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Scientific Reports Jul 2015Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions...
Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ± 1-2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.
Topics: Antibodies, Viral; Antigen-Antibody Reactions; Dynamic Light Scattering; Influenza A Virus, H1N1 Subtype; Nanotechnology; Optical Tweezers; Staining and Labeling
PubMed: 26160194
DOI: 10.1038/srep12087 -
Analytical Chemistry Sep 2014Molecular diagnostics based on the polymerase chain reaction (PCR) offer rapid and sensitive means for detecting infectious disease, but prohibitive costs have impeded...
Molecular diagnostics based on the polymerase chain reaction (PCR) offer rapid and sensitive means for detecting infectious disease, but prohibitive costs have impeded their use in resource-limited settings where such diseases are endemic. In this work, we report an innovative method for transforming a desktop computer and a mobile camera phone--devices that have become readily accessible in developing countries--into a highly sensitive DNA detection system. This transformation was achieved by converting a desktop computer into a de facto thermal cycler with software that controls the temperature of the central processing unit (CPU), allowing for highly efficient PCR. Next, we reconfigured the mobile phone into a fluorescence imager by adding a low-cost filter, which enabled us to quantitatively measure the resulting PCR amplicons. Our system is highly sensitive, achieving quantitative detection of as little as 9.6 attograms of target DNA, and we show that its performance is comparable to advanced laboratory instruments at approximately 1/500th of the cost. Finally, in order to demonstrate clinical utility, we have used our platform for the successful detection of genomic DNA from the parasite that causes Chagas disease, Trypanosoma cruzi, directly in whole, unprocessed human blood at concentrations 4-fold below the clinical titer of the parasite.
Topics: Cell Phone; Chagas Disease; Coloring Agents; DNA, Protozoan; Electrophoresis, Polyacrylamide Gel; Humans; Microcomputers; Polymerase Chain Reaction; Software; Trypanosoma cruzi
PubMed: 25223929
DOI: 10.1021/ac5022419 -
Scientific Reports Nov 2016We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open...
We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is "fit-for-all", well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.
Topics: Animals; Axin Protein; Breast Neoplasms; Chromatography, Liquid; Exosomes; Female; Humans; Hydroxycholesterols; Mice; Peptides; Proteomics; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry
PubMed: 27897190
DOI: 10.1038/srep37507