation of new treatment…
Oncology
Development of a Spatial Metabolic Map of Immunotherapy Sensitive and Resistant Cutaneous Skin Carcinoma
Poster Background Ultrahigh-Plex Single-cell Spatial Phenotyping has revealed valuable insights into the tumor immune microenvironment (TiME) for biomarker discovery and stratification of clinical responses. Metabolic reprogramming is a key hallmark…
Spatial Immunometabolic Profiling of Non-Small Cell Lung Cancer Reveals Distinct Immune Exclusion Patterns and Functional Niches
Analytical validation of a multiplex immunofluorescence 7-Biomarker Immunoprint® assay on the Akoya PhenoImager HT platform for stage I/II Melanoma
Neurology
Neurodegenerative biomarkers in different chambers of the eye relative to plasma: an agreement validation study
Alzheimer’s Research and Therapy | August 26, 2024 Sampani K, Ness S, Tuz-Zahra F, Aytan N, Spurlock EE, Alluri S, Chen X, Siegel NH, Alosco ML, Xia W, Tripodis Y,…
Neurology
Clinical validation of the LucentAD p-Tau 217 as a lab developed test (LDT) for clinical use
Poster The intersection of new therapeutic options for Alzheimer’s disease (AD), the emergence of accurate blood-based tests for AD, and the anticipated health system log jam for confirmatory diagnostic testing…
Neurology
Development of thresholds and a visualization tool for use of a blood test in routine clinical dementia practice
Alzheimer’s & Dementia | August 3, 2024 Verberk IMW, Jutte J, Kingma MY, Vigneswaran S, Gouda MMTEE, van Engelen MP, Alcolea D, Arranz J, Fortea J, Lleó A, Chevalier C,…
Neurology
Discovering novel plasma biomarkers for ischemic stroke: Lipidomic and metabolomic analyses in an aged mouse model
Journal of Lipid Research | August 2, 2024 Becktel DA, Frye JB, Le EH, Whitman SA, Schnellmann RG, Morrison HW, Doyle KP. J Lipid Res. 2024 https://doi.org/10.1016/j.jlr.2024.100614 Abstract Ischemic stroke…